HK1169709B - Identification of small molecules recognized by antibodies in subjects with neurodegenerative diseases - Google Patents

Description

Identification of small molecules recognized by antibodies in subjects with neurodegenerative diseases

Background

This application claims priority over U.S. provisional application serial No. 61/183,260 filed on 6/2 2009 and U.S. provisional application serial No. 61/318,655 filed on 29/3 2010, each of which is incorporated herein by reference in its entirety.

The invention was carried out with government support from the national institute for heart, lung and blood (grant NO1-HV28185) and the national institutes of health (grant DP10D 00066301). The government has certain rights in the invention.

I. Field of the invention

The present invention relates generally to the fields of molecular biology, immunology and neurobiology. More specifically, the invention relates to the identification of peptoids that are recognized by Neurodegenerative Disease (ND) specific antibodies. These peptoids can be used to identify subjects having or at risk of having ND.

Description of the related Art

Alzheimer's Disease (AD) is a progressive fatal brain Disease with up to 530 million Americans suffering. AD destroys brain cells, causing memory, thinking, and behavior problems. These symptoms worsen over time and ultimately the disease is fatal. Currently, 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 dementia cases. Unfortunately, despite the treatment of symptoms, there is no cure.

Diagnosing alzheimer's disease is an empirical process involving several types of assessments, and can take many days or weeks to complete. Assessment includes an understanding of detailed medical history and physical examinations. In addition, standard laboratory tests (including blood, urine and CSF tests) were designed primarily to help rule out 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. Depression detection should also be included. Finally, brain imaging scans are recommended to exclude brain tumors or blood clots in the brain that cause symptoms. In summary, there is currently no single test that exactly diagnoses alzheimer's disease, and alzheimer's disease may be diagnosed definitively only by examining brain tissue after death.

Parkinson's Disease (PD) is another degenerative disease of the brain (central nervous system) that often impairs motor ability, speech and other functions. It affects movement (motor symptoms), but other typical symptoms include mood, behavior, thought, and sensory disturbances (non-motor symptoms). Individual patients may vary considerably in symptoms and the progression of the disease also varies from individual to individual. Symptoms of PD are due to loss of pigmented dopamine-secreting (dopaminergic) cells (spontaneous or genetic, toxic or traumatic loss) in the substantia nigra pars compacta. These neurons affect the striatum, and their absence results in altered activity of the basal ganglia neural circuit that regulates movement, essentially inhibiting the direct pathway and exciting the indirect pathway.

Diagnosis of PD presents similar but slightly different challenges. When performing a neurological examination to assess patients with any dyskinesia, the physician should understand the medical history and perform a physical examination. In addition, neurological examinations are performed to fully assess the nervous system, including the patient's motor, coordination, and balance status. Laboratory testing of the blood of patients with typical symptoms of Parkinson's disease rarely reveals any abnormalities. Electroencephalography (EEG's) records some states of brain electrical activity, but is less effective in determining PD. Brain MRI and CAT scans produced clear and fine autopsy pictures, but the brain of PD-diseased patients showed normal even under such fine examinations because the changes associated with PD were microscopic in level and these scans could not be shown. Since there is no definitive diagnostic test to provide a specific answer, the doctor must make a PD diagnosis based on the judgment.

Therefore, there remains a need for diagnostic methods for these two diseases and other neurological diseases: it is (i) accurate and objective, (ii) simple and reproducible, and (iii) useful in both early and late stage cases.

Summary of The Invention

The present invention provides compositions comprising a peptoid bound to an antibody indicative of a neurodegenerative disease and methods of detecting an antibody in a sample comprising an antibody comprising contacting the sample comprising an antibody with a support having the peptoid attached thereto. The peptoids of the present invention have the formula:

wherein R is¹And R⁴Independently selected from hydrogen(ii) a An alkyl group; an allyl group; a methyl group; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; n-butylamine; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; an aryl group; a heteroaryl group; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAzolyl (isoxazoyl);an azole group; piperonyl (piperonyl); a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; pyrimidinyl (pyrimidyl); a pyrimidine base (pyrimidinyl); a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAzolyl (isoxazolyl); an isoquinoline cycloalkyl group; an alkenyl group; a cycloalkenyl group; a phenyl group; a pyridyl group; a methoxyethyl group; (R) -methylbenzyl; unsubstituted or substituted by NH₂OH or SH substituted C_1-6An alkyl group; unsubstituted or substituted by NH₂OH or SH substituted C_2-6An alkynyl group; lysyl groups; carboxy-includes one or more chemical groups described in tables 1 and 2 below. R²、R³And R⁵Independently selected from C_0-6An alkylaryl 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 (alkynl)) or R¹⁶(wherein R is¹⁶Is selected from H or C_1-6Alkynyl (alkynl)); 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.

Or

Wherein R is₆Selected from hydrogen; an alkyl group; an allyl group; a methyl group; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; n-butylamine; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; an aryl group; a heteroaryl group; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAn azole group; an isoquinoline cycloalkyl group; an alkenyl group; a cycloalkenyl group; a phenyl group; a pyridyl group; a methoxyethyl group; (R) -methylbenzyl; unsubstituted or substituted by NH₂OH or SH substituted C_1-6An alkyl group; unsubstituted or substituted by NH₂OH or SH substituted C_2-6An alkynyl group; lysyl groups; a carboxyl group; hydroxyl-comprises one or more chemical groups described in tables 1 and 2 below. R⁷And R⁸Independently selected from C_0-6An alkylaryl 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.

Or

Wherein R is⁹、R¹¹And R¹³Independently selected from hydrogen; an alkyl group; an allyl group; a methyl group; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; n-butylamine; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; an aryl group; a heteroaryl group; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzene ring openerAn azole group; quinoline; different from each otherAn azole group; an isoquinoline cycloalkyl group; an alkenyl group; a cycloalkenyl group; a phenyl group; a pyridyl group; a methoxyethyl group; (R) -methylbenzyl; unsubstituted or substituted by NH₂OH or SH substituted C_1-6An alkyl group; unsubstituted or substituted by NH₂OH or SH substituted C_2-6An alkynyl group; lysyl groups; a carboxyl group; and hydroxyl-includes one or more chemical groups described in tables 1 and 2 below. R¹⁰、R¹²And R¹⁴Independently selected from C_0-6An alkylaryl 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.

Or

Wherein, in one embodiment, R of formula 1D¹⁷-R²⁰And R²³Independently selected from hydrogen; an alkyl group; an allyl group; a methyl group; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; n-butylamine; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; an aryl group; a heteroaryl group; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAn azole group; an isoquinoline cycloalkyl group; an alkenyl group; a cycloalkenyl group; a phenyl group; a pyridyl group; a methoxyethyl group; (R) -methylbenzyl; unsubstituted or substituted by NH₂OH or SH substituted C_1-6An alkyl group; unsubstituted or substituted by NH₂OH or SH substituted C_2-6An alkynyl group;lysyl groups; a carboxyl group; hydroxyl-comprises one or more chemical groups described in tables 1 and 2 below. R²¹、R²²And R²⁴Independently selected from C_0-6An alkylaryl 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.

In another embodiment, R of formula 1D¹⁷-R²⁰、R²³And R²⁴Independently selected from hydrogen; an alkyl group; an allyl group; a methyl group; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; n-butylamine; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; an aryl group; a heteroaryl group; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAn azole group; an isoquinoline cycloalkyl group; an alkenyl group; a cycloalkenyl group; a phenyl group; a pyridyl group; a methoxyethyl group; (R) -methylbenzyl; unsubstituted or substituted by NH₂OH or SH substituted C_1-6An alkyl group; unsubstituted or substituted by NH₂OH or SH substitutedC_2-6An alkynyl group; lysyl groups; a carboxyl group; and hydroxyl-includes one or more chemical groups described in tables 1 and 2 below. R²¹And R²²Independently selected from C_0-6An alkylaryl 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.

In yet another embodiment, R of formula 1D¹⁷、R¹⁹、R²⁰And R²³Independently selected from hydrogen; an alkyl group; an allyl group; a methyl group; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; n-butylamine; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; an aryl group; a heteroaryl group; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAn azole group; an isoquinoline cycloalkyl group; an alkenyl group; a cycloalkenyl group; a phenyl group; a pyridyl group; a methoxyethyl group; (R) -methylbenzyl; unsubstituted or substituted by NH₂OH or SH substituted C_1-6An alkyl group; unsubstituted or substituted by NH₂OH orSH-substituted C_2-6An alkynyl group; lysyl groups; a carboxyl group; and hydroxyl-includes one or more chemical groups described in tables 1 and 2 below. R¹⁸、R²¹And R²⁴Independently selected from C_0-6An alkylaryl 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.

In formula 1A, R2, R3, R4, and R5 may be additionally selected from those groups shown in tables 1 and 2 provided by the equivalent letter designations for the positions of specific peptoid monomers in ADP1-3 and PDP1-3 oligomers. For example, in formula 1A, the R variables selected from R2, R3, and R5 can be independently selected from table 1 and/or 2 for the particular peptoid ADP-1, A, B or C, and those side chains or monomers shown for ADP2(D, E or F) and ADP3(G, H or I).

