HK1197088B

HK1197088B - Biomarker discovery in complex biological fluid using bead or particle based libraries and diagnostic kits and therapeutics

Info

Publication number: HK1197088B
Application number: HK14110612.1A
Authority: HK
Inventors: M．R．穆拉; J．席尔克
Original assignee: 欧科制药有限责任公司
Priority date: 2011-03-24
Filing date: 2012-03-22
Publication date: 2019-07-05

Description

Biomarker discovery in complex biological fluids using bead or particle based libraries and diagnostic kits and therapeutics

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applications 61/467,256, 61/491,717, and 61/583,881, filed on 3/24/2011, 5/31/2011, and 1/6/2012, respectively, all of which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to novel screening methods and diagnostic and therapeutic products derived therefrom. In particular, a novel large bead-based library comprising a rich diversity of small molecules, peptides, peptoids, and/or other oligomers is used to screen biological samples for disease-associated biomarkers. The present invention allows rapid and direct screening of plasma, serum or other biological fluids to find disease-associated antibodies in a range of diseases and also to find antibody-specific molecules that can serve as diagnostic tools or as therapeutics for the diseases. Diagnostic kits comprising such antibody-specific molecules can be prepared for essentially any disease state with an antibody or immunogenic component, such as autoimmune diseases, central nervous system disorders, and cancer. Such kits can be made from essentially any known carrier system, provided that the system can support or bind to an antibody-specific molecule such as a peptoid or other ligand binding moiety. Similarly, any known detection method, including ELISA or other known detection methods, can be used to detect antibodies after an initial screen to find putative hits, and/or to use such putative hits in diagnostic assays after a diagnostic screen. Such methods may also be used to screen for other biomarkers, including proteins and/or other biomarkers on the surface of cells, to distinguish cells expressing disease-related markers from healthy cells that do not express such markers.

Background

U.S. patent publication 2007/0003954 discloses protein and antibody profiling using small molecule microarrays. Ligands that bind to ligand binding moieties are disclosed, wherein the ligands are arranged in a synthetic molecular array for screening biomarkers and molecular fingerprints. The specific arrays described therein include, for example, peptoid microarrays having 7680 different compounds bound to the array. In that disclosure, a bead-based library is used as a starting method for preparing a peptoid that is subsequently transferred to a microarray to screen biological fluids with addressable locations on the microarray. Screening results in a unique pattern or molecular fingerprint on the array for any particular protein in a complex biological mixture. U.S. patent application 2010/0303805, which is incorporated herein by reference, discloses specific peptoids and diagnostic arrays that can be used to screen biomarkers associated with central nervous system disorders in biological fluids. The specific monomers disclosed therein for use in forming arrays therein may also be used in the novel screening methods of the invention, provided that the library is expanded to a much larger number of beads/peptoids or beads/ligands, e.g., greater than 100K to 150 MM.

The present inventors have found that significantly larger bead-based libraries (as opposed to microarray-based screens for antibody biomarkers or bead-based screens for cells) can be used to directly screen complex biological samples under appropriate conditions to find disease-associated biomarkers and significantly larger libraries of ligands that bind to such ligand-binding moieties. This significantly larger library includes a significantly improved number of high affinity ligands, which serve as diagnostic tools as well as potential therapeutics. This approach also allows for significantly improved screening rates for any particular complex biological fluid, as the need to prepare microarrays or similar addressable carrier systems is avoided in the first place. Once the screening is performed, microarrays or other carrier systems containing hits found in the screening can be fabricated, including diagnostic arrays, and are included within the scope of the invention.

Disclosure of Invention

the term "random" includes those libraries having a rich diversity of side chains on monosubstituted amines that form any particular disease or condition, and each library may be used for screening against a different disease or condition, or the same library may be used for screening for a variety of disease states or conditions the term "random" includes those libraries having a rich diversity of side chains on monosubstituted amines that form any particular monomer in an oligomeric chain, even if some of the chemical and/or physical characteristics on any particular monomer are, for example, functional/soluble portions of a desired character or characteristic of a target oligomer, this diversity of R groups on amine starting materials is also "random". for plasma-based screening or serum screening, for example, any particular ligand that is desired to bind to a bead has solubility characteristics that facilitate interaction with a ligand binding moiety such as an antibody in solution, in addition, the size of the oligomer is also considered in forming a ligand, when the target moiety is, such as an antibody or protein, the ligand may be bound to a bead binding moiety such as a glycine-binding moiety, or peptide-binding moiety, such as a peptide-amino acid-amino acid-amino acid-.

Beads/vectors with ligands comprising the library are also subsequently obtained in the process of the invention. The invention encompasses a process for screening for disease-associated biomarkers in a biological fluid comprising the steps of screening a biological control sample and a biological diseased sample with at least one bead-based ligand library, and using such screening to discover disease-associated biomarkers. The invention encompasses a process for screening a complex biological sample for the presence of a disease-associated biomarker comprising exposing the sample to a plurality of ligand-containing carriers, wherein at least one ligand detectably binds to the disease-associated biomarker. The invention also encompasses a method of screening for disease-associated biomarkers in a complex biological sample comprising the steps of (1) exposing a random ligand library to a control sample to identify and remove any non-specific ligand hits, and (2) exposing the remaining ligand library to a diseased sample to identify any ligands associated with the disease-associated biomarkers in the diseased sample. In particular, the invention encompasses a process for screening a biological sample for disease-associated biomarkers comprising (1) pretreating a ligand-containing bead (LBB) library with a suitable solvent to form treated LLBs; (2) exposing the treated LLB to a Normal Control (NC) biological sample having a control sample ligand binding moiety; (3) treated LBB from control samples was exposed to Dynabead screening (iron-labeled anti-IgG antibody) and hits were removed; (4) washing the remaining LBB library and exposing the library to an NC biological sample with any remaining control sample ligand binding moieties using quantum dot-labeled secondary anti-IgG antibodies, and taking hits; (5) washing the remaining LBB library and exposing the library to a biological sample from a patient with a disease; (6) exposing treated LBB from diseased samples to a Dynabead screen and taking hits; (7) washing the remaining LBB and exposing the library to a biological sample from a patient with a disease; (8) adding quantum dot labeled secondary anti-IgG antibodies to the washed LBB and identifying disease-associated ligand binding moieties that bind to the ligand on the LBB, and optionally, after washing the dynabeads from step (6), repeating step (8) using the Dynabead hits from step (6) and identifying Dynabead Qdot hits. In a preferred embodiment, Tentagel beads (with an embedded PEG linker) are used in the preparation of LBB. Alternative beads and/or particles having different and/or optional linkers may also be used in conjunction with alternative detection methods. The beads may also be selected from, for example, Luminex beads. In a preferred procedure, the Dynabead step is not utilized, except as an initial confirmation step to confirm a Qdot hit.

in a preferred embodiment, the ligand is a peptoid and the peptoid is sequenced to identify and/or confirm or reconfirm the identity of a putative diagnostic probe, which may also be used in a diagnostic kit or as the basis for a therapeutic drug or vaccine candidate, depending on the particular disease or condition.

The invention also encompasses diagnostic kits for using the ligands (or modified forms thereof) identified in the biological sample screening methods. The ability to specific screens leads to the rapid identification of a significant number of actual hits that are also used in such diagnostic kits. The term "fast" in this case means that the present process avoids complex and unnecessary steps of constructing a microarray before having to analyze complex biological fluids, which thus results in a significant saving in time. Furthermore, the present method allows a much larger number of molecules to be screened against complex biological fluids at any one time, rather than being limited to a small number on a microarray. In addition to the significant number of hits found, the ligands found included a significantly greater number of high affinity binders relative to those found using previous screening methods that did not use such bead or particle based techniques to directly screen or assay complex biological fluids. Such ligands may be used in a multiplex disease platform comprising a first peptoid screened for disease or condition a and at least one additional peptoid screened for disease or condition B.

in addition, diagnostic arrays constructed from peptoids, alpha-substituted peptoids, or ligands found in the initial screen can be used in clinical trials to identify or help identify patient stratification and/or disease progression in any particular patient population or subpopulation.

Other features and advantages of the present invention will become apparent from the following detailed description of examples and the accompanying drawings. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Drawings

The following drawings form part of the present specification.

FIG. 1 shows a basic chemical schematic of the preparation of a Tentagel bead (KN1B) library for screening of Alzheimer's serum samples. Figure 1A shows starting from polystyrene beads with amino groups as reactants (PEG or equivalent or alternative linkers can be formed between the beads and the terminal amino groups). Figure 1B shows the starting amino acid as methionine on the beads and which is subsequently reacted to form the compound shown in B. FIG. 1C shows the sub-monomers (monomeric amines and haloacetic acids) used to form an oligomer library of compounds.

FIG. 2 shows a basic chemical schematic of the preparation of a Tentagel bead (JC3B) library also used to screen Alzheimer's serum samples. Figure 1A shows starting from polystyrene beads with amino groups as reactants (PEG or equivalent or alternative linkers can be formed between the beads and the terminal amino groups). Figure 1B shows the starting amino acid on the beads as methionine, and which is subsequently reacted to form the compound shown in B, and figure 1C shows the sub-monomers (monomeric amines and haloacetic acids) used to form the oligomer library of compounds. JC3B was also used to screen pancreatic cancer sera (data not shown).

FIG. 3 shows a basic chemical schematic of the preparation of a Tentagel bead (JC4B) library for screening of Alzheimer's serum samples. Figure 1A shows starting from polystyrene beads with amino groups as reactants (PEG or equivalent or alternative linkers can be formed between the beads and the terminal amino groups). Figure 1B shows the starting amino acid as methionine on the beads and which is subsequently reacted to form the compound shown in B. FIG. 1C shows the sub-monomers (monomeric amines and haloacetic acids) used to form an oligomer library of compounds.

FIG. 4 shows a basic chemical schematic of the preparation of a Tentagel bead (JC5B) library for screening of Alzheimer's serum samples. Figure 1A shows starting from polystyrene beads with amino groups as reactants (PEG or equivalent or alternative linkers can be formed between the beads and the terminal amino groups). Figure 1B shows the starting amino acid as methionine on the beads and which is subsequently reacted to form the compound shown in B. FIG. 1C shows the sub-monomers (monomeric amines and haloacetic acids) used to form an oligomer library of compounds. JC5B monomers include isobutylamine, 2-methoxyethylamine, diaminobutane, furfurylamine, cyclohexylamine, R-methylbenzylamine, piperonylamine and 4- (aminoethyl) benzenesulfonamide.

FIG. 5 shows a basic chemical schematic of the preparation of a Tentagel bead (JC7B) library for screening serum samples. Figure 1A shows starting from polystyrene beads with amino groups as reactants (PEG or equivalent or alternative linkers can be formed between the beads and the terminal amino groups). Figure 1B shows the starting amino acid as methionine on the beads and which is subsequently reacted to form the compound shown in B. FIG. 1C shows the sub-monomers (monomeric amines and haloacetic acids) used to form an oligomer library of compounds.

FIG. 6 shows a schematic of the inventive process for screening complex biological samples using a bead-based peptoid ligand library.

Figure 7 shows the Normal Control (NC) Dynabead hits following QDot addition in peptoid libraries (JC3B) prepared to be screened against alzheimer's normal control serum samples and alzheimer's diseased serum samples. Hits are picked and the remaining ligand-bound beads are used in a disease-based screen.

Figure 8 shows the Tentagel bead screen of diseased serum from blood samples from alzheimer patients after NC hits were removed. Hits are shown in red, which are Qdot secondary antibodies bound to disease-associated biomarkers (antibodies) in serum, which are bound to a peptoid attached to a bead by a PEG linker.

Figure 9 shows the reproducibility test using a normal control sample (NC 030093) after SDS wash and QDOT addition. Arrows show which NC peptoid hits were picked for the sequences.

Figure 10 shows the reproducibility test using a normal control sample (NC050047) after SDS wash and QDOT addition.

Figure 11 shows a reproducibility test using diseased samples after SDS wash and QDOT addition.

Figure 12 shows the peptoid sequences from JC3B library selected from the alzheimer's screen for putative hits. The C-terminus is on the right side of the sheet and the N-terminus is on the left side.

Figure 13 shows the chemical structure of a preferred high affinity hit from alzheimer's screening from JC3B library. In this example, the structures shown have cysteine residues and are synthesized after determining the initial hit structure in the preliminary screen. The JC3B library contained similar peptoids, but with a methionine residue instead of a cysteine residue at the C-terminus.

FIG. 14 shows competition experiments between high affinity ligand (ADTG1) in solution versus ADTG-1-ADTG-42 on microarray supports. This competition experiment shows that ADTG1 in solution binds to the same antibody that has bound to the peptoids ADTG-1, ADTG14, ADTG24, ADTG25, ADTG31, ADTG35, and ADTG40 on the microarray. Similar experiments were performed on each peptoid to find four sets of peptoids that bound four different alzheimer's autoantibodies (data not shown).

FIG. 15 shows four different classes of peptides that bind to different autoantibodies in the Alzheimer's screen. Each group on the figure has a higher affinity adhesive on top.

Figure 16A shows AD test data (blinded) for a cohort of patients using P1aag1(JC3B-1) peptoids, and figure 16B shows test data (blinded) for the same AD patient cohort using P1aag2(JC 3B-21). Each peptoid is presented on a microarray.

Figure 17A shows AD test data (blinded) for a cohort of patients using P1aag3(JC3B-7) peptoids, and figure 17B shows test data (blinded) for the same AD patient cohort using P1aag4(JC 3B-5). Each peptoid is presented on a microarray.

Figure 18A shows AD test data (blinded) for a cohort of patients using P1aag5(JC3B-R8) peptoids, and figure 18B shows test data (blinded) for the same AD patient cohort using P1aag6(JC 3B-R12). Each peptoid is presented on a microarray.

Figure 19 shows ADP2 microarray data in the same patient population for testing with P1aag 1-6. Fig. 19B shows comparative data using P1aag4 for the same patient group. This data shows a clear correlation between the results achieved with previously identified ADP2 and newly identified P1aag4 in the same patient population.

Fig. 20A shows microarray data for ADP3 in the same patient group for tests performed using P1aag1-6, and fig. 20B shows comparative data using P1aag2 for the same patient group. This data shows a clear correlation between the results achieved with previously identified ADP3 and newly identified P1aag2 in the same patient population.

Figure 21 shows P1aag5 (putative hit 5 or JC3B-R8) confirmation on TentaGel beads in comparison of diseased AD sera at 40ug/mL versus healthy controls (pooled).

Figure 22A shows peptoid hits in pancreatic cancer screens using QDot655 and using JC5B library. FIGS. 22B and C show hit reconfirmation using QDot655 (arrows point to hits).

Figure 23 shows pancreatic peptoid hit confirmation and comparison of disease serum addition and detection with QDot655 versus normal serum addition.

Fig. 24 shows hit confirmation by mixing the AD tag and the PC tag. The data show that PC labeling was detected while no detectable antibodies were present on the AD peptoid beads in the pancreatic cancer serum (serum 1).

Figure 25 shows pancreatic cancer screening hits from JC3B library.

Figure 26 shows pancreatic cancer screening hits from JC5B library.

Fig. 27A, B and C show the results of SLE (lupus) screening. A is a normal control, and B and C are SLE sera from two different groups 1 and 2. The arrow points to a hit.

Fig. 28 shows SLE hits from KN1B library. The C-terminus is on the right side of the sheet.

FIG. 29 shows the hit confirmation for the peptoid KN 1B-20. Group 1 was pooled diseased serum at a concentration of about 0.374mg/mL (left panel) (hits are shown on beads in red shade). Non-diseased pooled sera (middle panel) were provided at a concentration of about 0.378mg/mL, and the right-most panel shows a serum-free control.

Figure 30 shows the binding/detection of one of the SLE (lupus) peptoids to an ELISA plate using two different binding methods using fluorescein labeling with peptoids at different concentrations.

FIG. 31 shows competition assays between plate-bound KN 1B-20-biotin-fluorescein versus free KN 1B-20-biotin at different concentrations in solution. Signal dampening occurs as binding from equimolar concentrations increases relative to free KN 1B-20-biotin concentration.

FIG. 32 shows ELISA plates with peptoids at different concentrations and clearly shows the difference between diseased serum (AD) (column 1P) and normal control serum (column 3) [1:200 doubling to 1:400, 1:800, 1:1,600, 1:3,200, 1:6,400, 1:12,800 per well ]. Arrows point to 1:800 dilution in 1XTBST buffer. The concentration of peptoid in the wells was 10 mM. Figure 32 also shows confirmation of the TentaGel bead platform to distinguish between diseased and control sera.

Figure 33 shows ELISA plates with 10mM ADP3 and at different dilutions of AD serum versus control serum. Arrows point to 1:800 dilution.

FIG. 34 shows ELISA plates with 10mM SLE-KN1B-20 and different dilutions of AD serum versus control serum. Arrows point to 1:800 dilution.

Figure 35 shows AD serum ELISA plots using 10mM ADP3 prepared in binding buffer at different serum dilutions. Separation between normal and diseased serum occurs over a dilution range of 1:200 up to about 1:10,000. The initial dilution was 1:200 (group 1 with. 394mg/mL AD serum and. 386mg/mL non-diseased serum).

Figure 36 shows SLE serum ELISA profiles using 10mm kn1B-20 prepared in binding buffer at different serum dilutions. Separation between normal and diseased serum occurs over a dilution range of 1:200 up to about 1:10,000. The initial dilution was 1:200 (SLE serum at.375 mg/mL and non-diseased serum at.396 mg/mL for group 1).

Figure 37 shows SLE serum ELISA profiles using 10mM KN1B-20 prepared in DMSO at different serum dilutions. Separation between normal and diseased serum occurs over a dilution range of 1:200 up to about 1:10,000. The initial dilution was 1:200 (SLE serum at.367 mg/mL and non-diseased serum at.322 mg/mL for group 1).

Figure 38 shows FACS platform for Tentagel bead hit confirmation.

Fig. 39 shows the degree of separation between beads with acetyl groups and beads with 2, 5-Dinitrophenyl (DNP) at different concentrations of serum (100 ug/mL to 1,000 ug/mL) and in response to treatment with secondary antibodies labeled with anti-DNP. Mean Fluorescence Intensity (MFI) separation is greatest at higher dilutions of 1,000ug/mL serum.

FIG. 40 shows that at a serum concentration of 1,000ug/mL, there is direct competition between free ethanolamine-DNP and binding of DNP (on the plate) to the anti-DNP antibody.