In formula 1B, R7 and R8 may additionally be selected from those groups shown in tables 1 and 2 provided by the equivalent letter designations for the positions of specific peptoid monomers in the ADP1-3 oligomer. For example, in formula 1B, the R variables selected from R7 and R8 can be independently selected from those side chains or monomers shown in tables 1 and/or 2 for the C of a particular peptoid ADP-1 and for ADP2(E or F) and ADP3 (I).

In formula 1C, R10, R11, R12, and R14 may additionally be selected from those groups shown in tables 1 and 2 provided by the equivalent letter designations for the positions of specific peptoid monomers in the ADP1-3 oligomer. For example, in formula 1C, the R variables selected from R7 and R8 can be independently selected from those side chains or monomers shown in tables 1 and/or 2 for A, B or C for a particular peptoid ADP-1 and for ADP2(D, E or F) and ADP3(G, H or I).

In formula 1D, R21, R22, R23, and R24 may additionally be selected from those groups presented in tables 1 and 2 for equivalent letter designations of specific peptoid monomer positions in the ADP1-3 oligomer. For example, in formula 1D, the R variables selected from R7 and R8 can be independently selected from table 1 and/or 2 for A, B or C for a particular peptoid ADP-1 and those side chains or monomers shown for ADP2(D, E or F) and ADP3(G, H or I).

In the formulae described herein, Z is a coupling group, which may include one or more amino acids or functional groups; l is an optional linker moiety, M is a substrate or support or label; and m, n, o and/or p are 0 to 6.

The method further comprises (b) detecting the antibody bound to the peptoid.

In certain aspects, the peptoids described herein may comprise a terminal functional group at the carboxy-terminus or the amino-terminus; the functional group can be coupled to a support, linker moiety, label, or other moiety. In certain aspects, a terminal cysteine residue is coupled to the peptoid and can provide a sulfhydryl group that further couples the peptoid to a substrate. In other aspects, the carboxy terminus can comprise NH₂OH or other chemical groups which may further react with the substrate (directly or indirectly) or the linker or other moiety.

A variety of linkers can be used. The simplest linker component is a bond between the peptoid and a second moiety (e.g., a matrix or other molecular entity). More generally, the linker will provide a single or multi-molecular backbone to covalently or non-covalently link one or more peptoids to one or more substrates or molecular moieties. Thus, attachment of the peptoids described herein to a desired substrate or molecular moiety may be performed by covalent or non-covalent means, which typically involves interaction with one or more functional groups or a second molecular entity located on the peptoid and/or substrate. Examples of chemically reactive functional groups that may be used for this purpose include amino, hydroxyl, mercapto, carboxyl and carbonyl groups, as well as sugar, vicinal diol, thioether, 2-aminoalcohol, 2-aminothiol, guanidino, imidazolyl and phenolic groups.

The method further comprises obtaining a sample from the subject. The method further comprises diagnosing the subject from which the sample was obtained as having alzheimer's disease if binding of the antibody to the peptoid is greater than that observed in a control non-diseased patient. In yet another aspect, the method may further comprise formulating a specific drug or therapy for the subject.

In certain aspects, the peptoid may be selected from AD1(APD1), AD2(APD2), and AD3(APD3), wherein Z is as described above. The sample can be contacted with more than one peptoid of formulae 1A-1D, e.g., three structurally distinct peptoids (e.g., AD1, AD2, and AD 3).

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

In certain aspects, peptoids of the present invention may comprise one of the following formulas:

(i)(X)_0-4(methylbenzyl) (n-butylamine)

(ii)(X)_0-4(allyl) (n-butylamine) (X), or

(iii) (X) (Piper (X)_0-2(n-butylamine) (X)_0-2(glycine);

wherein X may be hydrogen; an alkyl group; an allyl group; a methyl group; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; n-butylamine; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; or a hydroxyl group. In a preferred embodiment, the monomeric amine or amino acid is selected from the group consisting of glycine, cysteine, allylamine, ethanolamine, isobutylamine, diaminobutane, methylbenzylamine, piperonylamine, 4 (2-aminoethyl) benzenesulfonamide, furfurylamine, benzylamine, 3-Methoxypropylamine (MPOA), 2-methoxyethyl, and cyclohexylamine. These monomers can be used in any combination to form oligomers of 3-12mer length.

Some embodiments include peptoids of 2, 3, 4, 5, 6, 7,8 or more monomeric units. In certain aspects, the peptoid may comprise a peptoid sequence from positions 1, 2, 3, 4, 5, 6 to 8 or from positions 8, 7, 6, 5, 4,3, 2 to 1, or any 2, 3, 4, 5, 6, 7 monomers therebetween.

The monomers may include various combinations of R groups, such as hydrogen; an alkyl group; an allyl group; a methyl group; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; n-butylamine; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; an aryl group; a heteroaryl group; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAn azole group; an isoquinoline cycloalkyl group; an alkenyl group; a cycloalkenyl group; a phenyl group; a pyridyl group; a methoxyethyl group; (R) -methylbenzyl; unsubstituted or substituted by NH₂OH or SH substituted C1-C6 alkyl; unsubstituted or substituted by NH₂OH or SH substituted C2-C6 alkynyl; lysyl groups; a carboxyl group; or hydroxy-including one or more chemical groups described in tables 1 and 2 below.

In certain aspects, certain monomeric units of formula ADP1, ADP2, and/or ADP3 can be selected groups in tables 1-3 and at specific positions. Some of the most preferredEmbodiments are specific compounds shown as ADP1, ADP2, ADP3, and also include those active compounds identified in the sarcosine scan (sarcosine scan) which identifies a specific monomer referred to as the a-I position in the ADP1-3 structure shown above. Tables 1 and 2 below provide the names of specific positions of ADP1-3 in the left column, ADP1-3 may additionally have side chains as shown in table 1 (using amines with said side chains) or alternative amines as shown in table 2, and they are located at these specific preferred positions. Residues a to I of ADP1, ADP2 and ADP3 may also be substituted by groups independently selected from: hydrogen; an alkyl group; an allyl group; a methyl group; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; n-butylamine; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; an aryl group; a heteroaryl group; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAn azole group; an isoquinoline cycloalkyl group; an alkenyl group; a cycloalkenyl group; a phenyl group; a pyridyl group; a methoxyethyl group; (R) -methylbenzyl; unsubstituted or substituted by NH₂OH or SH substituted C_1-6An alkyl group; unsubstituted or substituted by NH₂OH or SH substituted C_2-6An alkynyl group; lysyl groups; carboxy-includes one or more chemical groups described in tables 1 and 2 below. R₂、R₃And R⁵Independently selected from C_0-6An alkylaryl 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.

TABLE 1 side chain modification of peptoids

TABLE 2 List of amines that can be used for the synthesis of the peptoids described herein

In another embodiment, there is provided a peptoid that binds antibodies indicative of parkinson's disease and a method of detecting antibodies in a sample comprising antibodies, comprising (a) contacting the sample comprising antibodies with a support having the peptoid attached thereto, the peptoid having the formula:

in one embodiment, R²⁷Is an alkylaryl group. In certain aspects, R²⁷Is a methylbenzyl group. R of formula 2A²⁵、R²⁶、R²⁸-R³²Independently selected from hydrogen; an alkyl group; an allyl group; a methyl group; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; n-butylamine; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; an aryl group; a heteroaryl group; a furyl group; an indolyl group; a thienyl group;a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAn azole group; an isoquinoline cycloalkyl group; an alkenyl group; a cycloalkenyl group; a phenyl group; a pyridyl group; a methoxyethyl group; (R) -methylbenzyl; c_0-6An alkylaryl 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 of the chemical groups described in tables 1 and 2. In certain aspects, R²⁶、R²⁹、R³¹And/or R³²Is an alkylamine, such as N-butylamine. In yet another aspect, R³⁰Is an alkenyl group, such as allyl.

In another embodiment, the peptoid may have formula 2B, wherein R³⁴Is an alkylamine, such as N-butylamine. In yet another embodiment, R³⁵Is an alkenyl group, such as allyl. In certain embodiments, R³⁷Is an alkoxy group such as ethanol. In certain aspects, R of formula 2B³³、R³⁶And R³⁸-R⁴⁰Independently selected from hydrogen; an alkyl group; an allyl group; a methyl group; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; n-butylamine; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; an aryl group; a heteroaryl group; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAn azole group; an isoquinoline cycloalkyl group; an alkenyl group; a cycloalkenyl group; a phenyl group; a pyridyl group; a methoxyethyl group; (R) -methylbenzyl; c_0-6An alkylaryl 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 of the chemical groups described in tables 1 and 2.

In the formulae described herein, Z is a coupling group, which may include one or more amino acids or functional groups; l is an optional linker moiety, M is a substrate or support or label; and m, n, o and/or p are 0 to 6.

In certain aspects, the method further comprises (b) detecting an antibody that binds to the peptoid.

The method further comprises obtaining a sample from the subject. The method can further comprise diagnosing the subject from which the sample was obtained as having Parkinson's disease if binding of antibody to the peptoid is greater than that observed in a control non-diseased patient. In yet another aspect, the method may further comprise formulating a drug or therapy for the subject.

In certain aspects, the peptoid may be selected from PD1, PD2, and PD 3. The sample can be contacted with more than one peptoid of formula 2A-2B, e.g., three structurally distinct peptoids (e.g., PD1, PD2, and PD 3).

In certain aspects, the support can be a bead, a plate, a dip stick, a filter, a membrane, a needle, or a well. The sample may be blood, serum, saliva or CSF. Detection may include RIA, FIA, ELISA, Western blot, flow cytometry, FRET or surface plasmon resonance.

In yet another embodiment, a method of treating a subject suspected of having a Neurodegenerative Disease (ND) is provided comprising (a) contacting a sample comprising antibodies from the subject with one or more supports having a peptoid attached thereto, the peptoid comprising a peptoid unit having formula (1A), formula (1B), formula (1C), formula (1D), formula (2A), and/or formula (2B), (B) detecting antibodies bound to the peptoid; and (c) making a treatment decision based on the results of step (b). The method may further comprise obtaining a sample from the subject. The method may further comprise diagnosing Parkinson's disease in the subject from which the sample is obtained if binding of the antibody to the PD class peptide is greater than that observed in a control non-diseased patient, or diagnosing Alzheimer's disease in the subject from which the sample is obtained if binding of the antibody to the AD class peptide is greater than that observed in a control non-diseased patient. In yet another aspect, the method may further comprise making a treatment decision for the subject. The sample may be contacted with more than one peptoid of formulae 1A-1D and/or 2A-2B. The sample may be contacted with three structurally distinct peptoids reactive with respective antibodies to PD and AD. The support may be a bead, plate, dip stick, filter, membrane, needle or well. The sample may be blood, serum, saliva or CSF. Detection may include RIA, FIA, ELISA, Western blot, flow cytometry, FRET or surface plasmon resonance.

In other embodiments, neurodegenerative diseases include, but are not limited to, alzheimer's disease; parkinson's disease; multiple Sclerosis (MS), amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease); dementia; motor neuron disease; prion disease (prion disease); huntington's disease; tauopathies (Tauopathies); dementia with chromosome 17; hereditary neuropathies and diseases involving cerebellar degeneration.

Another embodiment relates to an antibody composition isolated from a biological fluid indicative of a neurodegenerative disease. In certain embodiments, the antibody is isolated by contacting a sample having the antibody with a peptoid composition that specifically binds to or is associated with an indicator of a neurodegenerative disease. Antibodies can be removed, isolated or purified from other non-antibody or non-ND specific components. The antibody can then be washed and/or dissociated from the peptoid capture agent.

In certain embodiments, the peptoid array is hybridized to a biological sample supplemented with a bacterial lysate (e.g., an e. The biological samples include control samples and samples with markers for central nervous system disease. For example, microarray slides were covered with hybridization cassettes and equilibrated with 1XTBST (50mM Tris, pH 8.0, 150mM NaCl, 0.1% Tween20) for about 15 minutes. The slides were 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, incubated with about 1ml of the biological sample in bacterial lysate (having a protein concentration of about 5, 10, 15, 20, or 25 μ g/ml, including all ranges and values therebetween) and gently shaken. The microarray is then washed with 1XTBST and labeled with anti-IgG antibody (e.g., 1: 400 dilution). The slides are then washed with the appropriate buffer. The slides are dried by centrifugation (e.g., spinning at 1500rpm for 5 minutes) and scanned on a microarray scanner, for example using a 635-nm laser at 100% power and photomultiplier tube magnification of 600 or 650. 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 disease sample with an E.coli lysate and contacting said samples with an array of peptoids or ligands. It is contemplated that the method may be used to perform any array that is used to detect and distinguish antibodies in serum when comparing a control sample to a disease sample.

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

In the claims and/or the specification, the term "comprising" may mean "a" when there is no quantitative limitation, but may also mean "one or more", "at least one", and "one or more than one".

It is contemplated that any embodiment discussed in the specification can be practiced with reference to any method or composition of the invention and vice versa. In addition, the compositions and kits of the invention can be used to perform the methods of the invention.

In this application, the term "about" is used to indicate an inherent variation in error of an instrument, a method for determining a value, or a variation existing between study objects.

Brief Description of Drawings

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. 1A-B-Structure of peptoids found to capture disease-specific antibodies. (FIG. 1A) peptides AD1-AD3 identified antibodies to Alzheimer's disease. (FIG. 1B) peptoid PD1-PD3 identifies Parkinson's disease antibodies. Z represents a functional group that may be further coupled to a substrate, a linker or a second molecular entity.

FIGS. 2A-C-antibody profiles that distinguish subjects with Alzheimer's disease and Parkinson's disease from controls. (FIG. 2A) serum antibody binding profile of peptoids AD1-AD 3. (FIG. 2B) serum antibody binding profile of the peptoid PD1-PD 3. (FIG. 2C) serum antibody binding profile of ADPD1-ADPD 3.

Figure 3 removal of AD serum samples (confirmed by autopsy). ADP1 bound antibodies in serum samples were removed by passing through a column with excess immobilized ADP 1. The removed sera were then evaluated for binding to ADP 1-3. The figure shows that the peptoids ADP1 and ADP3 bind the same antibody, while the peptoid ADP2 binds a different antibody.

Figure 4 validation of ADP1 peptoids as selectable markers for alzheimer's disease on microarrays.

Figure 5 validation of ADP2 peptoids as selectable markers for alzheimer's disease on microarrays.

Figure 6 validation of ADP3 peptoids as selectable markers for alzheimer's disease on microarrays.

Figure 7 validation of the intermediate subject AD12 after 5 years.

FIG. 8 Luminex for Alzheimer's disease versus normal controlAnd (4) titrating. (FIG. 8A) autopsy confirmation of AD samples compared to normal controls. (FIG. 8B) AD5 vs. NC 9. (FIG. 8C) AD8 vs. NC 11. (FIG. 8D) autopsy confirmation of AD samples compared to normal control pools.

FIG. 9 different Normal controlsLuminex of samplesAnd (4) titrating. (FIG. 9A)2 different normal controls were compared to the normal control pool. (FIG. 9B) AD8 vs. NC 11. (FIG. 9C) autopsy confirmation of AD samples compared to normal control pools.

FIG. 10 shows a sample obtained from LuminexValidation of ADP1 peptoids on beads as selectable markers for alzheimer's disease.

FIG. 11 shows a sample obtained from LuminexValidation of ADP2 peptoids on beads as selectable markers for alzheimer's disease.

FIG. 12 shows a sample obtained from LuminexValidation of ADP3 peptoids on beads as selectable markers for alzheimer's disease.

FIG. 13 Luminex for intermediate samplesAnd (4) titrating.

FIG. 14 sarcosine scan results of ADP 1-3.

FIG. 15 comparison of peptoid arrays incubated with or without bacterial lysate blocking agent.

Description of illustrative embodiments

I. Neurodegenerative diseases

Neurodegenerative Diseases (ND) include a variety of debilitating conditions of the central and peripheral nervous systems. However, most affect the CNS. These diseases include Alzheimer's disease, Pick's disease, senile dementia, Parkinson's disease, multiple sclerosis, multiple system atrophy, Lewy body dementia, Huntington's disease, progressive supranuclear palsy, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, dementia, motor neuron disease, prion disease, Huntington's disease, tauopathies disease, chromosomal dementia No. 17, hereditary neuropathy, and diseases involving cerebellar degeneration.

Since there is generally no single definitive test for these diseases, and since many of these diseases show coincident symptoms, physicians spend weeks and millions of dollars in testing for the purpose of "exclusion," which ultimately provides little or no diagnostic benefit. There is therefore a great need for simple and accurate detection.

The present inventors have identified a number of small molecule "peptoids" that are specific ligands for antibodies specifically produced by subjects with parkinson's disease or alzheimer's disease. Peptoids or N-substituted oligoglycines are particular sub-types of peptidomimetics. They are closely related to their natural peptide counterparts, but differ chemically in that their side chains are attached along the backbone of the molecule at the nitrogen atom, rather than at the alpha-carbon (as in amino acids). Since these peptoids exhibit sensitive selectivity for disease-specific antibodies, simple immunoassay-based assays, which are inexpensive and easy to use, can now be used to provide definitive diagnosis for at least some PD and AD subjects. These and other aspects of the invention are set forth in detail below.

A. Parkinson's disease

Parkinson's Disease (PD) is one of the types of disorders classified as dyskinesias. It is chronic and progressive. Parkinson's disease occurs when substantia nigra cells begin to malfunction and eventually die. This results in the loss of dopamine, a chemical messenger that transports signals to the brain portion that controls motor initiation and coordination. The initial symptoms are tremors, stiffness or rigidity of the limbs and trunk; bradykinesia or bradykinesia; and postural instability or impaired balance and coordination. Secondary symptoms include altered language, loss of facial expression, difficulty swallowing, watery mouth, pain, dementia or confusion, sleep disorders, depression, fear or anxiety, memory difficulties, urinary problems, fatigue and pain, and weakness. However, the symptoms vary widely, and the disease may progress rapidly or slowly.

As many as 1 million americans suffer from PD. Although about 15% of patients are diagnosed with disease before the age of 40, the incidence increases with age. The etiology is unknown, and although there is no cure available, there are many treatment options (e.g., drugs and surgery) to address the symptoms. The degree of success of each treatment varies from individual to individual, as does the length of time that the treatment option remains effective.