Figure 41 ADP 3-bound anti-antibodies from pooled normal control sera and pooled AD sera. The data show good separation at serum concentrations ranging from 20 to 140ug/mL using two different secondary antibodies (goat anti-human Dylight649 and goat anti-human Alexa 647).

Figure 42 shows ADP 3-bound autoantibodies from normal control and AD sera after background subtraction at different serum concentration ranges. There was a significant degree of separation at most serum concentration ranges of less than 20ug/mL to 120ug/mL or greater.

Figures 43 and 44 show the structure of a peptoid ligand hit for SLE (lupus) resynthesis.

Figure 45 shows ADP3 preparation on 10um Tentagel beads and subsequent cleavage using CNBr along with the mass spectrometry readings of the lactones shown.

Figure 46 shows ADP 3-bound autoantibodies from normal control and alzheimer's disease sera at different concentrations. The beads were pre-blocked with 1X TBST for 3 hours and subsequently detected using goat anti-human Alexa647 secondary antibody.

Figure 47 shows ADP 3-bound autoantibodies from normal control and alzheimer's disease sera at different serum concentrations, and also shows DNP values.

Figures 48 and 49 show ADP 3-bound autoantibodies from normal controls versus alzheimer's disease serum using pre-blocking conditions such as e.coli (e.coli) lysate and lysine.

FIG. 50 shows a simple schematic of the preparation and differentiation between peptoids used in microarrays relative to those placed on ELISA plates. Schematic diagram of how peptoid microarrays were prepared: individual beads were separated into the wells of a microtiter plate, and peptoids were cleaved from the beads to prepare concentrated stock solutions. It should be noted that each well will now contain a single species of peptoid. Several thousand peptoids were then spotted on a chemically modified glass microscope slide in such a way that they were covalently bound to the surface. Thousands of slides can be generated with high reproducibility from a single synthetic library. ELISA production is similar except that no PEG chains are present on the surface, but the peptoid density on the ELISA plate may be different from the density on the microarray.

FIG. 51 shows an ELISA assay with a clear distinction between normal control and diseased serum at a serum dilution of 1:800 using horseradish peroxidase linked to a secondary antibody that detects disease-associated antibody-peptoid complexes. A colorless substrate was added and changed color (blue) upon reaction with the bound HRP enzyme.

Figure 52 shows the titration data for a plurality of AD peptoids compared in an ELISA assay at different serum dilutions of diseased serum (a) relative to control serum (B). When the concentration was increased from 1:12,800 to 1:200, there was no signal intensity in normal serum, but there was a clear distinction and intensity for all AD peptoids.

Figure 53 provides a simplified diagram confirming the correlation between clinical diagnosis of an informed AD patient serogroup at various stages of alzheimer's disease (or not) versus data obtained from the same patient serum sample (blinded) and screened against ADP 3-like peptides to detect disease-associated antibodies. Results shown are from a blinded serum sample study by Mayo clinical Jacksonville. UND = undefined. The graph is derived from obtaining a single serum concentration (1:800) dilution. Reads >1 were considered positive, reads between 1 and 0.7 were considered indeterminate, and reads below 0.7 were considered negative.

Figure 54 provides a simplified diagram confirming the correlation between clinical diagnosis of an informed AD patient serogroup at various stages of alzheimer's disease (or not) versus data obtained from the same patient serum sample (blinded) and screened against multiple AD peptoids (the graph is the average of the results for 9 peptoids) to detect disease-associated antibodies. Results shown are from a blinded serum sample study by MayoClinic Jacksonville. UND = undefined. The graph is derived from obtaining a single serum concentration (1:800) dilution. Reads >1 were considered positive, reads between 1 and 0.7 were considered indeterminate, and reads below 0.7 were considered negative.

Figure 55 provides a simplified diagram confirming the correlation between clinical diagnosis of an informed AD patient serogroup at various stages of alzheimer's disease (or not) versus data obtained from the same patient serum sample (blinded) and screened against multiple AD peptoids of the present invention to detect disease-associated antibodies. Results shown are from a blinded serum sample study by Mayo clinical Jacksonville. UND = undefined. The graph is derived from obtaining a single serum concentration (1:800) dilution. Reads >1. are considered positive, reads between 1 and 0.7 are considered indeterminate, and reads below 0.7 are considered negative. The data also show the performance for other dementias, with MCI/depression samples labeled and lewy body dementia samples labeled as well. The data show that at least three MCI patients have serum samples with detectable amounts of more than 1 captured antibody by the AD selective peptoids of the invention.

Fig. 56A-D provide data on a subset of samples from patients with inconsistencies between Opko Health peptoid diagnostic assays using multiple AD peptoids versus clinical diagnosis when this information was provided blindly. FIG. 56A shows data on the peptoid ADP3 and others as shown for patients who were clinically diseased but for which the Opko peptoid Plaag4 was below 1.0 (UND at a single point; positive for titration of AD). All other Opko peptoids were positive for AD (i.e., above 1.0). Figure 56B shows that all Opko peptoids are positive for disease-associated antibodies in patients currently diagnosed as normal (non-demented), suggesting pre-AD. Figure 56C shows that none of the Opko AD peptoids showed an intensity above 1 at any dilution point in patients clinically diagnosed with AD suggesting that this patient has some other form of dementia. Figure 56D shows that in clinically positive AD patients, multiple Opko AD peptoids were not positive for disease-associated antibodies, but two peptoids (P1 aag6 and P1aag 4) were positive, thus UND at a single point and UND even after titration.

Figure 57 shows a clustering diagram generated from previous AD samples using microarrays on point ADP 3. There was a clear correlation between disease in the microarray data and the data generated using the ELISA platform versus the control. Figure 57 also shows selection of ADP3 for disease-associated antibodies that are associated with alzheimer's disease but not with parkinson's or lupus (SLE).

Figure 58 provides a summary of ELISA analysis using a total of 106 serum samples tested.

FIG. 59 provides the chemical structure of P1aag 7-9.

Detailed Description

The present invention represents a significant advance in diagnostic and therapeutic discovery. In particular, the present inventors have found that screening for complex biological samples exceeds the screening methods of previous methods. In particular, there is a need for the discovery of disease-associated biomarkers and improved methods for the preparation of diagnostic kits comprising high affinity ligands for such biomarkers. The present invention relates to methods of screening for such biomarkers and diagnosing disease and disease progression using ligands that detect such biomarkers.

The invention includes compositions comprising a library of particle-based compounds selected from peptoids, peptides, oligomers, small molecules, and any naturally derived or synthetically prepared molecule, and which may be placed on a carrier system such as beads or small particles. This "library" is then pre-treated and exposed to a complex biological fluid, such as plasma or serum, in which disease-associated biomarkers or other target biomarkers, such as antibodies or proteins or other markers, such as cell surface proteins, are "screened" for the presence or absence under appropriate conditions and after exposure to a control plasma or serum sample to allow for the removal of non-selective ligands. Blood samples or other biological fluid samples are obtained from patients who may or may not have a particular disease, and the results generated by the screening are compared to results obtained from control healthy patients or control diseased patients.

The primary screen results in a significant number of high affinity ligands for any particular disease-associated biomarker, such as antibodies. The invention also encompasses processes for generating high affinity ligands that can be used in diagnostic settings for such disease states and/or the ligands themselves can be used as, for example, therapeutic vaccines or drugs that can target the disease-associated antibodies located in specific regions of the body or body tissue. Such drugs may be linked to other moieties such as chemotherapeutic agents or other agents that generate or can generate a localized immune response to remove and/or degrade autoantibodies.

Alzheimer's Disease (AD) is a progressive and fatal brain disease affecting up to 5.3 million americans. AD destroys brain cells, causing problems with memory, thinking, and behavior. These symptoms go worse over time and, ultimately, the disease is fatal. Today, it is the sixth leading cause of death in the united states and is the most common form of dementia, accounting for 50-70% of all cases of dementia. Unfortunately, while treatments exist for symptoms, there is no cure.

Diagnosing alzheimer's disease is an empirical process that involves several types of assessments and can take many days to weeks to complete. Assessment includes obtaining detailed medical history and physical examination. In addition, standard laboratory tests, including blood, urine and CSF tests, are primarily designed to help eliminate other possible conditions. Neuropsychological testing is also performed using a variety of tools to assess memory, problem solving, attention, visual motor coordination, and abstract thinking. Tests for depression should also be included. Finally, brain imaging scans are recommended to exclude brain tumors or blood clots in the brain as the cause of symptoms. In summary, there is currently no single test that accurately diagnoses alzheimer's disease, where a definitive diagnosis of alzheimer's disease may only be by examining brain tissue after death.

Parkinson's Disease (PD) is another degenerative disease of the brain (central nervous system) that often impairs motor skills, speech and other functions. It affects movement (motor symptoms), but other typical symptoms include mood, behavior, thinking, and perceptual disturbances (non-motor symptoms). The individual symptoms of a patient can be quite different and the progression of the disease is obviously also individual. Symptoms of PD result from the loss (idiopathic or genetic, toxic or traumatic) of pigmented dopamine-secreting (dopaminergic) cells in the pars compacta region of the substantia nigra (literally "black matter"). These neurons project to the striatum and their loss results in a change in the activity of the neural circuits within the basal ganglia, which regulate movement, essentially inhibiting the direct pathway and firing the indirect pathway.

Diagnosis of PD presents similar, albeit somewhat different, challenges. When performing a neurological examination to assess a patient with any dyskinesia, the physician should obtain a medical history and perform a physical examination. In addition, neurological examinations are performed to make an adequate assessment of the nervous system, including observations of the patient's motor, coordination, and balance aspects. Laboratory testing of the blood of patients with typical symptoms of parkinson's disease only rarely reveals any abnormalities. Electroencephalography (EEG's) record certain aspects of brain electrical activity, but they are ineffective in recognizing PD. MRI and CAT scans of the brain produce significant and precise anatomical images, but the brain of individuals with PD disease looks normal even under this scrutiny, since the changes associated with PD are microscopic and cannot be revealed by these scans. No definitive diagnostic test is provided for a particular answer, and the physician must base his or her diagnosis of PD on judgment.

Thus, there remains a need for diagnostic procedures for both these diseases and other neurological diseases that are (i) accurate and objective, (ii) simple and reproducible, and (iii) applicable in early and late cases.

in accordance with the present invention, there is provided a composition comprising a peptoid that binds antibodies indicative of neurodegenerative diseases and a method of detecting antibodies in an antibody-containing sample comprising contacting the antibody-containing sample with a carrier having a peptoid attached thereto, the ligand library can comprise a compound of formula I wherein the R groups on the amine side chain or α carbon are independently selected from hydrogen, alkyl, allyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-butyl amine, sec-butyl, tert-butyl, pentyl, hexyl, isopentyl, aryl, heteroaryl, furyl, indolyl, thiophenyl, thiazolyl, imidazolyl, isoxazolyl, oxazolyl, piperonyl, pyrazolyl, pyrrolyl, pyrazinyl, pyridyl, pyrimidinyl, pyrimidyl, purinyl, cinnolinyl, benzofuranyl, benzothienyl, benzotriazolyl, benzoxazolyl, quinoline, isoxazolyl, isoquinolinyl, alkenyl, cycloalkenyl, phenyl, pyridyl, methoxyethyl, (R) -methylbenzyl, C_0-6An alkaryl group; c_0-6An alkyl heteroaryl group; c substituted by a group selected from_1-6Alkyl groups: OH, SH, halogen, OR¹⁵、COOR¹⁵、NR¹⁵(wherein R is¹⁵Is selected from H or C_1-6Alkyl or C_1-6Alkynyl) or R¹⁶(wherein R is¹⁶Is selected from H or C_1-6Alkynyl groups); OC_1-6An alkyl group; c_2-6An alkenyl group; c_2-6An alkynyl group; c_2-6An alkenyl group; and C_2-6Alkynyl-includes one or more chemical groups described in tables 1 and 2 below.

Preferred ligand libraries of the invention for screening for Alzheimer's disease use comprise random ligand libraries for screening complex biological fluids comprising a compound of formula I on a carrier,

wherein R is₁Selected from the electron rich amino acid side chain Y;

R₂is selected from H;

and R is₃-R₆Independently selected from H, -C₁-C₆Alkyl, -C₁-C₆Alkyl SCH₃、-C₀-C₆Alkyl radical C₂-C₆Alkenyl, -C₀-C₆Alkyl radical C₂-C₆Alkynyl, -C₁-C₆COOH、-C₁-C₆Alkyl OH, -C₁-C₆Alkyl N (R)₂、-C₃-C₈Cycloalkyl, -C₁-C₆Alkylaryl, -C₁-C₆Alkyl heteroaryl, -C₁-C₆Alkyl radical NC (O) C₁-C₆Alkyl, -C₁-C₆An alkylcycloamide, wherein any of said aryl or heteroaryl groups can be independently replaced by-OH, Cl, F, Br, -OCH₃、-SO₂NH₂or-O-CH₂-O-substitution.

Other peptoid libraries suitable for screening for alzheimer's disease include those comprising:

random ligand libraries for screening complex biological fluids comprising a compound of formula I on a carrier,

wherein the compound is produced by a process comprising the use of a reactant selected from the group consisting of:

(A) furfuryl amine; 3, 4-dimethoxyethanolamine; benzylamine; n- (2-aminoethyl) acetamide; n- (3-aminopropyl) -2-pyrrolidone; ethanolamine; glycine; diaminobutane; allylamine; piperonyl amine; methylbenzylamine; isobutylamine; 4- (2-aminoethyl) benzenesulfonamide or cyclohexylamine; or

(B) (ii) a methoxyethylamine; piperonyl amine; cyclohexylamine; diaminobutane; methylbenzylamine; isobutylamine; furfuryl amine or 4- (2-aminoethyl) benzenesulfonamide; or

(C) Furfuryl amine, ethanolamine; glycine; diaminobutane; allylamine; piperonyl amine; methylbenzylamine; isobutylamine or 4- (2-aminoethyl) benzenesulfonamide; or

(D) Furfuryl amine, N- (2-aminoethyl) acetamide; n- (3-aminoethyl) -2-pyrrolidone; ethanolamine; glycine; diaminobutane; allylamine; piperonyl amine; methylbenzylamine; isobutylamine; 4- (2-aminoethyl) benzenesulfonamide; or

(E) Cysteine, glycine, allylamine, ethanolamine, isobutylamine, methylbenzylamine, piperonylamine, methionine, cyclohexylamine, 3, 4-dimethoxyphenethylamine, benzylamine, N- (2-aminoethyl) acetamide, N- (3-aminopropyl) -2-pyrrolidone, 4- (2-aminoethyl) benzenesulfonamide and furfurylamine; and

wherein

R₁Is selected from- (C)₁-C₆)SCH₃；

R₂Is selected from H;

R₃and R₅Independently selected from H, -C₁-C₆Alkyl, -C₁-C₆Alkyl SCH₃、-C₀-C₆Alkyl radical C₂-C₆Alkenyl, -C₀-C₆Alkyl radical C₂-C₆Alkynyl, -C₁-C₆COOH、-C₁-C₆Alkyl OH, -C₁-C₆Alkyl N (R)₂、-C₃-C₈Cycloalkyl, -C₁-C₆Alkylaryl, -C₁-C₆Alkyl heteroaryl, -C₁-C₆Alkyl radical NC (O) C₁-C₆Alkyl, -C₁-C₆An alkylcycloamide, wherein any of said aryl or heteroaryl groups can be independently replaced by-OH, Cl, F, Br, -OCH₃、-SO₂NH₂or-O-CH₂-O-substitution;

R₄selected from furfuryl or- (C)₁-C₆Alkyl) NR₇R₈，

R₆Selected from H, 1-yl-allyl, 1-yl-2-hydroxyethyl, isobutyl, 1-yl-n-butylamine, methylbenzyl, piperonyl, cyclohexyl, 1-yl-2- (3, 4-dimethoxyphenyl) ethyl, benzyl, 1-yl-2- (acetamide) ethyl, 1-yl-3-2-pyrrolidone, 1-yl-2- (4-benzenesulfonamide) ethyl or furfuryl, and

n is 3 to 11.