Levodopa is a dopamine precursor, which is considered a breakthrough in PD therapy. Unfortunately, patients experience debilitating side effects, including severe nausea and vomiting, and as the dose increases and the time of use increases, patients experience other side effects, including abnormal movement. Xinemet (Sinemet) (levodopa + carbidopa) showed a significant improvement, and the addition of carbidopa prevented the metabolism of levodopa in the intestine, liver and other tissues, allowing more levodopa to reach the brain. Therefore, only small doses of levodopa are needed and severe nausea and vomiting are greatly reduced.

Stalewo (carbidopa + levodopa + entacapone) is a combination tablet for patients experiencing signs and symptoms of "decline" from the end of dose. Tablets combine carbidopa/levodopa with entacapone. While carbidopa reduces the side effects of levodopa, entacapone prolongs (by up to 10%) the activity of levodopa in the brain.

Symmetrel (amantadine hydrochloride) activates dopamine release from storage sites and may prevent dopamine reuptake to the nerve endings. It also blocks glutamate receptor activity. Its dopaminergic action results in its effectiveness in reducing motor abnormalities caused by levodopa and is therefore known as an indirect-acting dopamine agonist, which is widely used in early monotherapy, adding more powerful restin when needed.

Anticholinergic drugs (trihexyphenidyl, benztropine mesylate, propidin, etc.) do not act directly on the dopaminergic system. They act to reduce the activity of another neurotransmitter, acetylcholine. There is a complex interaction between acetylcholinergic levels and dopamine levels in the brain. Many clinicians find that the addition of anticholinergic drugs is generally effective if the agonist or levodopa does not alleviate tremor. Side effects include blurred vision, dry mouth and urinary retention. These drugs are contraindicated for elderly patients because they can lead to vague and hallucinations of consciousness.

Other drugs include Selegiline (Selegiline) or deprenyl (eldepreyl), which have been shown to delay the need for phenine when used in the earliest stages of PD. Dopamine agonists are drugs that directly activate dopamine receptors and can be administered alone or in combination with a statin. These include bromocriptine (Parlodel), pergolide (Permax), pramipexole (Mirapex) and ropinirole (Requip). COMT inhibitors such as tolcapone (tasimar) and entacapone (Comtan) prolong the duration of remission by blocking the action of enzymes that degrade levodopa.

Surgery is an option in some patients where the drug is not sufficient. The patient should discuss the procedure in detail with his neurologist before making any decisions. Two older invasive procedures are pallidotomy and thalamotomy. Globulotomy can relieve the symptoms of stiffness and bradykinesia, and thalamotomy helps control tremors. Doctors rarely perform both procedures because both permanently damage parts of the brain and have serious side effects. The injury may render future surgeries, such as brain tissue transplantation, impossible.

Deep Brain Stimulation (DBS) is safer and more effective and therefore replaces these approaches. It is the preferred surgical option because it has the same (if not better) effect as pallidotomy and thalamotomy. DBS also leaves the possibility for other procedures that may be possible in the future. Like any surgery, it is risky and side-effects. The main advantage of DBS surgery is the reduction of motor fluctuations, i.e. the rise and fall caused by the reduction of the effectiveness of restin. Electrodes are typically placed on one side of the brain. DBS electrodes implanted on the left side of the brain control symptoms on the right side of the body and vice versa. In some cases, the patient needs to place electrodes on both sides of the brain.

B. Alzheimer's disease

Dementia is a brain disease that seriously affects a person's daily activities. Alzheimer's Disease (AD) is the most common form of dementia in elderly people. Scientists believe that up to 4 million americans suffer from AD. The disease usually develops after age 60, with the risk increasing with age. Although young people may also suffer from AD, it is much less common. Approximately 3% of men and women between the ages of 65 and 74 suffer from AD, and half of those aged 85 and older are likely to suffer from this disease. Despite being the subject of a large number of studies, the exact cause of AD is unknown and there is no cure.

AD affects part of the brain that controls thought, memory and language. It is named after the german doctor AloisAlzheimer. In 1906, Alzheimer's doctors noted changes in brain tissue in a woman who died from uncommon mental illness. He found abnormal plaques (now called amyloid plaques) and entangled fiber bundles (now called neurofibrillary tangles). These plaques and tangles in the brain are considered to be hallmarks of AD today.

Scientists also find other brain changes in AD patients. Loss of nerve cells in areas of the brain critical for memory and other mental capabilities. The level of chemicals that carry complex information back and forth between nerve cells in the brain is low. Thus, AD can disrupt normal thinking and memory by inhibiting (physiologically and chemically) information transfer between neural cells.

AD is progressive and characterized by memory loss, impaired speech, impaired visuospatial ability, poor judgment, apathy, but retained motor ability. As mentioned above, AD usually begins at age 65, but its onset may be as early as age 40, initially manifested as memory decline, and impairment of cognitive, personality, and performance over the years. Confusion and instability may also occur. The type, severity, sequence and progression of mental changes vary widely. Early symptoms of AD, including amnesia and attention loss, can be easily overlooked due to natural signs similar to aging. Similar symptoms may also be due to fatigue, sadness, depression, illness, loss of vision or hearing, alcohol abuse, or use of certain medications, or simply be unrecoverable when too many details are available.

There is no cure for AD and no means to slow disease progression. For people in the early or middle stages of the disease, drugs such as tacrine may alleviate some cognitive symptoms. Aricept (donepezil) and esmosergine (rivastigmine) are reversible acetylcholinesterase inhibitors suitable for the treatment of mild to severe Alzheimer's dementia. Also, some medications may help control behavioral symptoms such as insomnia, burning, wandering, anxiety, and depression. These treatments are intended to make the patient more comfortable.

The course of the disease varies from individual to individual. Some people only have an illness in the last 5 years of life, while others may have an illness for up to 20 years. The most common cause of death in AD patients is infection.

The molecular aspects of AD are complex and still not fully established. As mentioned above, AD is characterized by the formation of amyloid plaques and neurofibrillary tangles in the brain, particularly in the central hippocampal region of memory processing. Several molecules contribute to these structures: amyloid beta protein (a β), Presenilin (PS), cholesterol, apolipoprotein e (apoe), and Tau protein. Where a β appears to play an important role.

A β comprises about 40 amino acid residues. Forms of 42 and 43 residues are much more toxic than the 40 residue form. A β is produced from Amyloid Precursor Protein (APP) by sequential proteolytic cleavage. One of these enzymes lacks sequence specificity and therefore produces a β of various lengths (39-43). Toxic forms of a β cause aberrant events such as apoptosis, free radical formation, aggregation and inflammation.

Presenilins encode proteases responsible for cleavage of APP into a β. There are two forms: PS1 and PS 2. Mutation of PS1 results in production of a β₄₂And is a typical cause of early AD onset.

Cholesterol lowering agents are said to have the ability to prevent AD, although there is no established evidence to link elevated cholesterol with increased risk of AD. However, the discovery that a β contains a sphingolipid binding domain provides further evidence for this theory.

Similarly, ApoE, which is now thought to be involved in cholesterol redistribution, is now thought to promote the development of AD. Individuals showing the epsilon 4 allele with minimal cholesterol efflux are more likely to develop AD.

Tau protein, which is associated with microtubules in the normal brain, forms Paired Helical Filaments (PHFs) in the brain with AD, which are the main components of neurofibrillary tangles. Current evidence suggests that a β protein may lead to hyperphosphorylation of Tau protein, leading to its dissociation from microtubules and aggregation into PHF.

For AD, drugs have been used to limit the progression of the disease and to alleviate or ameliorate some of the associated symptoms. These drugs generally belong to the group of cholinesterase inhibitors, muscarinic agonists, antioxidants or anti-inflammatory agents. Galantamine (remininyl), tacrine (Cognex), selegiline, physostigmine, revistin, donepezil (Aricept), rivastigmine (exenert), mephospholate, melameilin, xanomeline, saleuzole, L-acetyl carnitine, idebenone, ENA-713 memric, quetiapine, neurostrol, and neuroidentify are some of the drugs proposed as AD therapeutics.

Diagnosis/prognosis/treatment decision of neurodegenerative diseases

The present invention provides for the first time a definitive diagnosis of diseases such as alzheimer's disease and parkinson's disease. This allows physicians confidence that they have correctly identified the physiological basis of the patient's symptoms, thus enabling early intervention and disease management.

Since treatment of AD is primarily a delay rather than a arrest in disease, and some PD treatments work in the same way, the ability to provide an early diagnosis of these diseases is critical to delaying the onset of more severe symptoms. This will also preserve the quality of life and prolong survival for these patients. Furthermore, it can limit the reduction of treatment costs, avoiding discomfort and possible injury to the patient, since patients can be provided with the correct medication to cope with their symptoms without "trial and error" sometimes caused by a wrong diagnosis.

These assays will all rely on the use of antibody-containing patient samples. The most commonly used biological sample is blood or serum, because of the high number of antibodies. However, other samples such as tears, saliva, sputum, spinal fluid, semen or urine may also be used.

In evaluating the antibody levels of a patient, the observed levels are compared to a standard. The criteria may depend on known values established for both disease and normal subjects, and thus may not require the user to provide anything other than a reaction control (i.e., the reagents and conditions required to indicate a positive reaction). Alternatively, an authentic control may be selected for testing, which comprises a similar sample obtained from an authentic human of known health or disease state. In addition, a series of samples obtained from the same subject over time can be assayed to look for a trend of elevated PD or AD antibody levels, which is indicative of disease progression.

Antibody-based diagnostic assays

As noted above, the application of this assay is (a) the identification of patients whose serum antibody profile indicates that they are at risk for developing or have suffered from ND; (b) identifying patients whose symptoms indicate that they may or may not suffer from ND (i.e., providing a definitive diagnosis of ND); (c) evaluating the effect of ND treatment; and (d) monitoring ND progression.

The detection method of the present invention is similar to antibody-based detection methods and thus includes formats such as enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), immunoradiometric assay, fluorescent immunoassay, chemiluminescent immunoassay, bioluminescent immunoassay, FACS, FRET, Western blot and the like. Various useful immunoassay procedures have been described in the scientific literature, such as Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al (1993) and Nakamura et al (1987). In general, the immunological binding method comprises obtaining a sample comprising an antibody, contacting the sample with a polypeptide of the invention under conditions effective to form a peptoid-antibody complex, and detecting the antibody bound to the peptoid.

The peptoid is typically attached to a solid support in the form of, for example, a column matrix, bead, filter, membrane, rod, plate, or well, and the sample is applied to the immobilized peptoid. The undesired components are eluted from the support, leaving the antibody complexed with the peptoid, and then detected in a variety of ways, such as the subsequent addition of an anti-Fc antibody linked to a detectable moiety.

Contacting the selected biological sample with the peptoid under effective conditions for a time sufficient to form a peptoid-antibody complex is a common practice of simply contacting the sample with the peptoid and incubating the mixture for a time sufficient for the antibody to bind to the peptoid. After this time, the sample-peptoid composition (e.g., ELISA plate, dot blot, or Western blot) is typically washed to remove any non-specifically bound antibody species, allowing detection of only those antibodies specifically bound to the immobilized antibody-peptoid complexes.

In general, detection of peptoid-antibody complex formation is well known in the art and can be achieved by performing a variety of methods. These methods are generally based on the detection of labels or markers, such as those of radioactive, fluorescent, biological and enzymatic labels. Patents directed to the use of these markers include U.S. Pat. nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Of course, the use of a second binding ligand, such as a second antibody and/or a biotin/avidin ligand binding configuration, as known in the art, may have additional advantages.

Numerous other forms are contemplated and are well known to those skilled in the art. The following discussion is intended to facilitate three specific assays of the invention.

A.ELISA

The simplest and straightforward meaning of immunoassays is binding assays. Certain immunoassays particularly useful in the present invention are the various types of enzyme-linked immunosorbent assays (ELISAs) and Radioimmunoassays (RIA) known in the art.

In an exemplary ELISA, a peptoid of the invention is immobilized on a selected surface, such as a well of a polystyrene microtiter plate, and a test composition suspected of containing the antibody is then added to the well. After binding and washing to remove non-specific binding complexes, bound antibodies can be detected. Detection may be by addition of another type of peptide linked to a detectable label. This type of assay is similar to a simple "sandwich ELISA" except that the labeled reagent binds to the Fab portion of the binding antibody. Detection can also be performed by adding a labeled antibody that binds to any binding antibody (e.g., recognizes the Fc region of the binding antibody). Optionally, the antibody is unlabeled, and a second antibody having binding affinity for the first antibody is subsequently added, the second antibody being linked to a detectable label.

In another exemplary ELISA, a sample suspected of containing the antibody is immobilized on the surface of a well and then contacted with a labeled peptoid of the invention. After binding and washing to remove non-specifically bound immune complexes, the bound labeled peptoid can be detected. Alternatively, the peptoid is unlabeled and can be detected by an artificial antibody (non-sample) that specifically binds to the selected peptoid, and the second antibody should be linked to a detectable label to enable detection.

Whatever format is used, the ELISA has some common features such as coating, incubation and binding, washing to remove non-specifically bound species, and detection of bound immune complexes. These are discussed below.

In coating the plate with a peptoid or antibody, the wells of the plate are typically incubated with a solution of the peptoid or antibody overnight or for a specified number of hours. In certain aspects, the plate can be blocked with a bacterial lysate, such as an E.coli lysate (see example 1). The wells of the plate are then washed to remove incompletely adsorbed species. Any remaining available surface in the wells is then coated with non-specific proteins that are antigenically neutral with respect to the test antisera. These include Bovine Serum Albumin (BSA), casein or milk powder solutions. The coating blocks non-specific adsorption sites on the immobilization surface, thereby reducing the background caused by non-specific binding of antisera to the surface. Alternatively, due to the simple and predictable chemical structure of peptoids, they can be bound to the support by specific chemical reactions.

In an ELISA, it may be more common to use a second or third detection modality rather than a direct method. Thus, after the peptoid or antibody is bound to the well, coated with a non-reactive substance to reduce background, and washed to remove unbound substances, the immobilized surface is contacted with the biological sample or peptoid to be detected under conditions sufficient for immune complex formation. Detection of the immune complex requires a labeled second binding peptoid or antibody, or a second binding peptoid or antibody that binds to a labeled third antibody (specific for the Fc region of the antibody or peptoid).

By "under conditions sufficient for immune complex (antigen/antibody) formation" is meant that the conditions preferably include dilution of the antigen and/or antibody with a solution such as BSA, Bovine Gamma Globulin (BGG), or Phosphate Buffered Saline (PBS)/Tween. These added reagents also help to reduce non-specific background.

"suitable" conditions also refer to incubation at a temperature or for a time sufficient to promote effective binding. The incubation step is typically about 1 to 2 to 4 hours or so, and the temperature is preferably about 25 ℃ to 27 ℃, or may be overnight at about 4 ℃.

After all incubation steps of the ELISA, the contact surfaces were washed to remove uncomplexed material. Preferred washing methods include washing with solutions such as PBS/Tween or borate buffer. The presence of even minute amounts of immune complexes can be detected after the formation of specific immune complexes between the test sample and the initially bound substances and subsequent washing.

The detection may use an enzyme which develops a color after incubation with a suitable chromogenic substrate. Thus, for example, it may be desirable to contact or incubate the first and second immune complexes with a urease, glucose oxidase, alkaline phosphatase or catalase conjugated antibody or peptoid for a period of time and under conditions conducive to further immune complex formation (e.g., incubation for 2 hours at room temperature in a solution containing PBS such as PBS-Tween).

After incubation with labeled antibodies or peptoids and subsequent washing to remove unbound material, the amount of label is determined, for example, by using a chromogenic material such as urea, bromocresol purple or 2, 2' -azino-bis- (3-ethyl-benzothiazoline-6-sulfonic Acid (ABTS) or H₂O₂(in the case of peroxidase as an enzyme label) incubation. Quantification may then be performed by measuring the degree of colour generation, for example by means of a visible spectrospectrophotometer.

B. Forster resonance energy transfer (Resonance Energy Transfer，FRET)

FRET is the phenomenon in which energy of an excited state in one molecule (called the donor) is transferred to another molecule through nonradiative coupling. The phenomenon is firstly caused byDescribed correctly and distinguished from other types of energy transfer, such as electron sharing (Dexter) or simple transfer (reabsorption of photons emitted by a donor by an acceptor). The Dexter mechanism requires physical contact of two molecules with very low probability of simple transfer. In contrast, when two molecules are presentWithin a radius (defined for any given fluorophore pair)There is a high probability of the mechanism.

The overall FRET potency depends onThe radius, and it is determined by several factors and directly related to the amount of coincidence of the absorption spectrum of the acceptor molecule and the emission spectrum of the donor molecule. The amount of FRET also depends on the orientation of the donor and acceptor molecules, although a large proportion will beThe biotransformation system has no rigid orientation. The effectiveness of FRET is also affected by the ability of the acceptor molecule to absorb light (indicated by its molar absorption coefficient) and the overall stability of the excited state of the donor molecule (indicated by the probability of absorption leading to fluorescence (quantum yield) and the lifetime of the excited state).

FRET between two different fluorophores can be measured by several methods: observing a change in fluorescence color, measuring the fluorescence lifetime of a donor, detecting a change in donor or acceptor upon photobleaching, or as we show in this new invention: by measuring the fluorescence polarization of the acceptor. Regardless of the method used, most of these assays share common instrumental characteristics.

The type of microscope used for measuring FRET may be appropriately selected according to the purpose. If frequent observation is required for monitoring the change over time, a common incident light fluorescence microscope is preferred. Confocal laser microscopy is preferred if resolution is improved (e.g., when monitoring specific intracellular locations). For microscope systems, inverted microscopes are preferred for measurements of most living cells because of their ability to maintain the physiological state of the cells and prevent contamination. When using an upright microscope, the lens may be submerged using water when using a high power lens.

The filter set may be appropriately selected according to the fluorescence wavelength of the fluorescent protein. For observing GFP, a filter with excitation light of about 470-490nm and fluorescence of about 500-520nm is preferably used. For viewing YFP, a filter with excitation light at about 490-510nm and fluorescence at about 520-550nm is preferred. For observing CFP, a filter with excitation light of about 425nm and fluorescence of about 460-500nm is preferably used. For the purposes of the present invention, there are no particular requirements on the microscope and the filter, except in the case where it is desired to minimize the polarizing elements in the light path. In both transmission and reflection microscopes, microscope manufacturers sell deformation-free optical elements for polarized light measurements, which optical elements also aid in these polarized fluorescence measurements.