In a more preferred embodiment, such libraries and/or compounds are selected from:

a compound having the formula Ia

Wherein the compound is selected from compounds of formula Ia, wherein

(a)R₉Is n-butylamine; r₁₀Is 1-yl-n-butylamine; r₁₁Is piperonyl; r₁₂Is a methyl benzyl group; r₁₃Is piperonyl; r₁₄Is a methyl benzyl group; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is 1-yl-2-methoxyethyl;

(b)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is 1-yl-n-butylamine; r₁₂Is cyclohexyl; r₁₃Is 1-yl-2-methoxyethyl; r₁₄Is 1-yl-2, 2-dimethylethyl (isobutyl); r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is a methyl benzyl group;

(c)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-n-butylamine; r₁₁Is piperonyl; r₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is 1-yl-n-butylamine; r₁₄Is a methyl benzyl group; r₁₅Is methylbenzyl, and R₁₆Is cyclohexyl;

(d)R₉is piperonyl; r₁₀Is 1-yl-n-butylamine; r₁₁Is an isobutyl group; r₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is a methyl benzyl group; r₁₄Is cyclohexyl; r₁₅Is isobutyl, and R₁₆Is 1-yl-n-butylamine;

(e)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-n-butylamine; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-2-methoxyethyl; r₁₃Is cyclohexyl; r₁₄Is cyclohexyl; r₁₅Is methylbenzyl, and R₁₆Is piperonyl;

(f)R₉is piperonyl; r₁₀Is 1-yl-n-butylamine; r₁₁Is isopropyl; r₁₂Is isopropyl; r₁₃Is 1-yl-2-methoxyethyl; r₁₄Is cyclohexyl; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is 1-yl-2- (4 (benzenesulfonamide) ethyl;

(g)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-n-butylamine; r₁₁Is piperonyl; r₁₂Is a methyl benzyl group; r₁₃Is piperonyl; r₁₄Is a methyl benzyl group; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is cyclohexyl;

(h)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-n-butylamine; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-2-methoxyethyl; r₁₃Is cyclohexyl; r₁₄Is cyclohexyl; r₁₅Is methylbenzyl, and R₁₆Is a Chinese fiddleA pepper base;

(i)R₉is 1-yl-n-butylamine; r₁₀Is a methyl benzyl group; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is 1-yl-n-butylamine; r¹⁴Is cyclohexyl; r¹⁵Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R¹⁶Is cyclohexyl;

(j)R₉is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₀Is a methyl benzyl group; r₁₁Is a methyl benzyl group; r₁₂Is cyclohexyl; r₁₃Is 1-yl-n-butylamine; r₁₄Is a methyl benzyl group; r₁₅Is isobutyl, and R₁₆Is 1-yl-n-butylamine;

(k)R₉is 1-yl-2-methoxyethyl; r₁₀Is an isobutyl group; r₁₁Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₂Is piperonyl; r₁₃Is 1-yl-n-butylamine; r₁₄Is cyclohexyl; r₁₅Is methylbenzyl, and R₁₆Is 1-yl-2-methoxyethyl;

(l)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is 1-yl-n-butylamine; r₁₂Is cyclohexyl; r₁₃Is 1-yl-2-methoxyethyl; r₁₄Is an isobutyl group; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is a methyl benzyl group;

(m) R9 is 1-yl-2-methoxyethyl; r10 is 1-yl-n-butylamine; r11 is 1-yl-n-butylamine; r12 is 1-yl-2-methoxyethyl; r13 is methylbenzyl; r14 is 1-yl-n-butylamine; r15 is furfuryl, and R16 is furfuryl;

(n) R9 is cyclohexyl; r10 is cyclohexyl; r11 is 1-yl-n-butylamine; r12 is furfuryl; r13 is 1-yl-2-methoxyethyl; r14 is 1-yl-2-methoxyethyl; r15 is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R16 is furfuryl;

(o) R9 is 1-yl-n-butylamine; r10 is piperonyl; r11 is 1-yl-2- (4 (benzenesulfonamide) ethyl; R12 is 1-yl-2-methoxyethyl; R13 is methylbenzyl; R14 is 1-yl-n-butylamine; R15 is 1-yl-2-methoxyethyl; and R16 is methylbenzyl;

(p) R9 is cyclohexyl; r10 is cyclohexyl; r11 is piperonyl; r12 is 1-yl-n-butylamine; r13 is 1-yl-n-butylamine; r14 is 1-yl-n-butylamine; r15 is 1-yl-n-butylamine and R16 is isobutyl;

(q) R9 is piperonyl; r10 is 1-yl-n-butylamine; r11 is 1-yl-2-methoxyethyl; r12 is 1-yl-2- (4 (benzenesulfonamide) ethyl; R13 is piperonyl; R14 is 1-yl-n-butylamine; R15 is methylbenzyl and R16 is methylbenzyl;

(R) R9 is methylbenzyl; r10 is methylbenzyl; r11 is methylbenzyl; r12 is 1-yl-n-butylamine; r13 is piperonyl; r14 is 1-yl-n-butylamine; r15 is piperonyl, and R₁₆Is 1-yl-n-butylamine;

(s)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-n-butylamine; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is a methyl benzyl group; r₁₄Is a methyl benzyl group; r₁₅Is 1-yl-2-methoxyethyl, and R₁₆Is piperonyl;

(t)R₉is a methyl benzyl group; r₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is 1-yl-n-butylamine; r₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is an isobutyl group; r₁₄Is 1-yl-n-butylamine; r₁₅Is methylbenzyl, and R₁₆Is 1-yl-2-methoxyethyl;

(u)R₉is 1-yl-2-methoxyethyl; r₁₀Is 1-yl-n-butylamine; r₁₁Is an isobutyl group; r₁₂Is an isobutyl group; r₁₃Is cyclohexyl; r₁₄Is 1-yl-n-butylamine; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is cyclohexyl;

(v)R₉is an isobutyl group; r₁₀Is 1-yl-n-butylamine; r₁₁Is 1-yl-n-butylamine; r₁₂Is a methyl benzyl group; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-n-butylamine; r₁₅Is piperonyl, and R₁₆Is piperonyl;

(w)R₉is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₀Is an isobutyl group; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-2-methoxyethyl; r₁₃Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₄Is an isobutyl group; r₁₅Is 1-yl-2-methoxyethyl, and R₁₆Is cyclohexyl;

(x)R₉is furfuryl; r₁₀Is furfuryl; r₁₁Is piperonyl; r₁₂Is cyclohexyl; r₁₃Is piperonyl; r₁₄Is 1-yl-n-butylamine; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is cyclohexyl;

(y)R₉is piperonyl; r₁₀Is piperonyl; r₁₁Is 1-yl-2-methoxyethyl; r₁₂Is 1-yl-2-methoxyethyl; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-n-butylamine; r₁₅Is 1-yl-n-butylamine, and R₁₆Is 1-yl-2-methoxyethyl;

(z)R₉is piperonyl; r₁₀Is 1-yl-n-butylamine; r₁₁Is an isobutyl group; r₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is a methyl benzyl group; r₁₄Is cyclohexyl; r₁₅Is isobutyl, and R₁₆Is 1-yl-n-butylamine;

(aa)R₉is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is a methyl benzyl group; r₁₂Is a methyl benzyl group; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-n-butylamine; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is 1-yl-2- (4 (benzenesulfonamide) ethyl;

(bb)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-2-methoxyethyl; r₁₁Is 1-yl-n-butylamine; r₁₂Is an isobutyl group; r₁₃Is cyclohexyl; r₁₄Is 1-yl-n-butylamine; r₁₅Is 1-yl-n-butylamine, and R₁₆Is piperonyl;

(cc)R₉is cyclohexyl; r₁₀Is a methyl benzyl group; r₁₁Is cyclohexyl; r₁₂Is piperonyl; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₅Is 1-yl-n-butylamine, and R₁₆Is 1-yl-2-methoxyethyl;

(dd)R₉is 1-yl-2-methoxyethyl; r₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is 1-yl-n-butylamine; r₁₂Is 1-yl-2-methoxyethyl; r₁₃Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₄Is 1-yl-2-methoxyethyl; r₁₅Is isobutyl, and R₁₆Is cyclohexyl;

(ee)R₉is 1-yl-2-methoxyethyl; r₁₀Is a methyl benzyl group; r₁₁Is 1-yl-n-butylamine; r₁₂Is 1-yl-n-butylamine; r₁₃Is piperonyl; r₁₄Is an isobutyl group; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is 1-yl-n-butylamine;

(ff)R₉is 1-yl-n-butylamine; r₁₀Is a methyl benzyl group; r₁₁Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₂Is a methyl benzyl group; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is cyclohexyl;

(gg)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-2-methoxyethyl; r₁₁Is 1-yl-n-butylamine; r₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is 1-yl-2-methoxyethyl; r₁₄Is a 1-radical-2-methoxyethyl; r₁₅Is 1-yl-n-butylamine, and R₁₆Is a methyl benzyl group;

(hh)R₉is cyclohexyl; r₁₀Is cyclohexyl; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is a methyl benzyl group; r₁₄Is cyclohexyl; r₁₅Is methylbenzyl, and R₁₆Is 1-yl-n-butylamine;

(ii)R₉is 1-yl-n-butylamine; r₁₀Is furfuryl; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is furfuryl; r₁₄Is cyclohexyl; r₁₅Is methylbenzyl, and R₁₆Is cyclohexyl;

(jj)R₉is 1-yl-n-butylamine; r₁₀Is a methyl benzyl group; r₁₁Is 1-yl-n-butylamine; r₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is 1-yl-2-methoxyethyl; r₁₄Is a methyl benzyl group; r₁₅Is 1-yl-2-methoxyethyl, and R₁₆Is an isobutyl group;

(kk)R₉is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₀Is 1-yl-n-butylamine; r₁₁Is 1-yl-n-butylamine; r₁₂Is a methyl benzyl group; r₁₃Is a methyl benzyl group; r₁₄Is cyclohexyl; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is a methyl benzyl group;

(ll)R₉is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is 1-yl-n-butylamine; r₁₂Is 1-yl-n-butylamine; r₁₃Is a methyl benzyl group; r₁₄Is 1-yl-n-butylamine; r₁₅Is methylbenzyl, and R₁₆Is 1-yl-n-butylamine;

(mm)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₂Is 1-2-methoxyethyl radical; r₁₃Is 1-yl-n-butylamine; r₁₄Is cyclohexyl; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is a methyl benzyl group;

(nn)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-n-butylamine; r₁₁Is piperonyl; r₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is 1-yl-n-butylamine; r₁₄Is a methyl benzyl group; r₁₅Is methylbenzyl, and R₁₆Is cyclohexyl;

(oo)R₉is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₀Is a methyl benzyl group; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is a methyl benzyl group; r₁₄Is piperonyl; r₁₅Is 1-yl-n-butylamine, and R₁₆Is 1-yl-2-methoxyethyl;

(pp)R₉is piperonyl; r₁₀Is 1-yl-n-butylamine; r₁₁Is 1-yl-n-butylamine; r₁₂Is methylamine; r₁₃Is piperonyl; r₁₄Is 1-yl-n-butylamine; r₁₅Is piperonyl, and R₁₆Is 1-yl-2-methoxyethyl;

(qq)R₉is cyclohexyl; r₁₀Is cyclohexyl; r₁₁Is furfuryl; r₁₂Is 1-yl-2-methoxyethyl; r₁₃Is an isobutyl group; r₁₄Is cyclohexyl; r₁₅Is methylbenzyl, and R₁₆Is a methyl benzyl group;

(rr)R₉is piperonyl; r₁₀Is 1-yl-n-butylamine; r₁₁Is an isobutyl group; r₁₂Is an isobutyl group; r₁₃Is 1-yl-2-methoxyethyl; r₁₄Is cyclohexyl; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is 1-yl-2- (4 (benzenesulfonamide) ethyl;

(ss)R₉is cyclohexyl; r₁₀Is cyclohexyl; r₁₁Is 1-yl-n-butylamine; r₁₂Is a firstA benzyl group; r₁₃Is 1-yl-n-butylamine; r₁₄Is a methyl benzyl group; r₁₅Is cyclohexyl, and R₁₆Is piperonyl;

and pharmaceutically acceptable salts thereof.

In a more preferred embodiment and for screening disease-associated biomarkers using kits and/or diagnostic machines and/or instruments in patients with or suspected of having alzheimer's disease, the following compounds are preferred:

a compound having the formula:

wherein the compound is selected from compounds of formula II, wherein

(h)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-n-butylamine; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-2-methoxyethyl; r₁₃Is cyclohexyl; r₁₄Is cyclohexyl; r₁₅Is methylbenzyl, and R₁₆Is piperonyl;

(m)R₉is 1-yl-2-methoxyethyl; r₁₀Is 1-yl-n-butylamine; r₁₁Is 1-yl-n-butylamine; r₁₂Is 1-yl-2-methoxyethyl; r₁₃Is a methyl benzyl group; r₁₄Is 1-yl-n-butylamine; r₁₅Is furfuryl, and R₁₆Is furfuryl;

(n)R₉is cyclohexyl; r₁₀Is cyclohexyl; r₁₁Is 1-yl-n-butylamine; r₁₂Is furfuryl; r₁₃Is 1-yl-2-methoxyethyl; r₁₄Is 1-yl-2-methoxyethyl; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is furfuryl;

(o)R₉is 1-yl-n-butylamine; r₁₀Is piperonyl; r₁₁Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₂Is 1-yl-2-methoxyethyl; r₁₃Is a methyl benzyl group; r₁₄Is 1-yl-n-butylamine; r₁₅Is 1-yl-2-methoxyethyl, and R₁₆Is a methyl benzyl group;

(p)R₉is cyclohexyl; r₁₀Is cyclohexyl; r₁₁Is piperonyl; r₁₂Is 1-yl-n-butylamine; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-n-butylamine; r₁₅Is 1-yl-n-butylamine, and R₁₆Is an isobutyl group;

(q)R₉is piperonyl; r₁₀Is 1-yl-n-butylamine; r₁₁Is 1-yl-2-methoxyethyl; r₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is piperonyl; r₁₄Is 1-yl-n-butylamine; r₁₅Is methylbenzyl, and R₁₆Is a methyl benzyl group;

(r)R₉is a methyl benzyl group; r₁₀Is a methyl benzyl group; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is piperonyl; r₁₄Is 1-yl-n-butylamine; r₁₅Is piperonyl, and R₁₆Is 1-yl-n-butylamine;

(gg)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-2-methoxyethyl; r₁₁Is 1-yl-n-butylamine; r₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is a 1-radical-2-methoxyethyl; r₁₄Is 1-yl-2-methoxyethyl; r₁₅Is 1-yl-n-butylamine, and R₁₆Is a methyl benzyl group;

(mm)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is 1-yl-2-, (4 (benzenesulfonamide) ethyl; r₁₂Is 1-yl-2-methoxyethyl; r₁₃Is 1-yl-n-butylamine; r₁₄Is cyclohexyl; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is a methyl benzyl group;

(ss)R₉is cyclohexyl; r₁₀Is cyclohexyl; r₁₁Is 1-yl-n-butylamine; r₁₂Is a methyl benzyl group; r₁₃Is 1-yl-n-butylamine; r₁₄Is a methyl benzyl group; r₁₅Is cyclohexyl, and R₁₆Is piperonyl;

and pharmaceutically acceptable salts thereof.

In a more preferred embodiment, the following compounds are selected as peptoid ligands for alzheimer's disease screening and/or detection:

the compound of claim 35, wherein the compound of formula II is selected from having R₉-R₁₆The group of (a) or (b),

and pharmaceutically acceptable salts thereof.

Autoimmune diseases

The invention also provides for the identification of molecules that can bind to autoimmune T cells and/or antibodies from a variety of autoimmune diseases or conditions. Although the examples are directed to EAE, an animal model for MS, the present invention should be useful in the context of a variety of autoimmune diseases, some of which are discussed below. In particular aspects, the disease state includes, but is not limited to, diseases such as Acute Disseminated Encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, allergic asthma, allergic rhinitis, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune aplastic anemia, autoimmune familial autonomic abnormalities, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, Autoimmune Inner Ear Disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, Autoimmune Thrombocytopenic Purpura (ATP), autoimmune thyroid disease, axonal and neuronal neuropathy, Barlow's disease, Behcet's disease, bullous pemphigoid, autoimmune diseases, autoimmune, Cardiomyopathy, Cashmere's disease, celiac disease (non-tropical), Chagas ' disease, chronic fatigue syndrome, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Relapsing Multifocal Osteomyelitis (CRMO), Culex syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Kegen's syndrome, cold agglutinin disease, congenital heart block, Coxsackie viral myocarditis, CREST disease, idiopathic mixed cryoglobulinemia, demyelinating neuropathy, dermatomyositis, Devycke's disease (neuromyelitis optica), discoid lupus, Descemera syndrome, endometriosis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Venturi syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, Goodpasts syndrome, churg-dunker syndrome, Guinea syndrome, and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), chronic relapsing osteomyelitis (CRMO-induced by cold), chronic relapsing multiple sclerosis (CRMO, Graves ' disease, guillain-barre syndrome, hashimoto's encephalitis, hashimoto's thyroiditis, hemolytic anemia, anaphylactoid purpura, herpes gestationis, hypogammaglobulinemia, Idiopathic Thrombocytopenic Purpura (ITP), IgA nephropathy, immunomodulatory lipoproteins, inclusion body myositis, insulin-dependent diabetes mellitus (type 1), interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus, kawasaki syndrome, lang-yield syndrome, leukemia disruptive vasculitis, lichen planus, lichen sclerosus, wood-like conjunctivitis, linear IgA disease (LAD), lupus (SLE), lyme disease, meniere's disease, microscopic polyangiitis, Mixed Connective Tissue Disease (MCTD), morfan's ulcer, muckle-hadamary disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (de vek), myelitis optica, Neutropenia, ocular cicatricial pemphigoid, optic neuritis, recurrent rheumatism, PANDAS (childhood streptococcal infection-associated autoimmune neuropsychiatric disorder), paraneoplastic cerebellar degeneration, Paroxysmal Nocturnal Hemoglobinuria (PNH), Parson-Rodie syndrome, Parkinson-Turner syndrome, pantitis (periuveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, I, II and type III autoimmune polyglandular syndrome, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma, Pure red cell aplasia, Raynaud's phenomenon, reflex sympathetic dystrophy, Reiter's syndrome, recurrent polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, sjogren's syndrome, sperm and testis autoimmunity, stiff man syndrome, Subacute Bacterial Endocarditis (SBE), sympathetic ophthalmia, takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TPP), Toyohimbe's syndrome, transverse myelitis, ulcerative colitis, Undifferentiated Connective Tissue Disease (UCTD), uveitis, vasculitis, vesicular dermatitis (vesiculobullus dermatasis), vitiligo or Wenner's granulomatosis or chronic active hepatitis, primary biliary cirrhosis, dilated cardiomyopathy, Myocarditis, autoimmune polycystic endocrine disorder type I (APS-I), cystic fibrosis vasculitis, acquired hypoparathyroidism, coronary artery disease, pemphigus foliaceus, pemphigus vulgaris, rosmussen encephalitis, autoimmune gastritis, insulin hypoglycaemic syndrome (hiratagia), insulin resistance type B, acanthosis, Systemic Lupus Erythematosus (SLE), pernicious anemia, treatment-resistant lyme arthritis, polyneuropathy, demyelinating diseases, atopic dermatitis, autoimmune hypothyroidism, vitiligo, thyroid-related eye disease, autoimmune abdominal disease, ACTH deficiency, dermatomyositis, sjogren's syndrome, systemic sclerosis, progressive systemic sclerosis, hard spotting, primary antiphospholipid syndrome, chronic idiopathic urticaria, connective tissue syndrome, Necrotizing and Crescentic Glomerulonephritis (NCGN) Systemic vasculitis, raynaud's syndrome, chronic liver disease, visceral leishmaniasis, autoimmune C1 deficiency, membranoproliferative glomerulonephritis (MPGN), prolongation of clotting time, immunodeficiency, atherosclerosis, neuronal disease, pemphigus paraneoplastic, paraneoplastic stiff person syndrome, paraneoplastic encephalomyelitis, subacute autonomic neuropathy, cancer-associated retinopathy, paraneoplastic strabismus oculus clonus ataxia, lower motor neuron syndrome, and langerhans myasthenia syndrome.

The peptoid libraries of the invention for use in screening for lupus-associated antibodies comprise the libraries specified above and further comprise a random ligand library for screening complex biological fluids comprising a compound of formula I on a carrier,

wherein R is₁Selected from the electron rich amino acid side chain Y;

R₂is selected from H;

Other peptoid libraries suitable for screening SLE (lupus) include libraries comprising:

wherein

R₁Is selected from- (C)₁-C₆)SCH₃；

R₂Is selected from H;

R₄selected from furfuryl or- (C)₁-C₆Alkyl) NR₇R₈，

n is 3 to 11.

Preferred embodiments for initial screening use include:

a compound having the formula:

wherein in the compound of formula IIIa, R₉-R₁₆Is selected from

(a)R₉Is 1-yl-allyl; r₁₀Is 1-yl-3N- (2-pyrrolidone) propyl; r₁₁Is acetic acid; r₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is benzyl; r₁₄Is 1-yl-n-butylamine; r₁₅Is isobutyl, and R₁₆Is 1-yl-allyl;

(b)R₉is cyclohexyl; r₁₀Is 1-yl-n-butylamine; r₁₁Is 1-yl-n-butylamine; r₁₂Is 1-yl-2- (3, 4-dimethoxyphenyl) ethyl; r₁₃Is benzyl; r₁₄Is piperonyl; r₁₅Is 1-yl-n-butylamine, and R₁₆Is 1-yl-2- (4 (benzenesulfonamide) ethyl;

(c)R₉is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₀Is 1-yl-allyl; r₁₁Is 1-yl-n-butylamine; r₁₂Is benzyl; r₁₃Is a methyl benzyl group; r₁₄Is benzyl; r₁₅Is methylbenzyl, and R₁₆Is 1-yl-n-butylamine;

(d)R₉is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₀Is 1-yl-2- (3, 4-dimethoxyphenyl) ethyl; r₁₁Is 1-yl-n-butylamine; r₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is 1-yl-n-butylamine; r₁₄Is benzyl; r₁₅Is methylbenzyl, and R₁₆Is benzyl;

(e)R₉is piperonyl; r₁₀Is piperonyl; r₁₁Is 1-yl-n-butylamine; r₁₂Is cyclohexyl; r₁₃Is benzyl; r₁₄Is 1-yl-2- (3, 4-dimethoxyphenyl) ethyl; r₁₅Is 1-yl-n-butylamine, and R₁₆Is a methyl benzyl group;

(f)R₉is 1-yl-allyl; r₁₀Is a methyl benzyl group; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is piperonyl; r₁₄Is benzyl; r₁₅Is piperonyl, and R₁₆Is 1-yl-n-butylamine;

(g)R₉is 1-yl-2- (3, 4-dimethoxyphenyl) ethyl; r₁₀Is an isobutyl group; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is a methyl benzyl group; r₁₄Is benzyl; r₁₅Is piperonyl, and R₁₆Is 1-yl-n-butylamine;

(h)R₉is a methyl benzyl group; r₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is a methyl benzyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is a methyl benzyl group; r₁₄Is a methyl benzyl group; r₁₅Is 1-yl-n-butylamine, and R₁₆Is a methyl benzyl group;

(i)R₉is piperonyl; r₁₀Is benzyl; r₁₁Is piperonyl; r₁₂Is benzyl; r₁₃Is 1-yl-n-butylamine; r₁₄Is benzyl; r₁₅Is cyclohexyl, and R₁₆Is 1-yl-n-butylamine, and

a pharmaceutically acceptable salt thereof.

Preferred embodiments for use in kits and/or other diagnostic methods include:

a compound having the formula:

wherein in the compound of formula II, R₉-R₁₆Is selected from

(i)R₉is piperonyl; r₁₀Is benzyl; r₁₁Is piperonyl；R₁₂Is benzyl; r₁₃Is 1-yl-n-butylamine; r₁₄Is benzyl; r₁₅Is cyclohexyl, and R₁₆Is 1-yl-n-butylamine, and

a pharmaceutically acceptable salt thereof.

Cancer treatment

The invention may also be used to identify and/or characterize the presence or absence of biomarkers associated with cancer or a precancerous condition. These cancers are selected, for example, from the following:

acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, cerebellar or cerebral in childhood, basal cell carcinoma, cholangiocarcinoma, extrahepatic, bladder cancer, bone cancer, osteosarcoma/malignant fibrous histiocytoma, brain stem glioma, brain tumor, cerebellar astrocytoma, brain tumor, cerebromas/malignant gliomas, brain tumor, ependymoma, brain tumor, medulloblastoma, brain tumor, supratentorial primitive neuroectodermal tumor, brain tumor, optic pathway and hypothalamic glioma, breast cancer, bronchial adenoma/benign tumor, burkitt lymphoma, carcinoid tumor, childhood, carcinoid tumor, gastrointestinal cancer, carcinoma of unknown primary focus, central nervous system lymphoma, primary, cerebellar astrocytoma, primary carcinoma, cerebellar glioma, primary carcinoma, secondary carcinoma, primary carcinoma of the brain, and malignant glioma, Childhood, cerebral astrocytoma/glioblastoma malignance, childhood, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumors, endometrial cancer, ependymoma, esophageal cancer, ewing's sarcoma in ewing's family of tumors, extracranial germ cell tumors, childhood, extragonal germ cell tumors, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, eye cancer, retinoblastoma, gallbladder cancer, stomach (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors: extracranial, extragonadal or ovarian, gestational trophoblastic tumors, brain stem glioma, childhood cerebral astrocytoma, glioma, childhood visual pathway and hypothalamic, gastric benign tumors, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, childhood, intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia, acute lymphoblastic (also known as acute lymphocytic leukemia), leukemia, acute myeloid (also known as acute myelogenous leukemia), leukemia, chronic lymphocytic (also known as chronic lymphocytic leukemia), leukemia, chronic myelogenous (also known as chronic myelogenous leukemia), Leukemia, hairy cell, lip and mouth cancer, liver cancer (primary), lung cancer, non-small cell, lung cancer, small cell, lymphoma, AIDS-related, lymphoma, Burkitt's, lymphoma, cutaneous T cell, lymphoma, Hodgkin, lymphoma, non-Hodgkin's (old classification of all lymphomas except Hodgkin's), lymphoma, primary central nervous system, Marcus Whittle, malacebush disease, macroglobulinemia, Waldenstrom, malignant fibrous histiocytoma of bone/osteosarcoma, medulloblastoma, childhood, melanoma, intraocular (eye), Merck cell carcinoma, mesothelioma, adult malignancy, mesothelioma, childhood, metastatic squamous neck cancer of unknown primary, oral cancer, multiple endocrine tumor syndrome, childhood, multiple myeloma/plasma cell tumor, mycosis fungoides, multiple endocrine tumor, multiple myeloma/plasma cell tumor, multiple myeloma, Myelodysplastic syndrome, myelodysplastic/myeloproliferative disorders, myeloid leukemia, chronic, myeloid leukemia, adult acute, myeloid leukemia, childhood acute, myeloma, multiple (myeloid cancer), myeloproliferative disorder, chronic, nasal and paranasal sinus cancers, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma/osteochondral fibrocytoma, ovarian cancer, ovarian epithelial cancer (superficial epithelial-mesenchymal tumor), ovarian germ cell tumor, ovarian low-grade potential malignancy, pancreatic cancer, islet cells, paranasal and nasal cancer, parathyroid carcinoma, penile cancer, laryngeal cancer, pheochromocytoma, pinealoastrocytoma, pinealoblastoma and supratentorial primitive extraneural tumor, Childhood, pituitary adenoma, plasma cell tumor/multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, childhood, salivary gland carcinoma, sarcoma, ewing's family tumor, sarcoma, kaposi, sarcoma, soft tissue, sarcoma, uterus, sezary syndrome, skin cancer (non-melanoma), skin cancer (melanoma), skin cancer, merck cells, non-small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma-see skin cancer (non-melanoma), primary-unknown squamous neck cancer, metastatic, gastric cancer, supratentorial primary neuroectodermal tumors, childhood, T-cell lymphoma, cutaneous-see mycosis fungoides and sezary syndrome, Testicular cancer, laryngeal cancer, thymoma, childhood, thymoma and thymus, thyroid cancer, childhood, renal pelvis and ureteral transitional cell carcinoma, trophoblastic tumor, gestational cancer, unknown primary site, adult, unknown primary site, childhood, ureteral and renal pelvis, transitional cell carcinoma, urinary tract cancer, uterine cancer, endometrium, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic glioma, childhood, vulvar cancer, waldenstrom's macroglobulinemia, nephroblastoma (kidney cancer), childhood.

In addition to those libraries described above, the peptoid libraries of the invention for screening for pancreatic cancer use also comprise random ligand libraries for screening complex biological fluids comprising a compound of formula I on a carrier,

wherein R is₁Selected from the electron rich amino acid side chain Y;

R₂is selected from H;

Other peptoid libraries suitable for screening for cancer include libraries comprising:

wherein

R₁Is selected from- (C)₁-C₆)SCH₃；

R₂Is selected from H;

R₄selected from furfuryl or- (C)₁-C₆Alkyl) NR₇R₈，

n is 3 to 11.

Preferred examples for primary screening uses include peptoids comprising:

a compound having the formula:

wherein the compound is selected from the group consisting of compounds of formula IIa, wherein

(a)R₉Is 1-yl-n-butylamine; r₁₀Is piperonyl; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is cyclohexyl; r₁₄Is an isobutyl group; r₁₅Is methylbenzyl, and R₁₆Is 1-yl-n-butylamine;

(b)R₉is a methyl benzyl group; r₁₀Is 1-yl-n-butylamine; r₁₁Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is an isobutyl group; r₁₄Is 1-yl-n-butylamine; r₁₅Is methylbenzyl, and R₁₆Is 1-yl-n-butylamine;

(c)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-n-butylamine; r₁₁Is 1-yl-2-methoxyethyl; r₁₂Is 1-yl-2-methoxyethyl; r₁₃Is an isobutyl group; r₁₄Is 1-yl-2-methoxyethyl; r₁₅Is 1-yl-n-butylamine, and R₁₆Is 1-yl-2-methoxyethyl;

(d)R₉is a methyl benzyl group; r₁₀Is 1-yl-2- (4 (benzenesulfonamide)) An ethyl group; r₁₁Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₂Is a methyl benzyl group; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-n-butylamine; r₁₅Is cyclohexyl, and R₁₆Is an isobutyl group;

(e)R₉is 1-yl-n-butylamine; r₁₀Is a methyl benzyl group; r₁₁Is a methyl benzyl group; r₁₂Is a methyl benzyl group; r₁₃Is 1-yl-2-methoxyethyl; r₁₄Is 1-yl-n-butylamine; r₁₅Is cyclohexyl, and R₁₆Is a methyl benzyl group;

(f)R₉is cyclohexyl; r₁₀Is cyclohexyl; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-2-methoxyethyl; r₁₃Is a methyl benzyl group; r₁₄Is a methyl benzyl group; r₁₅Is 1-yl-n-butylamine, and R₁₆Is 1-yl-n-butylamine;

(g)R₉is furfuryl; r₁₀Is furfuryl; r₁₁Is an isobutyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is 1-yl-n-butylamine; r₁₄Is a methyl benzyl group; r₁₅Is methylbenzyl, and R₁₆Is a methyl benzyl group;

(h)R₉is a methyl benzyl group; r₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is cyclohexyl; r₁₂Is cyclohexyl; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-2-methoxyethyl; r₁₅Is 1-yl-n-butylamine, and R₁₆Is a methyl benzyl group;

(i)R₉is an isobutyl group; r₁₀Is a methyl benzyl group; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is 1-yl-n-butylamine; r₁₄Is an isobutyl group; r₁₅Is isobutyl, and R₁₆Is 1-yl-n-butylamine;

(j)R₉is an isobutyl group; r₁₀Is 1-yl-n-butylamine; r₁₁Is a methyl benzyl group; r₁₂Is a methyl benzyl group; r₁₃Is a methyl benzyl group; r₁₄Is cyclohexyl; r₁₅Is methylbenzyl, and R₁₆Is 1-yl-n-butylamine;

(k)R₉is cyclohexyl; r₁₀Is cyclohexyl; r₁₁Is a methyl benzyl group; r₁₂Is a methyl benzyl group; r₁₃Is 1-yl-n-butylamine; r₁₄Is an isobutyl group; r₁₅Is 1-yl-n-butylamine, and R₁₆Is an isobutyl group;

(l)R₉is cyclohexyl; r₁₀Is cyclohexyl; r₁₁Is an isobutyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is 1-yl-n-butylamine; r₁₄Is a methyl benzyl group; r₁₅Is 1-yl-n-butylamine, and R₁₆Is 1-yl-2-methoxyethyl;

and pharmaceutically acceptable salts thereof.

A preferred embodiment for use in a kit for screening for pancreatic cancer comprises:

a compound having the formula:

wherein the compound is selected from compounds of formula II, wherein

(d)R₉is a methyl benzyl group; r₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₂Is a methyl benzyl group; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-n-butylamine; r₁₅Is cyclohexyl, and R₁₆Is an isobutyl group;

(h)R₉is a methyl benzyl group; r₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is cyclohexyl; r₁₂Is cyclohexyl; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-2-methoxyethyl; r₁₅Is 1-yl-n-butylamine, andand R is₁₆Is a methyl benzyl group;

and pharmaceutically acceptable salts thereof.

Other alternative examples of primary screening uses for detecting pancreatic cancer autoantibodies include:

a compound having the formula:

wherein the compound is selected from the group consisting of compounds having R₉-R₁₆The compound of (a) to (b),

(a)R₉is piperonyl;R₁₀is cyclohexyl; r₁₁Is 1-yl-n-butylamine; r₁₂Is 1-yl-n-butylamine; r₁₃Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₄Is 1-yl-n-butylamine; r₁₅Is isobutyl, and R₁₆Is 1-yl-n-butylamine;

(b)R₉is piperonyl; r₁₀Is piperonyl; r₁₁Is cyclohexyl; r₁₂Is 1-yl-n-butylamine; r₁₃Is 1-yl-allyl; r₁₄Is an isobutyl group; r₁₅Is cyclohexyl, and R₁₆Is 1-yl-n-butylamine;

(c)R₉is a methyl benzyl group; r₁₀Is piperonyl; r₁₁Is cyclohexyl; r₁₂Is piperonyl; r₁₃Is piperonyl; r₁₄Is a methyl benzyl group; r₁₅Is 1-yl-n-butylamine, and R₁₆Is 1-yl-n-butylamine;

(d)R₉is an isobutyl group; r₁₀Is cyclohexyl; r₁₁Is an isobutyl group; r₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is 1-yl-allyl; r₁₄Is 1-yl-n-butylamine; r₁₅Is 1-yl-allyl, and R₁₆Is piperonyl;

(e)R₉is piperonyl; r₁₀Is an isobutyl group; r₁₁Is piperonyl; r₁₂Is cyclohexyl; r₁₃Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₄Is 1-yl-n-butylamine; r₁₅Is 1-yl-n-butylamine, and R₁₆Is piperonyl;

(f)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-n-butylamine; r₁₁Is 1-yl-n-butylamine; r₁₂Is piperonyl; r₁₃Is a methyl benzyl group; r₁₄Is 1-yl-n-butylamine; r₁₅Is 1-yl-n-butylamine, and R₁₆Is 1-yl-2-hydroxyethyl;

(g)R₉is 1-yl-2-hydroxyethyl; r₁₀Is a methyl benzyl group; r₁₁Is cyclohexyl; r₁₂Is 1-yl-n-butylamine; r₁₃Is piperonyl; r₁₄Is a methyl benzyl group; r₁₅Is methylbenzyl, and R₁₆Is 1-yl-n-butylamine;

(h)R₉is a methyl benzyl group; r₁₀Is piperonyl; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is a methyl benzyl group; r₁₄Is 1-yl-n-butylamine; r₁₅Is 1-yl-n-butylamine, and R₁₆Is piperonyl;

(i)R₉is a methyl benzyl group; r₁₀Is 1-yl-allyl; r₁₁Is piperonyl; r₁₂Is piperonyl; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₅Is 1-yl-n-butylamine, and R₁₆Is a methyl benzyl group;

(j)R₉is a methyl benzyl group; r₁₀Is a methyl benzyl group; r₁₁Is piperonyl; r₁₂Is a methyl benzyl group; r₁₃Is piperonyl; r₁₄Is 1-yl-n-butylamine; r₁₅Is methylbenzyl, and R₁₆Is 1-yl-n-butylamine;

(k)R₉is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₀Is a methyl benzyl group; r₁₁Is a methyl benzyl group; r₁₂Is a methyl benzyl group; r₁₃Is piperonyl; r₁₄Is 1-yl-n-butylamine; r₁₅Is 1-yl-n-butylamine, and R₁₆Is an isobutyl group;

(l)R₉is 1-yl-allyl; r₁₀Is a methyl benzyl group; r₁₁Is 1-yl-n-butylamine; r₁₂Is cyclohexyl; r₁₃Is piperonyl; r₁₄Is 1-yl-n-butylamine; r₁₅Is 1-yl-n-butylamine, and R₁₆Is piperonyl;

(m)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-2-hydroxyethyl; r₁₁Is an isobutyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is 1-yl-2-hydroxyethyl;R₁₄is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₅Is 1-yl-n-butylamine, and R₁₆Is cyclohexyl;

(n)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-2-hydroxyethyl; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-2-hydroxyethyl; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-n-butylamine; r₁₅Is piperonyl, and R₁₆Is 1-yl-2-hydroxyethyl; and

a pharmaceutically acceptable salt thereof.

Preferred embodiments for kits and diagnostic uses include:

a compound having the formula:

(a)R₉is piperonyl; r₁₀Is cyclohexyl; r₁₁Is 1-yl-n-butylamine; r₁₂Is 1-yl-n-butylamine; r₁₃Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₄Is 1-yl-n-butylamine; r₁₅Is isobutyl, and R₁₆Is 1-yl-n-butylamine;

(m)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-2-hydroxyethyl; r₁₁Is an isobutyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is 1-yl-2-hydroxyethyl; r₁₄Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₅Is 1-yl-n-butylamine, and R₁₆Is cyclohexyl;

a pharmaceutically acceptable salt thereof.