In addition, when live cells are observed over time by using a fluorescence microscope, the cells should be photographed in a short time, and thus a high-sensitivity cold CCD camera is used. By using a cold CCD camera, thermal noise can be reduced by cooling the CCD, and a clear weak fluorescence image can be obtained by short-time exposure. Confocal microscopy can also be used for live cell imaging, as long as care is taken to minimize exposure time.

C.Luminex bead

Luminex’s xMAPWith the aid of existing technology platforms including flow cytometry, microspheres, lasers, digital signal processing and traditional chemistry. xMAPIs characterized by a flexible, open architecture design that can be configured to perform a variety of bioassays quickly, economically, and accurately.

Luminex colors the microspheres into 100 different groups. Each set of spheres may be coated with a peptoid of the invention, thereby enabling capture and detection of specific antibodies from the sample. In a Luminex mini-analyzer, the laser excites the internal dye specific to each microspheroidal particle and any reporter dye captured in the assay. A number of readings were made for each set of balls and the results were confirmed. In this way, xMAP technology enables up to 100 independent assays to be performed in a single sample in multiple passes, and is fast and accurate.

xMAP technology has been applied to many branches of life sciences, including protein expression profiling, molecular and immunodiagnostics, HLA detection, and biological protection/environment.

Tentagel beads

In certain aspects, tentagel beads or resins can be used in compositions and methods for detecting antibodies associated with a particular ND. One of the most common microsphere formulations is tentagel, a styrene-polyethylene glycol copolymer. These microspheres do not swell in non-polar solvents such as hexane, but swell by about 20-40% in volume when exposed to more polar or aqueous matrices.

Peptoids can be attached to or synthesized on solid supports such as tantagel beads. Tentagel beads have a polystyrene core and adhered to the core are a plurality of polyoxyethylene arms, each arm having a primary amine at the free end. Peptoids can be synthesized using peptoid synthesis chemistry by sequential conjugation of each residue added to the peptoid. The beads produced by this split synthesis method each contain multiple copies of a single peptoid sequence.

Since the polyoxyethylene arms of tentagel beads are water soluble, the configuration of peptoids is largely determined by thermodynamics and their primary sequence.

E. Immunoassay kit

In still other embodiments, the invention relates to a test kit for use in the above method. The peptoids of the present invention will be contained in the kit. The immunoassay kit will therefore comprise in a suitable container one or more peptoids that bind to antibodies of alzheimer's disease, parkinson's disease or both, optionally attached to a detection agent and/or a support.

In certain embodiments where the peptoid is pre-bound to a solid support, the support is provided and comprises a column matrix, beads, a dip-stick, or wells of a microtiter plate. The immunodetection agents of the kit may take any of a variety of forms, including those in which a detectable label is bound or linked to a given peptoid or a second antibody. Exemplary second antibodies are those that have binding affinity for the sample antibody.

The container typically comprises at least one tube, test tube, bottle, cup, syringe or other container in which the peptoid may be placed, or preferably, the peptoid is appropriately aliquoted. The kits of the invention will also typically contain means for hermetically packaging the peptoids, antibodies, and any other reagent containers for sale. Such containers may include injection molded or blow molded plastic containers in which the desired tube is retained.

Certain embodiments relate to kits comprising and methods of using peptoids described herein. Kits and/or methods for detecting, visualizing, and/or classifying at least one disease state are disclosed. The steps performed include obtaining a sample from a subject (e.g., a human) and performing a peptoid binding molecule in the sample using a reagent or array matrix provided in the form of a kit. Thus, at least one antibody indicative of a disease state is isolated or identified in the sample. Antibodies that bind to a peptoid described herein are associated with at least one risk of developing a disease or the presence of a particular disease state.

In addition, the present invention contemplates the use of a variety of kits. One such kit provides for the determination of the presence of disease-specific antibodies, including one or more antibodies associated with alzheimer's disease and/or parkinson's disease. At least one peptoid capable of specifically binding to a disease-specific antibody is incorporated into the kit. In certain aspects, reagents for determining binding of a peptoid to an antibody may also be included. The peptoids described herein can be immobilized on a solid support or matrix and comprise at least one detection agent to determine whether an antibody binds to the peptoid. The sample for any of the kits may be a fractionated or unfractionated body fluid or tissue sample. Non-limiting examples of such fluids are blood, blood products, urine, saliva, cerebrospinal fluid and lymph.

Also contemplated are kits for diagnosing neurodegenerative disease states, determining risk assessments, and identifying therapeutic pathways related thereto. The kit comprises at least one peptoid capable of specifically binding to an antibody indicative of a disease state. Reagents for determining binding between the peptoid and the antibody may also be included.

Disease-specific antibodies analyzed according to the methods of the invention are released into the circulation and may be present in the blood or any blood product (e.g., plasma, serum and dilutions or preparations thereof) and other body fluids (e.g., cerebrospinal fluid (CSF), saliva, urine, lymph, etc.). The measured antibody level may be determined using any suitable direct or indirect assay. The assay may be a competitive assay, a sandwich assay, and the label may be selected from well-known labels such as radioimmunoassays, fluorescent or chemiluminescent immunoassays, or immuno-PCR techniques.

Antibody compositions

Certain embodiments include methods and compositions for characterizing antibodies characteristic of a particular ND, or antigenic determinants recognized thereby. For purposes of this specification and the appended claims, the terms "epitope" and "antigenic determinant" are used interchangeably to refer to the antigenic site of B and/or T cell response or recognition. B cell epitopes can be formed by both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed from tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3 (or more often, at least 5 or 8-10) amino acids in a unique spatial configuration. Methods for determining spatial configuration of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitophemiping Protocols (1996). Antibodies recognizing the same epitope can be identified in a simple immunoassay, in which the ability of one antibody to prevent the binding of another antibody to the target antigen is demonstrated.

T cells recognize a continuous epitope of about 9 amino acids on CD8 cells or about 13-15 amino acids on CD4 cells. T cells recognizing the epitope can be identified in an in vitro assay measuring antigen-dependent proliferation in response to the epitope by activated T cells³H-thymidine incorporation (Burke et al, 1994), antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al, 1996) or cytokine secretion.

The terms "antibody" or "immunoglobulin" are used interchangeably herein and in the claims to refer to any of several classes of structurally related proteins that function as part of the immune response of an animal or recipient, including IgG, IgD, IgE, IgA, IgM, and related proteins.

Under normal physiological conditions, antibodies are present in plasma and other body fluids and in the membranes of certain cells, and are produced by lymphocytes of the type designated B and their functional equivalents. IgG class antibodies are formed from 4 polypeptide chains, which are linked together by disulfide bonds. The 4 chains of a complete IgG molecule are 2 identical heavy chains, called H chains, and two identical light chains, called L chains.

The antibody may be bound to a solid support matrix or conjugated to a detectable moiety, or both, as is well known in the art. A general discussion of conjugation of fluorescent or enzymatic moieties is found in Johnstone et al (1982). The binding of antibodies to solid support matrices is also well known in the art (Harlow et al, 1988; Borrebaeck.1992).

Once an antigen or antibody indicative of ND is identified, recombinant techniques can be used to produce variants of the identified antigen and/or antibody, including monoclonal antibodies. For example, Single Chain Antibodies (SCAs) are genetically engineered proteins designed to expand the therapeutic and diagnostic applications that can be achieved with monoclonal antibodies. SCAs have the binding specificity and affinity of monoclonal antibodies, which in their native form is about one fifth to one sixth the size of a monoclonal antibody, which typically results in their very short half-life. Compared to most monoclonal antibodies, SCAs have several advantages, including their ability to be directly fused to polypeptides that can be used for detection (e.g., luciferase or fluorescent proteins). In addition to these advantages, fully human SCAs can be isolated directly from a human SCA library without the need for expensive and time-consuming "humanization" processes.

Single chain recombinant antibodies (scFVs) consist of antibody VL and VH domains linked by a designed flexible peptide tether (teter) (Atwell et al, 1999). Advantages of scFv compared to intact IgG are smaller volume and structural simplicity and comparable antigen binding affinity, they can be more stable than similar 2-chain Fab fragments (Colcher et al, 1998; Adams and Schier, 1999). The variable regions of both the heavy and light chains (VH and VL) are approximately 110 amino acids in length. They may be linked by a 15 amino acid linker, or by a longer sequence, for example one that is sufficiently flexible to allow the two domains to assemble into a functional antigen-binding pocket. In some specific embodiments, the addition of multiple signal sequences targets or secretes the scFv to different organelles in the cell. The addition of a light chain constant region (Ck) dimerizes it via disulfide bonds, resulting in increased stability and mobility. Thus, for single chain Fv (scFv) SCAs, although the two domains of the Fv fragment are encoded by separate genes, it has been demonstrated that synthetic linkers can be made by recombinant methods to form them as a single protein chain scFv (Bird et al, 1988; Huston et al, 1988). In addition, they are frequently used because of their ease of isolation from phage display libraries and their ability to recognize conserved antigens (for review see Adams and Schier, 1999). Thus, in some aspects of the invention, the antibody can be an SCA isolated from a phage display library rather than generated from the more traditional antibody generation techniques described above.

"substantially pure" in the context of a protein refers to a protein that is isolated from other contaminating proteins, nucleic acids, and other biological materials derived from the original source organism. Purity or "isolation" can be determined by standard methods and is normally at least about 50%, more normally at least about 60%, generally at least about 70%, more generally at least about 80%, usually at least about 85%, more usually at least about 90%, preferably at least about 95%, more preferably at least about 98%, and in some most preferred embodiments, at least 99%.

Methods for producing or isolating polyclonal antibodies are known to those skilled in the art. Typically, polyclonal antibodies are prepared by taking a source comprising the antibody of interest and fractionating the source to enrich for antibodies having the activity of interest (e.g., peptoid binding). See, e.g., Harlow and Lane (1988) Antibodies: a Laboratory Manual, CSH press, NY.

Briefly, examples of isolating an antibody that binds a particular peptoid may include, but are not limited to, obtaining a sample comprising the antibody; ammonium sulfate precipitation of the antibody in the sample; antibodies are isolated by immunoaffinity purification using standard techniques and one or more peptoids as affinity agents. The affinity resin used may be activated CH-sepharose coupled with a peptoid having the structure described herein. The antibody pellet may be loaded onto a column and washed with PBS or another suitable buffer or wash solution. The precipitate can then be eluted and collected. The concentration of the obtained antibody can be determined by total protein colorimetric assay (Bio-Rad).

The present invention is not intended to be limited to the use of this particular protocol for generating and purifying Antibodies, as a variety of Protocols are available and known to those of skill in the art (see, e.g., the eds. Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press [1989 ]; Harlow and Lane eds., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press [1988 ]; Ausubel et al, Current Protocols in Molecular biology, Ch.11, John Wiley & Sons, Inc., New York [1994 ]). The only criterion for the antibody production method used in the present invention is that it produces a sufficiently pure antibody preparation.

Definition of chemical groups

When used in reference to a chemical group, "hydrogen" refers to-H; "hydroxy" means-OH; "oxo" ("oxo") means ═ O; "halo" independently means-F, -Cl, -Br, or-I; "amino" means-NH₂(ii) a "hydroxylamino" refers to-NHOH; "nitro" means-NO 2; imino means NH; "cyano" means-CN; "azido" refers to-N₃(ii) a "mercapto" means-SH; "thio" means S; "thioether" means-S-; "sulfonamido" means-NHS (O) 2-; "Sulfonyl" means-S (O)₂-; "sulfinyl" means-S (O) -; and "silyl" refers to-SiH 3.

For the structures provided herein, the following subscripts in parentheses define the groups further as follows: "(Cn)" defines the exact number of carbon atoms in the group (n). For example, "alkyl group_(C2-10)"refers to those alkyl groups having from 2 to 10 carbon atoms, such as 2, 3, 4, 5, 6, 7,8, 9, or 10, or any range derived therein, such as 3-10 carbon atoms.

The term "alkyl", when used without the "substituted" modifier, refers to a straight or branched, cyclic, or acyclic, non-aromatic monovalent group having a saturated carbon atom as the point of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. group-CH₃(Me)、-CH₂CH₃(Et)、-CH₂CH₂CH₃(n-propyl), -CH (CH)₃)₂(isopropyl), -CH (CH)₂)₂(cyclopropyl), -CH₂CH₂CH₂CH₃(n-butyl), -CH (CH)₃)CH₂CH₃(sec-butyl), -CH₂CH(CH₃)₂(isobutyl), -C (CH)₃)₃(tert-butyl), -CH₂C(CH₃)₃(neopentyl), cyclobutyl, cyclopentyl, cyclohexyl and cyclohexylmethyl are all non-limiting examples of alkyl groups. The term "substituted alkyl" refers to a straight or branched chain, cyclic, or acyclic structure of a non-aromatic monovalent group having a saturated carbon atom as the point of attachment, no carbon-carbon double or triple bonds, and at least one atom independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. The following groups are non-limiting examples of substituted alkyls: -CH₂OH、-CH₂Cl、-CH₂Br、-CH₂SH、-CF₃、-CH₂CN、-CH₂C(O)H、-CH₂C(O)OH、-CH₂C(O)OCH₃、-CH₂C(O)NH₂、-CH₂C(O)NHCH₃、-CH₂C(O)CH₃、-CH₂OCH₃、-CH₂OCH₂CF₃、-CH₂OC(O)CH₃、-CH₂NH₂、-CH₂NHCH₃、-CH₂N(CH₃)₂、-CH₂CH₂Cl、-CH₂CH₂OH、-CH₂CF₃、-CH₂CH₂OC(O)CH₃、-CH₂CH₂NHCO₂C(CH₃)₃and-CH₂Si(CH₃)₃。

The term "alkenyl", when used without the "substituted" modifier, refers to a monovalent group of straight or branched, cyclic, or acyclic structure having a nonaromatic carbon atom as the point of attachment, having at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples of alkenyl groups include: -CH ═ CH₂(vinyl), -CH ═ CHCH₃、-CH＝CHCH₂CH₃、-CH₂CH＝CH₂(allyl), -CH₂CH＝CHCH₃and-CH ═ CH-C₆H₅. The term "substituted alkenyl" refers to a monovalent group of a straight or branched, cyclic, or acyclic structure having a nonaromatic carbon atom as the point of attachment, having at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and at least one atom independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. The radicals-CH ═ CHF, -CH ═ CHCl and-CH ═ CHBr are non-limiting examples of substituted alkenyl groups.

The term "alkynyl", when used without the "substituted" modifier, refers to a monovalent group of straight or branched, cyclic, or acyclic structure having a nonaromatic carbon atom as the point of attachment, having at least one carbon-carbon triple bond, and having no atoms other than carbon and hydrogen. The group-C.ident.CH, -C.ident.CCH₃、-C≡CC₆H₅and-CH₂C≡CCH₃Are non-limiting examples of alkynyl groups. The term "substituted alkynyl" refers to a monovalent group of linear or branched, cyclic, or acyclic structure having a nonaromatic carbon atom as the point of attachment, having at least one carbon-carbon triple bond, and at least one atom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. The group-C ≡ CSi (CH)₃)₃Are non-limiting examples of substituted alkynyl groups.

The term "aryl," when used without the "substituted" modifier, refers to a monovalent group having an aromatic carbon atom as the point of attachment, said carbon atom forming part of one or more six-membered aromatic ring structures in which the ring atoms are all carbon, and wherein the monovalent group contains no atoms other than carbon and hydrogen. Non-limiting examples of aryl groups include: phenyl (Ph), methylphenyl, (dimethyl) phenyl, -C₆H₄CH₂CH₃(ethylphenyl), -C₆H₄CH₂CH₂CH₃(propylphenyl), -C₆H₄CH(CH₃)₂、-C₆H₄CH(CH₂)₂、-C₆H₃(CH₃)CH₂CH₃(methylethylphenyl), -C₆H₄CH＝CH₂(vinylphenyl), -C₆H₄CH＝CHCH₃、-C₆H₄C≡CH、-C₆H₄C≡CCH₃Naphthyl and a monovalent group derived from biphenyl. The term "substituted aryl" refers to a monovalent group having an aromatic carbon atom as the point of attachment, said carbon atom forming part of one or more six-membered aromatic ring structures in which the ring atoms are all carbon, and wherein said monovalent group further has at least one atom independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. Non-limiting examples of substituted aryl groups include the following: -C₆H₄F、-C₆H₄Cl、-C₆H₄Br、-C₆H₄I、-C₆H₄OH、-C₆H₄OCH₃、-C₆H₄OCH₂CH₃、-C₆H₄OC(O)CH₃、-C₆H₄NH₂、-C₆H₄NHCH₃、-C₆H₄N(CH₃)₂、-C₆H₄CH₂OH、-C₆H₄CH₂OC(O)CH₃、-C₆H₄CH₂NH₂、-C₆H₄CF₃、-C₆H₄CN、-C₆H₄CHO、-C₆H₄CHO、-C₆H₄C(O)CH₃、-C₆H₄C(O)C₆H₅、-C₆H₄CO₂H、-C₆H₄CO₂CH₃、-C₆H₄CONH₂、-C₆H₄CONHCH₃and-C₆H₄CON(CH₃)₂。

The term "heteroaryl," when used without the "substituted" modifier, refers to a monovalent group having an aromatic carbon or nitrogen atom as the point of attachment, the carbon or nitrogen atom forming part of an aromatic ring structure in which at least one ring atom is nitrogen, oxygen, or sulfur, and wherein the monovalent group contains no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. Non-limiting examples of aryl groups include acridinyl, furyl, imidazoimidazolyl, imidazopyrazolyl, imidazopyridyl, imidazopyrimidinyl, indolyl, indazolinyl, methylpyridyl, oxazolyl, phenylimidazolyl, pyridyl, pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolinyl, quinoxalyl, tetrahydroquinolyl, thienyl, triazinyl, pyrrolopyridyl, pyrrolopyrimidyl, pyrrolopyrazinyl, pyrrolotriazinyl, pyrroloimidazolyl, benzopyranyl (where the point of attachment is one of the aromatic atoms), and chromanyl (where the point of attachment is one of the aromatic atoms). The term "substituted heteroaryl" refers to a monovalent group having an aromatic carbon or nitrogen atom as the point of attachment, said carbon or nitrogen atom forming part of an aromatic ring structure in which at least one ring atom is nitrogen, oxygen, or sulfur, and wherein said monovalent group further has at least one atom independently selected from the group consisting of non-aromatic nitrogen, non-aromatic oxygen, non-aromatic sulfur, F, Cl, Br, I, Si, and P.