The invention may also be used to screen for biomarkers associated with any other disease or condition. Such diseases and conditions range from the neurological diseases, autoimmune diseases and cancers identified above as well as any other diseases or conditions that have biomarkers, such as antibodies or other characterizing proteins or biomolecules, associated with the disease or disease progression. These diseases and conditions include in particular inflammatory diseases, infectious diseases, cardiovascular diseases and metabolic diseases. Specific infectious diseases include AIDS, anthrax, food poisoning, brucellosis, chancroid, chlamydial infection, cholera, coccidioidomycosis, cryptosporidiosis, cyclosporinosis, diphtheria, ehrlichiosis, arboviral encephalitis, enterohemorrhagic Escherichia coli (Escherichia coli), giardiasis, gonorrhea, dengue fever, Haemophilus influenzae (haemophilius influenza), Hansen disease (leprosy), Hantaan virus pulmonary syndrome, hemolytic uremic syndrome, hepatitis A, hepatitis B, hepatitis C, human immunodeficiency virus, Legionella disease, Listeria disease, Lyme disease, malaria, measles, meningococcal disease, mumps, pertussis (pertussis), plague, paralytic polio, parrot, Q-type fever, rabies, rocky mountain fever, rubella, congenital rubella syndrome (SARS), Shigella, smallpox-mediated by infection, food poisoning, brucellosis, chancroplanus, enterohemorrhagic septicemia, gial disease, epidemic, and epidemic disease, Streptococcosis (invasive group a), streptococcal toxic shock syndrome, streptococcus pneumoniae (streptococcus pneumoniae), syphilis, tetanus, toxic shock syndrome, trichinosis, tuberculosis, tularemia, nociception, vancomycin-mediated resistance staphylococcus aureus (staphylocosus aureus), chicken pox, yellow fever, variant creutzfeldt-jakob disease (vCJD), ebola hemorrhagic fever, echinococcosis, hendra virus infection, human monkeypox, influenza a, H5N1, lassa fever, marburg hemorrhagic fever, nipah virus, orbutzfeldt-jakob disease, rift-valley fever, venezuelan equine encephalitis, and west nile virus.

The large ligand libraries of the invention can be used directly in biological fluids under suitable experimental conditions to screen for such markers without the use of fewer carrier members (e.g., about 100,000 or less) or the need to transfer such peptides or ligands to a microarray prior to screening of the biological fluid. In addition, ligand libraries can be used to screen for cell-based receptors that are specifically associated with particular cell surface markers. Unlike previous methods, the present invention allows for the inclusion of a greater number of beads/resins, and therefore a larger library, in ligand binding agent screening or cell receptor screening, to screen complex biological samples directly. The invention therefore also includes a method of monitoring disease progression comprising the steps of: using a kit or apparatus or device having at least one ligand derived from the rapid screening method of the present invention, a biological sample of a patient is screened at time point 1, followed by screening the biological sample of the patient at time point 2 or any subsequent time to track and/or monitor the presence or absence of a disease-associated biomarker in the patient at any time point.

Ligands

As previously described with respect to microarray systems, essentially any molecule or compound can be used to construct a bead or resin based random library. These "molecules" or "compounds" may include natural products or artificial compounds or synthetically derived molecules. The sources of such molecules may be from biological systems as well as non-biologically derived sources. Preferred ligand moieties for initial screening uses using large bead libraries under the conditions claimed herein are made in part from a subunit selected from any known monomeric amine and any known acetate halide or substituted acetate halide. For example, table 1 provides a series of R groups on an alternative monosubstituted amine:

TABLE 1 side chain modifications for peptoids

Table 2a list of amines that can be used to synthesize the peptoids described herein.

Preferred monomers and/or sub-monomers for use in AD screening, pancreatic cancer screening and lupus screening (primary and/or diagnostic) are selected from cysteine, glycine, methionine, allylamine, ethanolamine, isobutylamine, diaminobutane, methylbenzylamine (racemic or enantiomeric), piperonylamine, cyclohexylamine, 3,4 dimethoxyphenethylamine, benzylamine, N- (2-aminoethyl) acetamide, N- (3-aminopropyl) -2-pyrrolidone, 4- (2-aminoethyl) benzenesulfonamide or furfurylamine.

Also useful as a subunit are acetic acid halides and/or R-substituted acetic acid halides, where R is selected from any amino acid side chain or any other group, including those groups or variables on the monosubstituted amines. Alternatively, any combination of amines and any acetic acid halides can be reacted to form monomers which are then reacted with another reactive monomer on the growing peptoid chain to form the oligomers of the present invention.

Combinatorial libraries of peptoids can be prepared as follows:

after addition of the amino acid (or any desired amino acid that may function or otherwise be used in the diagnosis of an oligomer or with the oligomer), the remaining monomers may be added using standard peptide chemistry or using bromoacetic acid (or α -substituted bromoacetic acid or similar reactants) and monosubstituted amines, wherein the amines are substituted with R groups may be selected from any known peptoid substituents, including, for example, those described in U.S. patent publication nos. 2010/0303805 or 2010/0303835 and/or zukermann and many Kodadek publications.

The process for preparing each peptoid generally involves (1) preparing an amino acid reactant on a support (including an optional linker on the support); (2) reacting the amino acid moiety on the support with an acid halide, such as bromoacetic acid or chloroacetic acid, to form a halo derivative, (3) reacting the halo derivative with a monosubstituted amine to form an amide, and (4) repeating steps (2) and (3) to form the peptoid. Methionine-containing peptoids are typically prepared in large libraries. Cysteine-containing peptides are typically prepared when larger scale quantities of high affinity peptoids are desired and after initial screening of large bead or resin libraries. In large bead-based libraries used for initial screening of complex biological fluids such as serum, long PEG linkers, which are generally necessary for microarray screening, are not required or required. The PEG linker may be on a bead or resin, provided that it is a short linker of less than about 10 monomer units. In diagnostic kits comprising beads less than about 50 microns (e.g., 10 microns) or tentagel beads, short PEG linkers (e.g., 2-10 PEG monomers) or longer PEG oligomers may be used.

the conditions for performing each step in the oligomer construction process utilize a solvent such as DMF or acetonitrile or dichloromethane.trifluoroacetic acid for cleavage purposes and piperidine or other suitable base as the base in the reaction between the bromo derivative and the amine in the preparation of the amino acid reactant a variety of protecting groups are used in the preferred embodiment, diaminobutane is used as the first amine subunit in the chain adjacent to the cysteine residue on the C-terminus of the peptoid in the first step of the process, the selected bead or resin (in gram or milligram quantities) is expanded in a suitable solvent such as DMF.

Combinatorial libraries of small molecules are commercially available or prepared using methods known in the art. See, e.g., Eichler et al 1995; cho et al, 1999; LePlae et al, 2002; ostergaard and Holm, 1997; yang et al, 1999). Furthermore, U.S. Pat. No. 6,344,334 and publication Gallop et al, (1994), Gordon et al, (1994); thompson and Ellman (1996) are also sources of such molecules and libraries.

Combinatorial libraries of peptides can be obtained commercially or prepared using methods known in the art. See, e.g., Stewart and Young (1984); tam et al (1983); merrifield (1986); and Barany and Merrifield (1979), each of which is incorporated herein by reference.

Combinatorial libraries of nucleic acids, including RNA or DNA, are commercially available or prepared using methods known in the art. Combinatorial libraries of oligosaccharides can be obtained commercially or prepared using methods known in the art.

In each case, a "ligand" or random ligand may be added to the support resin or bead to form a screening library that can be used under the conditions described herein to screen complex biological fluids for biomarkers. Preferably the ligand is a peptoid ligand.

In addition to constructing and/or using such libraries, it may be necessary or desirable to characterize, purify, and/or synthesize or resynthesize any such ligands. Such methods are known in the art and include the full range of purification methods such as HPLC via chromatography or purification methods via chemical methods; methods of characterization such as mass spectrometry or NMR or a combination of any of these methods. Such methods are also described, for example, in U.S. patent publication 2007/0003954, which is hereby incorporated by reference. In such cases, any such purified ligand can be referred to as a compound or a substantially purified compound.

In the initial screening method of the present invention, the beads and/or resin are used as a carrier device with oligomers operably coupled to the carrier. In diagnostic kits or other kits with "hits" or "putative hits" from such initial screens, the vector system can be expanded to essentially any vector system, including microarrays or any other known diagnostic platform. In these cases it is necessary to ensure that such kits or other carrier systems with putative hits also have or are adapted to have a detector or detection method to allow detection of ligands with ligand binding moieties attached to such ligands. Preferred detection methods include, for example, ELISA or other methods involving the use of labeled secondary antibodies.

The carrier may be made of any suitable material. Materials used to prepare such supports may include, for example, glass, plastic, ceramic, or polymeric resins or beads. The carrier may also comprise a material such as nickel, copper, steel or other metal or metal mixture. The support may also be adapted to have a linker and/or other means for binding or linking or reacting with the ligand or a reactive group on the ligand. Such groups are also described in U.S. patent publication No. 2007/0003954. In the present invention, the number of resins or beads having individual ligands bonded thereto or to linkers and subsequently to the support ranges from greater than 100K to about 150,000,000 (MM). The preferred number range utilized in the initial screening method of the invention is 1MM to 2MM ligand/resin.

Resins are most preferred for use in the large scale ligand screening methods of the invention. These resins are graft copolymers consisting of a low-crosslinked polystyrene matrix onto which polyethylene glycol (PEG or POE) is grafted. TentaGel resin is commercially available (Rapp Polymere GmbH). Because PEG is a "chameleon type" polymer with hydrophobic and hydrophilic properties, the graft copolymer exhibits modified chemistry. There are in principle two methods of introducing PEG onto modified polystyrene matrices, depending on the manufacturer. The simplest immobilization procedure is to couple PEG to chloromethylated polystyrene via one of its terminal hydroxyl groups according to conventional ether synthesisOr other bifunctional PEGs for coupling to solid supports. The manufacturer found that by means of anionic graft copolymerization, which builds PEG directly on the substrate in one step, PEG chains with molecular masses up to 20 kilodaltons have been immobilized to functionalized crosslinked polystyrene. Graft copolymers with PEG chains of about 2000-3000 daltons proved to be optimal in terms of kinetic rate, flowability, swelling and resin capacity. Since there is no procedure to obtain monodisperse PEG with more than 10 ethylene oxide units by any polymerization technique, there is theoretically no method to introduce monodisperse PEG chains with more than 10 ethylene oxide units into the resin, or to obtain monodisperse PEG by direct polymerization on a polystyrene backbone (monodisperse is defined as PEG without any molecular weight distribution). These graft copolymers are pressure stable and can be used in batch processes as well as under continuous flow conditions. The copolymer comprises about 50-70% PEG (w/w). The properties of these polymers are highly controlled by the PEG properties as well as the relative polystyrene matrix.

The number of beads available within a certain amount of resin and the capacity of a single bead must be known to set up a chemical library or peptide library by the "one bead-one compound" method. Table 2 summarizes certain particle sizes and correlates them with the respective capacities of the individual beads. The general load based on TentaGel beads was calculated to be in the range of 0.25-0.3 mmol/g. For analytical characterization, at least 5pmol of resin-bound peptide was required for sequencing on the beads. To estimate the optimal amount of resin for the library that can be economically processed, the bead size and bead capacity must be considered. All beads showed a very narrow particle size distribution with respect to homogeneity and kinetic rate of diffusion process as well as single bead analysis and single bead quantification.

TABLE 3

Resin composition	Size [ mu ] m]	Beads/g	Capacity/bead
				TentaGel NH₂	750μm	4.62x10³	65nmol
TentaGel NH₂	500μm	1.5x10⁴	19nmol
				TentaGel NH₂	300μm	6.4x10⁴	4nmol
TentaGel NH₂	200μm	2.15x10⁵	1.3nmol
				TentaGel NH₂	130μm	8.87x10⁵	280-330pmol
TentaGel NH₂	90μm	2.86x10⁶	80-100pmol
				TentaGel M NH₂	35μm	4.55x10⁷	5.5pmol
TentaGel M NH₂	20μm	2.4x10⁸	1.0pmol
				TentaGel M NH₂	10μm	1.95x10⁹	0.13pmol

Correlation of particle size, number of beads per gram of resin and capacity per single bead. The volume of the single beads is calculated based on the volume of 0.25 to 0.3mmol/g resin.

There are several types of TentaGel beads available that exhibit custom properties, depending on their application:

TentaGel S resin:

the PEG spacer is attached to the polystyrene backbone via an alkyl linkage. This bond is not sensitive to acids or bases. Such resins are standard types of resins used in peptide synthesis, solid phase organic synthesis or combinatorial chemistry.

TentaGel PAP resin:

PEG is attached to the polystyrene backbone via a benzyl ether linkage. This benzyl ether linkage is sensitive to harsh acid conditions such as 100% TFA or a mixture of TFA/TMSBr.

These specially tailored resins are used for immunization procedures or for the synthesis of PEG-modified derivatives (PEG attachment products). The PEG spacer is cleaved from the solid support along with the synthesized compound by applying solid phase conditions (e.g., PEG-modified peptide) using harsh acid conditions to obtain a soluble PEG-modified compound.

TentaGel N resin:

the PEG spacer is attached to the polystyrene backbone via a benzyl ether linkage. These custom resins are used in oligonucleotide chemistry for small and large scale oligonucleotide synthesis. The capacity was increased by a factor of 10 compared to CPG glass.

Since the TentaGel resin is a copolymer composed of polystyrene and polyethylene glycol, the chemical and physical properties of both base polymers must be considered.

PEG is itself a water-absorbing polymer. It is known from the literature that PEG esters are not very stable and are easily hydrolyzed. Depending on storage conditions and storage time, PEG itself can oxidize along the polyether chain to form peroxides or esters. Thus, acid treatment or base treatment hydrolyzed the PEG-ester formed, which resulted in a small amount of "PEG leakage". This leakage can be noticed by MS or NMR as PEG signal and impurities in the final product. This chemical behavior is true for all PEG and PEG-based polymers.

TABLE 4

In addition to the TentaGel beads, other resins and/or particles can be used to construct a ligand/bead library. For example, a slightly crosslinked polystyrene resin or polyamide resin may be used. The group attaching the substrate to the resin bead may be an essential part of solid phase synthesis. The linker is a special protecting group, which is only reproduced at the end of the synthesis, since most of the time the linker will block the functional group. The linker must not be affected by the chemical action of the compound used to modify or prolong the attachment. And the final cutting step should be performed easily and in good yield. Optimal linkers must allow for attachment and cleavage in quantitative yield.

In certain aspects, the support can be a bead, a plate, a dipstick, a filter, a membrane, a needle, or a wall. The sample may be blood, serum, saliva or CSF. The detection may comprise RIA, FIA, ELISA, Western blot, flow cytometry, FRET or surface plasmon resonance.

Carboxylic acid linker

The first linker group used for peptide synthesis has the name of the parent of solid phase synthesis. Chloromethyl resin is crosslinked polystyrene functionalized with chloromethyl groups. The carbonyl group is attached by nucleophilic displacement of the chloride in DMF by cesium carboxylate salts. Cleavage to regenerate the carboxylic acid is typically achieved by hydrogen fluoride.

The second type of linker for carboxylic acids is the Wang linker (Wang). This linker is typically attached to crosslinked polystyrene, TentaGel, and polyacrylamide to form a wang resin. It was designed to synthesize peptide carboxylic acids using the Fmoc protection strategy, and the carboxylic acid product was cleaved with TFA due to the activated benzyl alcohol design. The resin of Wang, which is the more acid labile form, has been developed. The SASRIN resin has the same structure as the wang linker, but with the addition of methoxy groups to stabilize the carbonium ions formed during acid catalyzed cleavage.

Formamide joint

rink linkers are generally preferred for generating primary carboxamides on solid phases. In the present invention, this linker is utilized when making or resynthesizing hits or putative hits from the primary screen of the present invention. In such cases, cysteine is the first monomer to react with the rink linker, and then the process involves subsequent monomer addition to construct the oligomer, or subsequent sub-monomer chemistry to construct the oligomer. The greater acid sensitivity in the rink linker is a result of two additional electrons donated by the methoxy group. In the generation of primary formamide, the starting material is attached as a carboxylic acid to the linker and, after synthetic modification, cleaved from the resin with TFA.

Rink resin was used to generate formamide after TFA catalyzed cleavage.

Alcohol linker

hydroxyl linkers based on Tetrahydropyran (THP) protecting groups have been developed by Thompson and Ellmann all types of alcohols are readily added to dihydropyrans, and the resulting THP protecting groups are stable to strong bases but are easily cleaved with acids.

Carbamate and amine linkers

Urethane linkers have been used to synthesize combinatorial libraries of 576 polyamines prepared in a search for inhibitors of trypanosome parasitic infections. Two linkers were studied. One based on hydroxymethylbenzoic acid 1 and the other, to which an electron donating group 2 has been added. The latter allows cleavage by TFA, while the former can be cleaved with strongly acidic conditions.

Very useful linkers have recently been developed for the formation of tertiary amines. (Tertiary amines are commonly used in drug molecules.) Primary and Secondary amines are introduced into the linker by Michael addition. The amine may be alkylated to give resin bound quaternary ammonium ions. Under weakly alkaline conditions, hofmann elimination occurs to give a high purity tertiary amine.

Traceless joint

In some cases, the starting material is loaded onto the resin in one form, such as a carboxylic acid, and cleaved in another form, such as formamide. This is fully acceptable if the target compound requires the function of release. (peptides always contain carboxylic acids or carboxamides). However, the growing interest in low molecular weight non-peptide combinatorial libraries has led to the need for new linkers. These linkers show non-specific function after cleavage. The traceless linker is so called because examination of the final compound does not reveal a trace of the point of attachment to the solid phase.

Sample (I)

As previously discussed, the complex biological fluids prepared for analysis in the processes of the present invention include or may include a wide variety of potential biomarkers, including markers expressed on cells (non-adherent cells, including T cells or other immune effector cells), microorganisms, proteins, peptides, lipids, polysaccharides, small molecules, organic molecules, inorganic molecules, biomolecules, and any detectable and reactable moieties in such complex environments. In preferred embodiments, such markers are antibodies, and in particular antibodies generated as a result of a disease or condition. In a preferred embodiment, a body fluid derived from a patient or animal or organism, such as serum, plasma, saliva or other liquid or sample, is the source of such markers. Each sample or tissue or biologically derived, or environmentally derived or obtained sample is conditioned, or treated or diluted, or otherwise disposed of, in order to expose the sample to an initial screening or any subsequent screening using putative hits or ligands that have affinity for such biomarkers. The sample is diluted according to the methods described herein to provide or allow sufficient discrimination between background levels or noise and signals associated with binding of the ligand and the ligand binding moiety.

The time and/or conditions required for exposure of the ligand/carrier to such a sample will depend on the particular sample and other factors. Preferred conditions for the process of the invention are also described herein. In almost all cases, washing or elution steps and other conditioning methods are utilized after exposure of biological fluids to large ligand libraries and/or ligands or kits derived from such libraries. Aqueous solutions are utilized including buffered solutions such as HEPES buffer, Tris buffer or phosphate buffered saline. The support system may also be treated with an energy absorbing material to promote desorption or ionization of the "complex" from the support surface. Chemical methods are also used to decouple or remove the ligand-ligand binding moiety complex from the support.

Detection methods for detecting ligand-ligand binding moiety complexes on a support include photometric and non-photometric assays. Such methods include methods that ensure that the process includes detecting and measuring absorbance, fluorescence, refractive index, polarization, or light scattering. These include direct and/or indirect methods of measuring such parameters. Methods involving fluorescence include the addition of fluorescent labels in immunological methods such as ELISA or sandwich assays. Methods involving refractive indices include Surface Plasmon Resonance (SPR), grating coupling methods (e.g., sensor uniform grating couplers, wavelength interrogating light sensors (WIOS), and chirped grating couplers), resonant mirrors, and interferometric techniques. Methods involving polarization include ellipsometry. Light scattering methods may also be used. Other methods for tagging and/or separation and/or detection may also include magnetic methods. Magnetic resonance imaging, gas phase ion mass spectrometry, MRI can all be used.

Analysis of the generated data generally involves quantification of the signal due to the detected biomarker relative to a control or reference. The data may be analyzed by any suitable method. Computers and computer programs may be used to generate and analyze data. The beads and/or other vectors may be computer-coded or coded for identification purposes. Data analysis includes signal intensity analysis under specific conditions of the analysis or detection method. The ligand, ligand binding or reference moiety and/or secondary detection moiety may be labelled or radiolabelled or tagged with a detectable moiety. One of ordinary skill in the art can assess the differences and/or distinctions between biological fluid samples having disease-associated biomarkers relative to those of control or healthy patient samples that do not contain such markers. One of ordinary skill in the art can also determine the presence of false positives or other hits that are or can be found in control samples, in accordance with the methods described herein, to account for and/or retrieve such "hits," and one of ordinary skill in the art can continue the process of determining or finding disease-related biomarkers in patient samples having any disease or condition, in accordance with the methods described herein. In all cases, "detection" of such hits is accomplished by means of detecting the binding of a ligand binding moiety, such as a disease-associated biomarker or other marker, to a ligand in a ligand library, such as those described herein.

Biomarkers associated with the diseases and/or conditions described herein will vary depending on the particular stage of the disease and/or condition of the particular patient or animal or other organism being evaluated. In most cases, it is the ligand of the putative neutralizing compounds described herein that is expected to mimic the natural antigen, which first initiates the immune response and/or the formation of antibodies or immune cells. The invention and screening procedures claimed and described herein do not require knowledge of the particular antigen or the antibodies generated in response to the antigen. However, in addition to being useful in the screening and diagnostic methods described herein, the ligands themselves may be useful as vaccines or drug candidates. The invention thus includes compounds and pharmaceutical compositions.

And (3) peptide screening:

to screen a one-bead-one-compound (OBOC) combinatorial peptoid library, tens of thousands to millions of peptoid-loaded beads are prepared and subsequently mixed with a complex biological sample. The initial complex biological sample is preferably a control sample, and subsequent complex biological samples treated with the ligand library that has "removed" the control hits are then treated and/or screened against the diseased complex biological sample. The ligands/beads that interact with at least one disease-associated biomarker are then detected, identified and isolated and/or characterized. In a preferred embodiment, a Tentagel screening protocol is used, which includes (1) bead preparation, (2) screening of complex biological fluids and (3) detection of hits.

Peptide screening:

to screen a one-bead-one-compound (OBOC) combinatorial peptide library, tens of thousands to millions of peptide-bearing beads are prepared and then mixed with a complex biological sample according to the processes described herein. Beads that interact with disease-associated biomarkers are then identified and isolated for compound structure determination. For example, OBOC peptide library screening can be performed using Streptavidin (SA) as the probe protein, labeled with a red fluorescent dye, and a COPAS BIO-bed flow sorting device to separate fluorescent from non-fluorescent BEADs. See Marani et al, j.comb.chem.,2009,11(1), pages 146-150. Red dyes that can be used are ATTO590 and texas red. After incubation of the library with SA-red fluorescent dye conjugate, positive beads caused by peptide-SA interaction were obtained. The beads were analyzed by matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF MS). Thus, the peptide library may be used in a manner similar to the process described herein for peptoids, wherein an initial control biological fluid sample is used to remove any ligand/bead hits from the starting compound library, and wherein the remaining members of the library are subsequently used to screen for any hits in the diseased complex biological fluid sample. These hits are putative hits that subsequently advance any diagnostic kit.

In a similar manner, any ligand can be screened on a bead or carrier using the procedures described herein. In addition to peptoids or peptides, these ligands include nucleic acid oligomers, polysaccharides, small molecules, and/or any combination thereof, which can be constructed into libraries and used to screen complex biological fluids under the conditions described herein.

Kit and diagnostic tool

Any of the compounds or compositions described herein can also be used in diagnostic kits in a clinical or laboratory setting. These kits can range from simple point-of-care diagnostic assays to complex and multiplex instruments or probes. The carrier system and the "package" surrounding the core carrier/ligand system may be selected from current commercial kits designed to include putative hits and or hits that are resynthesized and installed on such a suitable platform, or they may be used for newly designed diagnostic kits. The kit will generally be accompanied by any suitable reagents and instructions for using the kit to screen and/or diagnose a particular disease or condition for which the kit is designed. Any such kit or method includes at least one putative hit or ligand that has been identified according to the screening methods described herein. The ligand or ligands may be selected from the same ligand or a mixture of ligands comprising a compound of the invention. Ligands can be selected based on their affinity for a particular disease state or a set or range of disease-associated biomarkers of a disease or condition. Preferably the ligand is a peptoid ligand. The kit also includes instructions for the physician to diagnose a particular disease or condition and specific markers for the particular kit and disease state or condition. The invention thus includes a combination of kits that include all of the necessary components thereof, e.g., putative peptoids or ligands found from initial screening using any of the libraries disclosed herein and/or known according to the specific methods and labeling instructions described herein. It is also contemplated that the specific processes and materials disclosed herein can be used in clinical and laboratory settings under the supervision of a skilled operator. The kit and/or apparatus or device includes a ligand, e.g., a peptoid, specific for the disease-associated antibody and/or cell. The "kit" may include a complete diagnostic kit or screening kit, or the "kit" may include components or subcomponents containing or comprising a diagnostic peptoid, an antibody discovered and characterized by such peptoid, or a natural antigen discovered and purified and/or characterized and discovered by an autoantibody as a result of interaction with the autoantibody. Such antibodies and purified antigens comprise portions of the invention.

Diagnostic method

The ligand libraries of the invention are useful for discovering and assaying ligands that bind to disease-associated biomarkers. Such ligands are then used in the kits and/or methods generally described above to assess, screen, or diagnose a disease state or condition. These diagnostic methods generally involve screening for and finding disease-associated biomarkers, including antibodies and/or other biomarkers. As noted above, these antibodies can also be identified and characterized on a suitable column using the ligands of the invention to extract or remove such antibodies from a blood sample. Antibodies in turn can be used to detect and find natural antigens that bind to such antibodies. The invention thus includes antibodies and purified antigens that bind to such antibodies and are discovered, isolated and characterized using the methods of the invention.

Kits and/or other devices for screening and/or diagnosing disease states or conditions must first be evaluated against a patient sample. These patient samples may be derived from a normal control sample or a patient sample, wherein the patient has been identified as a patient having or suspected of having the disease or condition. In addition to the "presence" of disease-associated biomarkers, the patient may also have other symptoms associated with the disease. The patient may be in an early stage of the disease, may not have the disease or condition at all, or may be in an advanced stage of the particular disease. In any clinical setting and under appropriate guidance and control, patients and clinical samples may be provided blindly and subsequently evaluated using the compounds of the invention. The data generated as a result of the screening can then be analyzed post-informed to find or not find statistically significant results or associations with known or potential data about any particular patient or group of patients. The invention encompasses methods of screening for the presence of a disease or condition comprising (1) screening a biological sample from a patient with at least one compound of the invention, (2) screening a control biological sample using the at least one compound under the same conditions, and (3) comparing the healthy control data against the patient data to determine the presence or absence of a disease-associated biomarker. A group of patients or patient samples having or suspected of having disease X may be screened against kits or diagnostic probes having at least one compound of the invention, and the data generated for each patient may be used to confirm or confirm the disease state or condition or lack thereof on a case-by-case basis. Such data generated herein may be used in combination with general information known about that particular patient to assess the condition of the patient and provide guidance to a medical practitioner who provides treatment options. The "information" generated as a result of any such screening can be used in a clinical trial setting to evaluate individual patients taking drug therapies. The invention thus includes a method of assessing the progress of a clinical trial comprising the use of a screen performed according to the methods described herein. In a preferred embodiment, the present invention relates to a method of screening for or targeting an early disease state comprising using a screening or compound as claimed herein to detect disease-associated biomarkers. The invention is particularly useful in the context of early cancer intervention, where detection of such biomarkers is expected to occur appropriately prior to the invasive progression of the disease. In another context, early intervention in cardiovascular and/or metabolic diseases and neurological diseases is expected to save lives and prevent or may be used to prevent further development of such diseases without the need for early medical intervention or treatment.

The invention also includes methods of increasing differential resolution or efficiency between a control or standard solution and a complex biological fluid comprising disease-associated biomarkers. For example, the method includes pre-conditioning or pre-treating or pre-blocking the system/serum with buffers or conditioning agents such as e.coli lysate and/or lysine.

In yet another embodiment, a method of treating a subject suspected of having a disease is provided comprising (a) contacting an antibody-containing sample from the subject with one or more carriers having a peptoid attached thereto, the peptoid comprising a peptoid having a formula described herein, (b) detecting antibodies that bind to the peptoid; and (c) making a treatment decision based on the results of step (b). The method may further comprise obtaining the sample from a subject. The method can further comprise making a diagnosis of a disease in the subject from which the sample was obtained when antibody binding to the peptoid is greater than that observed for a control non-diseased patient. The method may additionally comprise making a treatment decision regarding said subject. The sample may be contacted with more than one peptoid having the formula described herein. The sample may be contacted with multiple platforms for the purpose of diagnosing multiple disease states or conditions. The support may be a bead, plate, dipstick, filter, membrane, needle or wall. The sample may be blood, serum, saliva or CSF. The detection may comprise RIA, FIA, ELISA, Western blot, flow cytometry, FRET or surface plasmon resonance.

Further embodiments relate to antibody compositions isolated from biological fluids indicative of disease. In particular embodiments, the antibodies are isolated by contacting a sample having such antibodies with a peptoid composition that specifically binds to the antibodies indicative of or associated with a disease. The antibody may be removed, separated or purified from other non-antibody and non-D specific components. The antibody can then be washed and/or dissociated from the peptoid capture reagent.

In particular embodiments, peptoid arrays made from peptoids found in the processes described herein are hybridized to biological samples that have been supplemented with bacterial lysates, such as e. Biological samples include control samples and samples with markers for central nervous system disorders. For example, microarray slides were covered with hybridization chambers and equilibrated with 1XTBST (50mM Tris, pH8.0, 150mM NaCl, 0.1% Tween20) for about 15 minutes. The slides are then blocked with bacterial lysate at a concentration of at least, at most, or about 0.5, 1, 1.5, 2mg/ml lysate. The lysate was removed and the slide incubated with about one milliliter of biological sample (having an approximate protein concentration of 5, 10, 15, 20, or 25 μ g/ml, including all ranges and values therebetween) in the bacterial lysate with gentle shaking. The microarray is then washed with 1XTBST and hybridized with labeled anti-IgG antibody (e.g., at 1:400 dilution). The slides are then washed with a suitable buffer. The slides were dried using a centrifuge (e.g., spinning at 1500rpm for 5 minutes) and scanned on a microarray scanner, e.g., using a 635-nm laser at 100% power and 600 or 650 photomultiplier gain. The invention therefore also relates to a method of reducing background antisera noise in a diagnostic assay comprising treating a control plasma sample and a diseased sample with an e. This process is believed to be useful in supporting the processing of any array used to detect and distinguish antibodies in serum against a background comparing control samples to diseased samples.

It is contemplated that any method or composition described herein can be performed with respect to any other method or composition described herein.

The use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one" but it is also consistent with the meaning of "one or more", "at least one" and "one or more than one".

It is contemplated that any embodiment discussed in this specification can be performed with respect to any method or composition herein, and vice versa. In addition, the compositions and kits of the invention can be used to practice the methods of the invention

Throughout this application, the term "about" is used to indicate that a value includes variations inherent in the device, the method used to determine the value, or variations present in the subject under study.

Vaccines and medicaments

It is to be understood that any of the putative hits or peptoids discovered by the processes described herein may also be therapeutic drug or vaccine candidates. The present invention thus relates to a process for discovering a drug candidate or vaccine comprising using a screen according to the methods described herein.

Example 1 library preparation

Protocols for peptoid synthesis (cysteine-peptoid or methionine-peptoid)

The following examples demonstrate how the peptoid libraries of the present invention are generated. Materials utilized in this example include reaction flasks or beakers, plastic tubes, 10-15 syringes with 3ml needles. Latex gloves, 10-15 polypropylene test tubes of 15ml and micropipettes with solvent safety caps (1000. mu.l), glass pipette and resin beads. Chemicals and/or reagents utilized include N, N dimethylformamide, bromoacetic acid (BMA), anhydrous dimethylformamide, piperidine, acetonitrile, 3-Diisopropylcarbodiimide (DIC), trifluoroacetic acid, 5(6) -carboxyfluorescein, Dichloromethane (DCM), and 4-methylmorpholine (NMM). Various amines utilized in each library preparation were also used, as well as HBTU (tetramethyluronium hexafluorophosphate) and triethylsilane.

Peptoid preparation

The concentration of each amine used in the process was calculated using the formula: v = FW/d/1000x2Mx5ml

The procedure is as follows:

step 1

Expansion of the resin beads.

(a) 250 mg of resin beads were placed in a clean dry reaction flask and 5ml of anhydrous DMF was added to the beads, allowing the beads to expand over a period of one hour or less. The beads were then washed multiple times with DMF under vacuum (2 or 3 ×).

Step 2

When "unprotected beads" (e.g., Tenta-Gel) are used, steps (b), (c) and (d) are omitted.

A 20% piperidine (base) solution using anhydrous DMF as the solvent was used in the following procedure:

when "protected beads" are used, the following process comprising steps (b), (c) and (d) is carried out2 times (one time)。

(b) 2.5ml of 20% piperidine solution was added to the protected beads;

(c) after addition of the piperidine, the reaction flask was placed on a shaker/incubator set at 200rpm at 25 ℃ for 20 minutes.

(d) The reaction flask was then washed 8-10 times with anhydrous DMF using 5ml DMF.

The following solutions were also prepared:

1. 468mg Fmoc-Cys (Trt) -OH (solution A) in dry DMF (2 ml volume).

2. 161.6mg NMM in anhydrous DMF2ml

3. 303.2mg HBTU was added to NMM vial (solution B).

Addition of HBTU/NMM and Fmoc-Cys

1ml each of solution A and solution B- (HBTU/NMM) and Fmoc-Cys (Trt) -OH) was added to the beads and shaken for 1 hour.

The beads were washed 5-10 times in DMF.

The remaining 1ml of solutions a and B were added to the beads, which were shaken for a period of one hour and then washed again 5-10 times in DMF.

The following solutions were also prepared:

20% piperidine (in anhydrous DMF)

2M Bromoacetic acid

50%DIC/A.DMF

2M solutions of each amine

The following steps (a), (b) and (c) are performed2 times (one time). 2.5ml of 20% piperidine solution was added; (b) the reaction flask was shaken at 200rpm at 25 degrees celsius, and then (c) the beads were washed 8X to 10X with DMF.

10ml of a 2M bromoacetic acid solution were prepared.

Also prepared was 10ml of 50%3.2M DIC/anhydrous DMF (v/v) solution.

A 2M amine solution was prepared in each library and for each amine of each library.

For peptoid synthesis, 1ml of 2M stock solution was used each time, and the amine was added to the peptoid chain.

Step 3

(a) Adding 1ml of bromoacetic acid into a reaction vessel;

(b) 1ml of 50% DIC/DMF solution was then added and the resulting solution (c) was microwaved at 10% power for 15 seconds.

Step (c) was performed 2 times, with the flask left and right vortexed between microwave settings.

A white precipitate formed after each microwave step. The beads were subsequently washed 8-10 times with DMF.

Step 4

The first amine in the 1ml sequence was added to the reaction flask containing the bromine intermediate from the previous step and the vessel was shaken to evenly distribute the amine on the beads. The reaction was then initiated 2 times with microwaves at 10% power for 15 seconds. The reacted beads were then washed 8-10 times with anhydrous DMF.

Steps 3 and 4 are repeated until all amines are added to prepare the target peptoid.

Step 5

The beads were then washed 3 times with Dichloromethane (DCM) and allowed to dry.

Step 6

The peptoid was subsequently cleaved from the beads using 95% TFA solution (5 ml). The detached bead peptoids were then collected and the beads were washed with solvent (CH 3CN and water) to remove residual peptoids. Argon was used to remove any residual TFA. The peptoid is then lyophilized and characterized and purified as needed.

The reaction conditions specified above may be modified on an as-needed basis, depending on the number required for any particular bead composition.

Figures 1-5 generally demonstrate how the libraries of the invention can be prepared for AD diagnosis, pancreatic cancer diagnosis and lupus. In general, beads having amine moieties are attached to amino acid residues by a series of steps using standard peptide chemistry, the beads are then reacted with activated carbonyl moieties having halide groups, which are then reacted with monomeric amines having R groups. Steps 2 and 3 of the cycle are repeated as shown in the figure to generate a large peptoid library with 1MM-2MM different ligands. Initial screening libraries prepared on Tentagel resin or beads typically have a methionine amino acid as the first monomer in the chain. The present inventors used such amino acids to facilitate cleavage from beads or resins that do not have cleavable linkers. The Rink resin used to construct cysteine-containing peptides has a linker that does not require or require the use of methionine as the first amino acid. Cysteine-containing peptides are generally synthesized after the initial screening where putative hits are found. The cysteine sulfur group allows the peptide-like chain to react with another reactive moiety on, for example, a diagnostic platform substrate. The resynthesized peptoid also contains a 1-yl-n-butylamino moiety as the first side chain in the chain after the amino acid amine. This group is believed to be necessary for displaying the peptoid and for dissolving the peptoid in aqueous solution.

Example 2 general screening methods

160 micron Tentagel beads attached to the selected peptoids were expanded overnight in DMF. The beads were then washed ten times in the reaction vessel with Millipore water and vigorous shaking. Fresh Millipore water was added each time and at the 10 th wash, the beads were allowed to oscillate at 150-200rpm overnight. The following day, the beads were washed with 1 × TBST in the same manner and allowed to oscillate at 150-200rpm for at least 3 hours.

The beads were then divided evenly into 1X TBST in 15ml conical tubes, about 0.5 grams per tube. TBST was removed and 4ml of diluted normal human serum was added to each tube. Nano-dropwise (Nano-dropped) serum stock prepared in 1X TBST to obtain the desired concentration of 20 ug/ml. The tubes containing serum and beads were then tumbled overnight at 4 degrees celsius in the dark. The serum was then aspirated out of the tube and replaced with 4ml of 1X TBST. The tube was then slowly inverted to resuspend the beads and allowed to settle. TBST was removed and added two more times for a total of three TBST washes.

The secondary antibody solution was then prepared by preparing 5ul goat anti-human IgG Qdot655/1ml1X TBST. Once the last TBST addition was removed from the beads, 4ml of Qdot solution was added and the beads were tumbled at 4 degrees celsius for 2 hours in the dark. The beads were then allowed to settle and the Qdot solution was removed. The beads were then washed three times with 4ml of 1X TBST. The beads were then poured into a clean petri dish containing a DAPI filter and visualized under a UV microscope. All red-stained beads were removed.

After the first screening was completed, the beads were poured back into a 15ml conical tube and tumbled at 4 degrees celsius for at least 4 hours before the next serum sample addition. Disease serum was then added to the beads in the same manner as normal serum addition, except that the serum was diluted in a PBS starting block (starting block) as opposed to 1X TBST. However, the initial mother liquor was prepared in 1X TBST in order to obtain the appropriate concentration with a nanopipette. Serum addition and secondary antibody addition were identical to normal serum.

Once diseased "hits" were removed, they were pooled into 1.5ml eppendorf tubes and heated in 1% SDS at 95 degrees celsius for 25-30 minutes. SDS was then removed from the tube and replaced with Millipore water. The beads were then tumbled at 4 degrees celsius for 15 minutes. The water was then replaced with fresh water and the beads were tumbled for an additional 15 minutes. The water was then removed and replaced with a 50/50 acetonitrile/water solution and allowed to tumble for an additional 15 minutes. The beads were then separated into individual wells in a 96-well plate and allowed to dry.

A solution of 20-30mg cyanogen bromide, 500ul acetonitrile, 400ul glacial acetic acid and 100ul Millipore water was prepared and 20ul of the solution was added to each well containing the hit beads. The plates were covered and allowed to oscillate at 100rpm for 16 hours. The cover is then removed and the cutting solution is evaporated from the wells. The hit points were then plated onto MSMS plates and sequenced using a 4800MALDI/TOF analyzer.

Fig. 6 provides a general schematic of the screening methods disclosed and claimed herein.

Example 3 Alzheimer's disease screening

Tentagel magnetic screening

500 mg of 160 μm Tentagel beads (JC3B library) were added to a 15ml conical tube. 5ml of DMF was added to the tube and the beads were allowed to stand overnight to swell. The next day, DMF was aspirated off the tube and replaced with 5ml of 1X TBST. The tube was inverted to mix and then the beads were allowed to settle to the bottom and the 1X TBST was removed. 5ml of 1 × TBST was added and removed five more times.

Normal AD serum samples were prepared by adding 4ml PBS starter blocks to the tubes and 7ul of four separate AD samples each to the same tubes. Serum was added to the washed beads and the beads and serum were allowed to tumble overnight at 4 degrees celsius in the dark. The next morning, beads were removed from the bowl and allowed to settle before the serum was aspirated out of the tube. Add 4ml of 1X TBST to the tube and invert the tube for mixing. The TBST was then aspirated out of the tube and replaced with 4ml of fresh 1X TBST and removed again.

The DYNA-bead solution was then prepared by adding 50ul of well-mixed goat anti-human IgG DYNA beads to 4ml of 1 XTBST. The mixture was then added to the washed beads. The beads were then allowed to tumble at 4 degrees celsius for 2 hours in the dark.

The DYNA bead screening was performed without washing the beads. The tube was placed on a magnetic holder and filled to the edge with 1X TBST. The magnet and tube were gently agitated for 2 minutes and the beads were allowed to settle in the magnetic holder. TBST and free beads that settled to the bottom were carefully removed, not touching the hit beads that were attached to the side by magnet, and replaced with fresh 1X TBST. This process was repeated two to three times until no beads attached to the sides of the tube could be seen. The hit beads were then incorporated into one tube.

The remaining non-hit beads were dispensed into 1.5ml tubes, inverted and rapidly pulsed centrifuged. The supernatant was removed and replaced with fresh 1X TBST. This process was repeated 6-8 times until no more DYNA beads could be seen in the bead/TBST solution. Hit beads were washed in the same manner.

The beads were pooled back into a 15ml tube and normal serum was added to the beads in the same manner as previously described and allowed to tumble overnight at 4 degrees celsius in the dark. In addition, 3ml each of four normal AD samples were added to a 1ml PBS starting block, and this solution was added to the DYNA bead "hit" bead tube. The next day, the beads were washed in the same manner as normal serum addition.

20ul goat anti-human IgG quantum dots 655 were diluted into 4Ml1 XTST (20 ul Qdot in I Ml1XTBST for "hit" tube) and added to the beads. The solution was tumbled in the dark for 2 hours at 4 degrees celsius. Hit and non-hit tubes were washed four times with 1X TBST and screened for bright red beads under a UV microscope. The remaining beads were tumbled in 4ml of 1X TBST for 1 hour and disease AD serum was added in the same manner as normal serum. Magnetic screening and Qdot addition are performed in the same manner as previously described. Hits were then sequenced on a MALDI TOF/TOF mass spectrometer.

Figure 7 shows pooled normal control serum samples incubated overnight with beads from a peptoid library (JC 3B). A screening using DYNA beads, an initial secondary antibody of goat anti-human IgG tagged with DYNA beads, was performed to remove non-specific bead hits. These non-specific hits were subsequently re-confirmed using different secondary antibodies. In this case, goat anti-human IgG tagged by quantum dots 655(Invitrogen) was detected on a UV microscope containing a DAPI filter. Red beads indicate reconfirmed hits; blue beads indicate non-reconfirmed beads. The percentage of reconfirmed beads remained low, so the DYNA bead screen was finally discontinued.

Figure 8 shows a peptoid library incubated with serum from AD patients. Thereafter, the library was incubated with secondary detection antibodies (goat anti-human IgG tagged by quantum dots 655 (Invitrogen)). Hit beads were detected under a UV microscope containing a DAPI filter and beads that were red in color were picked as hits to be sequenced for further testing.

Figures 9, 10 and 11 show that after isolation of disease hits from the peptoid library, a 1% SDS wash was performed to strip hits from any residual antibody. The beads were then incubated with individual disease samples and then with goat anti-human IgG quantum dots 655. The beads were then visualized under a UV microscope to determine if the hits would be reconfirmed from individual samples. Consistent reconfirmed hits were selected for sequencing.

Figure 12 shows the sequences of all selected hits in an alzheimer's screen of JC3B library.

Figure 13 shows the chemical structure of putative hits selected in the alzheimer's screen.

Figure 14 shows competition experiments between peptoids ADTG1 in solution versus those designated ADTG1-42 on microarrays. The data show that the peptoids ADTG1, 14, 24, 25, 31, 35, and 40 bind to the same autoantibodies. The same experiment was performed for each peptoid on the array, and this procedure determined that the peptoid bound seven different autoantibodies against alzheimer's antigen.

Figure 15 shows the sequences of these 41 peptoids, designated more active to less active starting from the top row, and presented in four different groups based on the results of competition experiments that found ligands binding to four different antibody biomarkers for AD. ADP1-3 was previously found in microarray screening (data not shown, but previously described in U.S. patent publication No. 2010/0303805), and was found to bind to two different antibody biomarkers for AD. ADP1 and ADP3 were found to bind to the same antibody, while ADP2 bound to a separate antibody. The novel ligands found in the novel screening methods of the invention (all other ligands shown in figure 15 and as shown in figure 13) also bind to different antibodies in the panel. Two new antibody biomarkers were found and some of the new ligands bound either the same antibody to which ADP1 and ADP3 bound or the antibody to which ADP2 bound.

AD data analysis: microarray data with a single measurement

Microarrays were prepared as described in U.S. patent publication No. 2010/0303805, which is hereby incorporated by reference. Microarray slides were covered with hybridization chamber and equilibrated with 1XTBST (50mM Tris, pH8.0, 150mM NaCl, 0.1% Tween20) for 15 min. The slides were then blocked with 1ml of blocking buffer at 4 ℃ for 1 hour. The blocking buffer was removed and the slides were incubated with 1ml serum (20mg/ml) at 4 ℃ for 16 hours with gentle shaking. In an alternative method, slides were blocked with 1ml of E.coli lysate (1.5mg/ml) for 1 hour at 4 ℃. Coli lysates were removed and the slides were incubated with 1ml serum (15mg/ml) in E.coli lysates (1.5mg/ml) for 18 hours at 4 ℃ with gentle shaking. The microarray was then washed three times with 1XTBST and hybridized with Alexa-647 labeled anti-IgG antibody (5mg/ml) for 2 hours at 4 ℃ on an orbital shaker. The chamber box was removed from the microarray slide and washed with 1XTBST (3 x15 minutes) followed by 0.1XTBST (1x 10). The slides were then dried on a centrifuge (at 1500RPM5 minutes) and scanned on a microarray scanner (Gene pixauttoader 4200) using a 635-nm laser at 100% power and 600 or 650 photomultiplier tube gain. All scanned images were analyzed by Gene Pix Pro6.0 software and Genespring software.

The new peptoids or ligands were constructed on microarrays and tested against patient samples according to the methods described above in blinded studies. FIGS. 16-20 provide the results of these screening assays. Serum samples were obtained from a pool of 34 patients. Each sample resulted in the intensity shown in figures 16-18 or lack thereof. Two of the new peptoids (P1 aag4 and P1aag 2) were compared against the known peptoids ADP3 and ADP2 in the same patient pool. These results show a direct correlation between the new peptoid versus the previous peptoids that have been identified in informed studies demonstrating a direct correlation between the presence of AD-associated antibodies and the clinical symptoms or presence of the disease.

FIG. 16A shows data on a panel of serum samples (20ug/mL) from 34 patients screened with the peptoid P1aag1 on the microarray. Figure 16B shows data on a panel of serum samples from 34 patients screened with the peptoid P1aag2 on the microarray.

Figure 17A shows data on a panel of serum samples from 34 patients screened with peptoid P1aag3 on a microarray. Figure 17B shows data on a panel of serum samples from 34 patients screened with peptoid P4aag1 on a microarray.

Figure 18A shows data on a panel of serum samples from 34 patients screened with peptoid P1aag5 on a microarray. Figure 18B shows data on a panel of serum samples from 34 patients screened with peptoid P1aag6 on a microarray.

Each of these peptoids is otherwise shown in fig. 13. P1aag1 is JC 3B-1; p1aag2 is JC 3B-21; p1aag3 is JC 3B-7; p1aag4 is JC 3B-5; p1aag5 is JC3B-R8 and P1aag6 is JC 3B-R12.

Figure 19 shows ADP2 microarray data. FIG. 19B shows P1aag4 microarray data using patient sera from the same patient pool. There is at least 90% correlation between the data set using ADP2 on the left and the data set using P1aag4 on the right.

Figure 20A shows ADP3 microarray data. FIG. 20B shows P1aag2 microarray data using patient serum samples from the same patient pool. There is at least 90% correlation between the data set using ADP3 on the left and the data set using P1aag2 on the right.

FIG. 21 shows confirmation of the use of a peptoid (JC3B-R8) in a tentagel-based screen for Alzheimer's disease. 140 micron (um) beads were used and the serum concentration was 40 ug/mL. There was a clear distinction between diseased beads and non-diseased (normal control) beads.

FIG. 50 shows a simple schematic of the preparation and differentiation between peptoids used in microarrays relative to those placed on ELISA plates. Schematic diagram of how peptoid microarrays were prepared: individual beads were separated into the wells of a microtiter plate, and peptoids were cleaved from the beads to prepare concentrated stock solutions. It should be noted that each well will now contain a single species of peptoid. Several thousand peptoids were then spotted on a chemically modified glass microscope slide in such a way that they were covalently bound to the surface. Thousands of slides can be generated with high reproducibility from a single synthetic library. ELISA production is similar except that no PEG chains are present on the surface, but the peptoid density on the ELISA plate can be different from the density on the microarray.

Figure 53 provides a simplified diagram confirming the correlation between clinical diagnosis of an informed AD patient serogroup at various stages of alzheimer's disease (or not) versus data obtained from the same patient serum sample (blinded) and screened against ADP 3-like peptides to detect disease-associated antibodies. Results shown are from a blinded serum sample study by Mayo clinical Jacksonville. UND = undefined. The graph is derived from obtaining a single serum concentration (1:800) dilution. Reads >1. are considered positive, reads between 1 and 0.7 are considered indeterminate, and reads below 0.7 are considered negative.

Figure 54 provides a simplified diagram confirming the correlation between clinical diagnosis of an informed AD patient serogroup at various stages of alzheimer's disease (or not) versus data obtained from the same patient serum sample (blinded) and screened against multiple AD peptoids (the graph is the average of the results for 9 peptoids) to detect disease-associated antibodies. Results shown are from a blinded serum sample study by MayoClinic Jacksonville. UND = undefined. The graph is derived from obtaining a single serum concentration (1:800) dilution. Reads >3 were considered positive, reads between 1 and 0.7 were considered indeterminate, and reads below 0.7 were considered negative.

Figure 58 provides a summary of ELISA analysis using a total of 106 serum samples tested. Using this data, 9 samples could not be recalled from a single point of data (clinically 4 AD, 5 normal). Examination of the titration curves designated 3 of 9 to be consistent with the clinical data. 6 are still unclear (inconsistency among different classes of peptides).

FIG. 59 provides the chemical structure of P1aag 7-9.

The data presented above for the Opko diagnostic method for detecting disease-associated antibodies associated with alzheimer's disease confirm that the methods disclosed and described herein are powerful tools for confirming and/or predicting the onset of alzheimer's disease and/or confirming that neurological diseases or disorders are certain other neurological disorders such as mild cognitive impairment. The data clearly support earlier filed patent applications which claim and disclose specific peptoids and methods for detecting disease-associated antibodies. Here, completely novel peptoids discovered by new and novel and more rapid screening processes were identified in a variety of platforms including microarrays and ELISAs for clinical data in patients with symptoms at various stages of alzheimer's disease and were significantly correlated with actual clinical data after informed.

Example 4 pancreatic cancer screening

Peptoid libraries were generated to perform screening of sera from pancreatic cancer patients. Two libraries were synthesized (JC3B and JC 5B). A total of 26 hits were obtained in the screen for pancreatic cancer biomarkers. The JC3B library contained an amine selected from: diaminobutane; r-methylbenzylamine; isobutylamine; cyclohexylamine; piperonyl amine; 4- (aminoethyl) benzenesulfonamide; furfuryl amine and 2-methoxyethylamine. The JC5B library was constructed from an amine selected from: isobutylamine; 2-methoxyethylamine; diaminobutane; furfuryl amine; cyclohexylamine; r-methylbenzylamine; piperonyl amine and 4- (aminoethyl) benzenesulfonamide. Pooled 6 samples of each pancreatic disease serum and control serum were used for screening. JC3B condition: 5ul each of the diseased serum samples in 4ml PBS blocking buffer was used, and 5ul each of the normal serum samples in 4ml1X TBST was used. JC5B condition: 10ug/ml of diseased serum in 4ml PBS blocking buffer was used, and 150ug/ml of control serum in 4ml1X TBST was used. Dnay bead screening: 50ul goat anti-human IgGDYNA beads were added to 1ml1X TBST. DYNA bead screening was performed to remove non-specific hits. Screening the quantum dots: adding 10ul/1 goat anti-human IgG quantum dots 655 to 1X TBST; screening using a UV microscope to retrieve hits. And (3) hit confirmation: JC 3B: 2ul of each control PC sample was added to 1ml of 1X TBST to screen hits; removing red (non-specific) beads; 2ul of each disease PC sample was added to 1ml of blocking buffer for screening hits; red beads were removed (actual hits) and cut for sequencing. This was performed for DYNA bead hits and Q-point hits. 12 hits were reconfirmed. JC 5B: 50ug/ml of pooled disease samples were added to 1ml of blocking buffer to hit; 15ug/ml of pooled control sample was added to 1ml of 1X TBST to hit; red beads were removed (actual hits) and cut for sequencing; this was performed for DYNA bead hits and Q-point hits. 14 hits were reconfirmed.

FIG. 22 (left panel) peptoid libraries were incubated with sera from pancreatic cancer patients. Thereafter, the library was incubated with secondary detection antibodies (goat anti-human IgG tagged by quantum dots 655 (Invitrogen)). Hit beads were detected under a UV microscope containing a DAPI filter and beads that were red in color were picked as hits to be sequenced for further testing.

(right panel): after isolation of pancreatic cancer disease hits from the peptoid library, 1% SDS washes were performed to strip hits from any residual antibodies. The beads were then incubated with individual serum samples from pancreatic cancer patients, and then with goat anti-human IgG quantum dots 655. The beads were then visualized under a UV microscope to determine if the hits would be reconfirmed from individual samples. Consistent reconfirmed hits were selected for sequencing.

FIG. 23: (left) hit confirmation images confirm the efficacy of the Tentagel screen. After isolation of pancreatic cancer disease hits from the peptoid library, 1% SDS washes were performed to strip hits from any residual antibodies. The beads were then incubated with individual serum samples from pancreatic cancer patients, and then with goat anti-human IgG quantum dots 655. The beads were then visualized under a UV microscope to determine if the hits would be reconfirmed from individual samples. Consistent reconfirmed hits were selected for sequencing.

(right) another SDS wash was performed on the hit beads, and then the beads were incubated with control serum, followed by goat anti-human IgG quantum dots 655. The beads were then visualized under a UV microscope to determine if the hits would be reconfirmed from individual samples. Consistent reconfirmed hits were selected for sequencing.

Figure 24 shows hit confirmation in pancreatic cancer screening by mixing. The data show that the markers are specific for a particular disease (PC versus AD).

Figure 25 shows pancreatic cancer ligands from JC3B library (putative hits).

Figure 26 shows pancreatic cancer ligands (putative hits) from JC5B library.

Example 5 Lupus screening

Figure 27 shows SLE hits in SLE library screening relative to normal controls. The KN1B library was used and found to be useful in this particular screen. Exactly the same protocol as described above for pancreatic cancer screening and AD screening was used in lupus screening, except for the library and serum samples.

Fig. 28 shows a hypothetical SLE hit.

Fig. 29 shows a hit confirmation study.

Diagnostic kit and method

Figure 30 shows the binding of one of the SLE (lupus) peptoids to an ELISA plate using two different binding methods. In the first case, biotin was used as the "part" (which is non-covalent) of the plate "binding" to the ELISA plate by streptavidin treatment. The peptoids have fluorescein tags. In the second case, the cysteines on the peptoids were covalently attached to the maleimide-treated ELISA plate. In both cases, the data show that significant fluorescence signals are visible at mM concentrations greater than about 3 mM.

FIG. 32 shows ELISA plates with peptoids at 10mM and shows clearly the differences between diseased serum (column 1) (AD) and normal control serum (column 3) at multiple serum dilutions [1:200 doubled to 1:400, 1:800, 1:1,600, 1:3,200, 1:6,400, 1:12,800 ]. Peptoid wells with AD peptoid ADP3 had a peptoid concentration of 10 mM. Similarly, figure 32 shows the clear distinction between diseased samples and normal control sera for the tentagel platform at a 40ug/ml diseased serum concentration on 140 micron beads.

FIG. 33 shows ELISA plates with peptoids at different concentrations and clearly shows the difference between diseased serum (AD) and normal control serum at multiple dilutions [1:200 to 1:12,800 ]. The arrows show data for 10mM ADP3 at 1:800 serum dilution.

FIG. 34 shows ELISA plates with peptoids at 10mM and clearly shows the difference between diseased Serum (SLE) and normal control serum at multiple serum dilutions [1:200-1:12,800 ]. The data show a clear distinction between diseased sera and controls using SLE-KN1B-20(10 mM). Plasma can also be used in all screening methods, provided that total protein is accounted for.

Figure 35 shows AD serum ELISA plots using 10mM ADP3 prepared in binding buffer at different serum dilutions. Separation between normal and diseased serum occurs over a dilution range of 1:200 up to about 1:10,000. The initial dilution was 1:200 (group 1AD serum.394 mg/mL and non-diseased serum at.386 mg/mL).

Figure 36 shows SLE serum ELISA profiles using 10mm kn1B-20 prepared in binding buffer at different serum dilutions. Separation between normal and diseased serum occurs over a dilution range of 1:200 up to about 1:10,000. The initial dilution was 1:200 (group 1SLE serum. 375mg/mL and non-diseased serum at. 396 mg/mL).

Figure 37 shows SLE serum ELISA profiles using 10mM KN1B-20 prepared in DMSO at different serum dilutions. Separation between normal and diseased serum occurs over a dilution range of 1:200 up to about 1:10,000. The initial dilution was 1:200 (group 1SLE serum. 367mg/mL and non-diseased serum at. 322 mg/mL).

Figure 38 shows FACS platform for Tentagel bead hit confirmation.

Figures 48 and 49 show ADP 3-bound autoantibodies from normal controls versus alzheimer's disease serum using pre-blocking conditions such as e.coli lysate and lysine.

ELISA protocol

96-well maleimide activated plates were obtained from Thermo Scientific and washed three times with 400 ul/well wash buffer (0.1M sodium phosphate, 0.15M sodium chloride, 0.05% Tween20, pH 7.2) using a plate washer from Beckman Coulter. The peptoid of interest was diluted to 10mM in PBS binding buffer (0.1M sodium phosphate, 0.15M sodium chloride, l0mM EDTA, pH 7.2) and 200ul of the peptoid solution was added to the appropriate wells. The plates were then allowed to incubate for 2 hours at room temperature in the dark with shaking at 500 rpm. The plate washer was then used to aspirate the peptoid solution from the wells and washed three times again with 400 ul/well wash buffer. L-cysteine HCL H20(Thermo Scientific) was diluted to 10ug/mL in binding buffer and added to 200 ul/well. The plates were then incubated in the dark at room temperature for 1 hour with shaking at 500rpm and washed three times. 200ul StartingBlock^TM(PBS) blocking buffer (Thermo Scientific) was added to the wells and the plates were incubated at 4 degrees Celsius for 1 hour in the dark with shaking at 500 rpm. The plates were washed three times with a plate washer and serum samples were prepared by serial dilution from 1:200 down in binding buffer. A 1:200 sample stock solution was concentrated using a nano-dropper (ThermoScientific) to ensure that they were similar. Each diluted sample was vortexed before the next dilution was prepared. 200ul of the appropriate dilutions for serum (disease and normal) were added to the plates, and binding buffer without serum was used as a control. The serum was allowed to incubate in the dark for 2 hours at room temperature with shaking at 500 rpm. The plate is washed again, and200ul of goat anti-human IgG HRP (Millipore) diluted 1:30,000 in binding buffer was added to the appropriate wells and incubated for 30 minutes at room temperature in the dark with shaking at 500 rpm. The plate was washed three times and 100ul of TMB (3, 3 ', 5, 5' -tetramethylbenzidine) solution was added to each well and the color was allowed to develop on the bench for 30 minutes in the dark. 100ul2M sulfuric acid stop solution was added to stop the reaction and the wells were read at 450 absorbance using a plate reader.

Thus, in each case and for each disease or any disease, the process of the invention can be used to rapidly discover disease-associated biomarkers and ligands that bind to such markers. These ligands-this larger library of ligands-can then be used for a variety of diagnostic and/or therapeutic purposes. Diagnostic platforms include microarrays, bead-based methods, and ELISA systems. The conditions utilized above encompass important aspects of the invention. These conditions include the range of dilutions for serum on beads or in wells and detection methods, as well as the concentration of a particular peptoid. The number of beads with peptoids on the beads may vary depending on the particular test kit or screening kit. These numbers may also vary depending on whether the beads/ligands are used in the initial screening protocols and methods described herein and/or in the test kits based on the discovery of high affinity ligands.

It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A random ligand library for screening complex biological fluids comprising a compound of formula I on a bead support,

wherein R is₁Selected from electron rich amino acid side chains;

R₂is selected from H;

and R is₃-R₆Is independently selected from-C₁-C₆Alkyl, aryl, heteroaryl, and heteroaryl,-C₁-C₆Alkyl SCH₃、-C₀-C₆Alkyl radical C₂-C₆Alkenyl, -C₀-C₆Alkyl radical C₂-C₆Alkynyl, -C₁-C₆COOH、-C₁-C₆Alkyl OH, -C₁-C₆Alkyl NH₂、-C₃-C₈Cycloalkyl, -C₁-C₆Alkylaryl, -C₁-C₆Alkyl heteroaryl, -C₁-C₆Alkyl radical NC (O) C₁-C₆Alkyl, -C₁-C₆An alkylcycloamide, wherein any of said aryl or heteroaryl groups can be independently replaced by-OH, Cl, F, Br, -OCH₃、-SO₂NH₂or-O-CH₂-an-O-substitution,

wherein n is 3-11, wherein the library comprises from 200,000 to 150 million different ligands, and wherein the library is for bead-based screening.

2. A random ligand library for screening complex biological fluids according to claim 1 comprising a compound of formula I on a bead support,

wherein

R₁Is selected from- (C)₁-C₆)SCH₃；

R₂Is selected from H;

R₃and R₅Is independently selected from-C₁-C₆Alkyl, -C₁-C₆Alkyl SCH₃、-C₀-C₆Alkyl radical C₂-C₆Alkenyl, -C₀-C₆Alkyl radical C₂-C₆Alkynyl, -C₁-C₆COOH、-C₁-C₆Alkyl OH, -C₁-C₆Alkyl NH₂、-C₃-C₈Cycloalkyl, -C₁-C₆Alkylaryl, -C₁-C₆Alkyl heteroaryl, -C₁-C₆Alkyl radical NC (O) C₁-C₆Alkyl, -C₁-C₆An alkylcycloamide, wherein any of said aryl or heteroaryl groups can be independently replaced by-OH, Cl, F, Br, -OCH₃、-SO₂NH₂or-O-CH₂-O-substitution;

R₄is selected from the group consisting of furfuryl,

R₆selected from 1-yl-allyl, 1-yl-2-hydroxyethyl, isobutyl, 1-yl-n-butylamine, methylbenzyl, piperonyl, cyclohexyl, 1-yl-2- (3, 4-dimethoxyphenyl) ethyl, benzyl, 1-yl-2- (acetamide) ethyl, 1-yl-3-2-pyrrolidone, 1-yl-2- (4-benzenesulfonamide) ethyl or furfuryl, and

n is 3 to 11.

3. The ligand library of claim 2, wherein the beads comprise a PEG linker of less than 10 monomer units.

4. The ligand library of claim 1, wherein the library comprises a compound of formula X

Wherein R is₁Selected from electron rich amino acid side chains;

R₂is selected from H;

and R is₃-R₆Is independently selected from-C₁-C₆Alkyl, -C₁-C₆Alkyl SCH₃、-C₀-C₆Alkyl radical C₂-C₆Alkenyl, -C₀-C₆Alkyl radical C₂-C₆Alkynyl, -C₁-C₆COOH、-C₁-C₆Alkyl OH, -C₁-C₆Alkyl NH₂、-C₃-C₈Cycloalkyl, -C₁-C₆Alkylaryl, -C₁-C₆Alkyl heteroaryl, -C₁-C₆Alkyl radical NC (O) C₁-C₆Alkyl, -C₁-C₆An alkylcycloamide, wherein any of said aryl or heteroaryl groups can be independently replaced by-OH, Cl, F, Br, -OCH₃、-SO₂NH₂or-O-CH₂-O-substitution, and

n is 3 to 10.

5. The ligand library of claim 4 wherein R₄Selected from-n-butylamine.

6. The ligand library of claim 5 wherein R₁Is selected from-CH₂CH₂SCH₃or-CH₂SH。

7. The ligand library of claim 1 wherein the library comprises a compound having formula Ia

Wherein the compound is selected from compounds of formula Ia, wherein

(f)R₉is piperonyl; r₁₀Is 1-yl-nButylamine; r₁₁Is isopropyl; r₁₂Is isopropyl; r₁₃Is 1-yl-2-methoxyethyl; r₁₄Is cyclohexyl; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is 1-yl-2- (4 (benzenesulfonamide) ethyl;

(y)R₉is piperonyl; r₁₀Is piperonyl; r₁₁Is 1-yl-2-methoxyethyl; r₁₂Is 1-yl-2-methoxyethylA group; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-n-butylamine; r₁₅Is 1-yl-n-butylamine, and R₁₆Is 1-yl-2-methoxyethyl;

(ee)R₉is 1-yl-2-methoxyethyl; r₁₀Is a methyl benzyl group; r₁₁Is 1-yl-n-butylamine; r₁₂Is 1-yl-n-butylamine; r₁₃Is piperonyl;R₁₄is an isobutyl group; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is 1-yl-n-butylamine;

(gg)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-2-methoxyethyl; r₁₁Is 1-yl-n-butylamine; r₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is 1-yl-2-methoxyethyl; r₁₄Is 1-yl-2-methoxyethyl; r₁₅Is 1-yl-n-butylamine, and R₁₆Is a methyl benzyl group;

(kk)R₉is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₀Is 1-yl-n-butylamine; r₁₁Is 1-yl-n-butylamine; r₁₂Is a methyl benzyl group; r₁₃Is a methyl benzyl group; r₁₄Is cyclohexyl; r₁₅Is 1-yl-2- (4 (benzenesulfonyl)Amine) ethyl, and R₁₆Is a methyl benzyl group;

(mm)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₂Is 1-yl-2-methoxyethyl; r₁₃Is 1-yl-n-butylamine; r₁₄Is cyclohexyl; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is a methyl benzyl group;

and pharmaceutically acceptable salts thereof.

8. The ligand library of claim 1 wherein the library comprises compounds having the formula:

wherein the compound is selected from compounds of formula II, wherein

(w)R₉is a 1-radical-2- (4 (benzenesulfonamide) ethyl group, R₁₀Is an isobutyl group; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-2-methoxyethyl; r₁₃Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₄Is an isobutyl group; r₁₅Is 1-yl-2-methoxyethyl, and R₁₆Is cyclohexyl;

(ii)R₉is 1-yl-n-butylamine; r₁₀Is furfuryl; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-2- (4 (benzenesulfonamide)An ethyl group; r₁₃Is furfuryl; r₁₄Is cyclohexyl; r₁₅Is methylbenzyl, and R₁₆Is cyclohexyl;

(oo)R₉is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₀Is a methyl benzyl group; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is a methylbenzyl radical；R₁₄Is piperonyl; r₁₅Is 1-yl-n-butylamine, and R₁₆Is 1-yl-2-methoxyethyl;

and pharmaceutically acceptable salts thereof.

9. The ligand library according to claim 8 wherein the compound of formula II is selected from the group consisting of compounds having the following R₉-R₁₆The group of (a) or (b),

(b)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-2- (4 (benzenesulfonamide)) An ethyl group; r₁₁Is 1-yl-n-butylamine; r₁₂Is cyclohexyl; r₁₃Is 1-yl-2-methoxyethyl; r₁₄Is 1-yl-2, 2-dimethylethyl (isobutyl); r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is a methyl benzyl group;

(f)R₉is piperonyl; r₁₀Is 1-yl-n-butylamine; r₁₁Is isopropyl; r₁₂Is isopropyl; r₁₃Is 1-yl-2-methoxyethyl; r₁₄Is cyclohexyl; r₁₅Is 1-yl-2- (4 (benzenesulfonamide) ethyl, and R₁₆Is 1-yl-2- (4 (benzenesulfonamide) ethyl,

and pharmaceutically acceptable salts thereof.

10. The ligand library of claim 1 wherein the library comprises compounds having the formula:

and pharmaceutically acceptable salts thereof.

11. The ligand library of claim 1 wherein the library comprises compounds having the formula:

wherein the compound is selected from compounds of formula II, wherein

(m)R₉Is 1-yl-n-butylamine; r₁₀Is piperonyl; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is cyclohexyl; r₁₄Is an isobutyl group; r₁₅Is methylbenzyl, and R₁₆Is 1-yl-n-butylamine;

(n)R₉is a methyl benzyl group; r₁₀Is 1-yl-n-butylamine; r₁₁Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₂Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₃Is an isobutyl group; r₁₄Is 1-yl-n-butylamine; r₁₅Is methylbenzyl, and R₁₆Is 1-yl-n-butylamine;

(o)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-n-butylamine; r₁₁Is 1-yl-2-methoxyethyl; r₁₂Is 1-yl-2-methoxyethyl; r₁₃Is an isobutyl group; r₁₄Is 1-yl-2-methoxyethyl; r₁₅Is 1-yl-n-butylamine, and R₁₆Is 1-yl-2-methoxyethyl;

(p)R₉is a methyl benzyl group; r₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₂Is a methyl benzyl group; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-n-butylamine; r₁₅Is cyclohexyl, and R₁₆Is an isobutyl group;

(q)R₉is 1-yl-n-butylamine; r₁₀Is a methyl benzyl group; r₁₁Is a methyl benzyl group; r₁₂Is a methyl benzyl group; r₁₃Is 1-yl-2-methoxyethyl; r₁₄Is 1-yl-n-butylamine; r₁₅Is cyclohexyl, and R₁₆Is a methyl benzyl group;

(r)R₉is cyclohexyl; r₁₀Is cyclohexyl; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-2-methoxyethyl; r₁₃Is a methyl benzyl group; r₁₄Is a methyl benzyl group; r₁₅Is 1-yl-n-butylamine, andand R is₁₆Is 1-yl-n-butylamine;

(s)R₉is furfuryl; r₁₀Is furfuryl; r₁₁Is an isobutyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is 1-yl-n-butylamine; r₁₄Is a methyl benzyl group; r₁₅Is methylbenzyl, and R₁₆Is a methyl benzyl group;

(t)R₉is a methyl benzyl group; r₁₀Is 1-yl-2- (4 (benzenesulfonamide) ethyl; R₁₁Is cyclohexyl; r₁₂Is cyclohexyl; r₁₃Is 1-yl-n-butylamine; r₁₄Is 1-yl-2-methoxyethyl; r₁₅Is 1-yl-n-butylamine, and R₁₆Is a methyl benzyl group;

(u)R₉is an isobutyl group; r₁₀Is a methyl benzyl group; r₁₁Is a methyl benzyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is 1-yl-n-butylamine; r₁₄Is an isobutyl group; r₁₅Is isobutyl, and R₁₆Is 1-yl-n-butylamine;

(v)R₉is an isobutyl group; r₁₀Is 1-yl-n-butylamine; r₁₁Is a methyl benzyl group; r₁₂Is a methyl benzyl group; r₁₃Is a methyl benzyl group; r₁₄Is cyclohexyl; r₁₅Is methylbenzyl, and R₁₆Is 1-yl-n-butylamine;

(w)R₉is cyclohexyl; r₁₀Is cyclohexyl; r₁₁Is a methyl benzyl group; r₁₂Is a methyl benzyl group; r₁₃Is 1-yl-n-butylamine; r₁₄Is an isobutyl group; r₁₅Is 1-yl-n-butylamine, and R₁₆Is an isobutyl group;

(x)R₉is cyclohexyl; r₁₀Is cyclohexyl; r₁₁Is an isobutyl group; r₁₂Is 1-yl-n-butylamine; r₁₃Is 1-yl-n-butylamine; r₁₄Is a methyl benzyl group; r₁₅Is 1-yl-n-butylamine, and R₁₆Is 1-yl-2-methoxyethyl;

and pharmaceutically acceptable salts thereof.

12. The ligand library of claim 1 wherein the library comprises compounds having the formula:

wherein the compound is selected from the group consisting of compounds having the following R₉-R₁₆The compound of (a) to (b),

(f)R₉is 1-yl-n-butylamine; r₁₀Is 1-yl-n-butylamine; r₁₁Is 1-yl-n-butylamine; r₁₂Is piperonyl; r₁₃Is a methyl benzyl group;R₁₄is 1-yl-n-butylamine; r₁₅Is 1-yl-n-butylamine, and R₁₆Is 1-yl-2-hydroxyethyl;

a pharmaceutically acceptable salt thereof.

13. The ligand library of claim 1 wherein the library comprises compounds having the formula:

a pharmaceutically acceptable salt thereof.

14. The ligand library of claim 1 wherein the library comprises compounds having the formula:

wherein in the compound of formula IIIa, R₉-R₁₆Is selected from

a pharmaceutically acceptable salt thereof.

15. The ligand library of claim 1 wherein the library comprises compounds having the formula:

wherein in the compound of formula II, R₉-R₁₆Is selected from

a pharmaceutically acceptable salt thereof.