The term "acyl" when used without the "substituted" modifier refers to a monovalent group having a carbonyl carbon atom as the point of attachment, and also having a straight or branched chain, cyclic, or acyclic structure, with no additional non-carbon or non-hydrogen atoms other than the carbonyl oxygen atom. The group-CHO, -C (O) CH₃(acetyl, Ac), -C (O) CH₂CH₃、-C(O)CH₂CH₂CH₃、-C(O)CH(CH₃)₂、-C(O)CH(CH₂)₂、-C(O)C₆H₅、-C(O)C₆H₄CH₃、-C(O)C₆H₄CH₂CH₃、-COC₆H₃(CH₃)₂and-C (O) CH₂C₆H₅Are non-limiting examples of acyl groups. Thus, the term "acyl" includes, but is not limited to, the radicals sometimes referred to as "alkylcarbonyl" and "arylcarbonylAnd (4) clustering. The term "substituted acyl" refers to a monovalent group having a carbonyl carbon atom as the point of attachment, which also has a linear or branched, cyclic, or acyclic structure, which also has at least one atom other than the carbonyl oxygen atom independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. group-C (O) CH₂CF₃、-CO₂H (carboxyl), -CO₂CH₃(methyl carboxyl), -CO₂CH₂CH₃、-CO₂CH₂CH₂CH₃、-CO₂C₆H₅、-CO₂CH(CH₃)₂、-CO₂CH(CH₂)₂、-C(O)NH₂(carbamoyl), -C (O) NHCH₃、-C(O)NHCH₂CH₃、-CONHCH(CH₃)₂、-CONHCH(CH₂)₂、-CON(CH₃)₂、-CONHCH₂CF₃-CO-pyridyl, -CO-imidazoyl and-C (O) N₃Are non-limiting examples of substituted acyl groups. The term "substituted acyl" includes, but is not limited to, "heteroarylcarbonyl" groups.

The term "alkoxy" when used without the "substituted" modifier refers to the group-OR, wherein R is the term alkyl as defined above. Non-limiting examples of alkoxy groups include: -OCH₃、-OCH₂CH₃、-OCH₂CH₂CH₃、-OCH(CH₃)₂、-OCH(CH₂)₂-O-cyclopentyl and-O-cyclohexyl. The term "substituted alkoxy" refers to the group-OR, wherein R is alkyl substituted as that term is defined above. For example, -OCH₂CF₃Is a substituted alkoxy group.

Similarly, the terms "alkenyloxy," "alkynyloxy," "aryloxy," "aralkyloxy," "heteroaryloxy," "heteroarylalkoxy," and "acyloxy," when used without the "substituted" modifier, refer to a group defined as-OR, wherein R is the term alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, and acyl, respectively, as defined above. When any of the terms alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, and acyloxy are modified by "substituted," it refers to the group-OR, where R is substituted alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, and acyl, respectively.

The term "alkylamino" when used without a "substituted" modifier refers to the group-NHR, where R is the term alkyl as defined above. Non-limiting examples of alkylamino groups include: -NHCH₃、-NHCH₂CH₃、-NHCH₂CH₂CH₃、-NHCH(CH₃)₂、-NHCH(CH₂)₂、-NHCH₂CH₂CH₂CH₃、-NHCH(CH₃)CH₂CH₃、-NHCH₂CH(CH₃)₂、-NHC(CH₃)₃-NH-cyclopentyl and-NH-cyclohexyl. The term "substituted alkylamino" refers to the group-NHR, where R is a substituted alkyl as that term is defined above. For example-NHCH₂CF₃Is a substituted alkylamino group.

The term "acylamino" (acylamino), when used without the "substituted" modifier, refers to the group-NHR, wherein R is the term acyl as defined above. A non-limiting example of an acylamino group is-NHC (O) CH₃. When the term acylamino is used with the modifier "substituted", it refers to a group defined as-NHR, wherein R is acyl substituted as defined above under the term substituted. The group-NHC (O) OCH₃And NHC (O) NHCH₃Are non-limiting examples of substituted amido groups.

The term "saturated" when referring to an atom means that the atom is only attached to other atoms by single bonds.

VI. examples

The following examples are included to demonstrate some preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1: validation of peptoids as selectable markers for Alzheimer's and Parkinson's disease

The microarray used by the present inventors comprised 2 copies of 4680 octameric peptide arrays along with multiple markers and control spots. Sera (blood) collected from 6 patients with alzheimer's disease and 6 normal controls were hybridized to the microarray. Serum samples were greatly diluted to provide a final total concentration of serum protein of 20. mu.g/ml, and the solution was hybridized to a peptoid microarray. After incubation and washing, the IgG binding pattern was revealed by subsequent incubation with Alexa-647 labeled secondary antibody. As a control, the array was contacted with the secondary antibody alone and any components that bound significant amounts of labeled secondary antibody were ignored in subsequent analyses.

GenePix analysis of all parts of the array revealed that reproducibly, several peptoids (figure 1A) were much brighter when exposed to AD serum compared to normal serum (intensity > 30,000 versus < 10,000 at this particular protein concentration) (figure 2A). Three objective peptoids show such characteristics. To detect the specificity of the peptoids, sera collected from Parkinson's disease patients were hybridized to these peptoids. However, only background levels of IgG antibodies were captured by peptoids from these serum samples, demonstrating that these peptoids are reagents that specifically capture antibodies produced by alzheimer's disease (fig. 2A).

After completing the training set, the inventors subsequently tested the ability of these three peptoids to differentiate AD by analyzing serum samples collected from patients (not included in the training set) (fig. 4-13). Peptoids were also validated in microarray and Luminex bead assays (fig. 7). All 3 peptoids identified in the experiment performed perfectly in blind experiments. The three peptoids were sequenced by mass spectrometry, re-synthesized and purified by HPLC.

In summary, the inventors have demonstrated that synthetic molecules capable of specifically capturing specific IgG of alzheimer's disease can be isolated by combinatorial library screening of peptoids.

TABLE 5 summary microarray analysis

Scoring	Prediction	Clinical diagnosis
			Is just	16	All right
Negative pole	16	All right
			Intermediate (II)	4	AD(2)NC(2)

All samples tested 36

TABLE 6 evaluation of assays using the Luminex platform

Scoring	Prediction	Clinical diagnosis
			Is just	47	All right
Negative pole	54	All right
			Intermediate (II)	9	AD(3)NC(2)PD(4)

All samples tested were 110 ═ d

Additional studies were performed by assaying binding properties of sera previously removed with APD1 peptoid on a peptoid array comprising ADP1-3 (fig. 3). Antibody removal experiments demonstrated that APD1 and APD3 bound similar antibody populations. Whereas ADP2 appears to bind to another antibody population.

Library synthesis and printing protocol: an 8-mer peptoid library with 1 constant residue (C-terminal cysteine) was synthesized on 500 μm polystyrene beads using a conventional split-and-pool method. Beads were placed in 96-well plates, one bead per well. The peptoid was then cleaved from the beads using a cleavage mixture of 95% trifluoroacetic acid, 2.5% water, and 2.5% triisopropylsilane. 50 (50. mu.l) of the mixture was added to each well and the beads were left in the mixture for 2 hours. The mixture was evaporated. After the mixture was evaporated, 40. mu.l of 50% acetonitrile and 50% water was added to each well.

The liquid contents of the 96-well plate (40. mu.l per well) were then transferred to a 384-well plate using a Tecan Genesis workstation 200. 34 (34. mu.l) of each plate were transferred to a new 384 well plate. Plates containing 6 μ l in each well were labeled as "residual" plates and left to evaporate the acetonitrile/water mixture. The plates were then blocked with adhesive film and stored in a-80 degree refrigerator ready for sequencing. The plate containing 34. mu.l of liquid was labeled "Master" and was also left to evaporate the acetonitrile/water mixture.

After evaporation of the liquid from the Master plate, 20 μ l DMSO (dimethyl sulfoxide) was added to each well using the Tecan Genesis workstation. Remove 5 (5. mu.l) from each well and place in another 384 well plate. To the plate each well was added 5. mu.l DMSO and was called a "replica" plate. The Master plate was then closed with an adhesive film and stored in a-80 degree refrigerator. The replica plates were stored in a 4 degree freezer ready for printing of microarrays.

The contents of the replica plates were then printed onto maleimide coated glass slides using a NanoPrint with MP946 Micro Spotting Pin. The pins were cleaned with a 10% ethanol and water mixture before printing and during each spotting cycle. There are multiple cleaning/sonic/drying cycles between each sampling and printing cycle. The complex array was printed with all 48 pins and 410 μm sized dots using a 10 by 10 dot matrix. Subarrays were printed with a single MP946 in Whatman Fast Frame in the form of 410 μm sized dots with 2 by 8 arrays on the slide and using a 10 by 10 lattice. The slide was left for 12 hours at 50% humidity before printing. After printing, the humidifier was turned off and the slides were placed for 12 hours and then scanned with a GenePix Autoloader4200 AL scanner. The slide was scanned with 500PMT to confirm the position and morphology of the spots.

The glass slide manufacturing scheme comprises the following steps: a diamond pen was used to mark one corner of each slide. The slides were then placed on glass slide racks, which were placed in a glass staining bath.

A glass beaker (2L) was placed in an ice bin and the surroundings were packed with ice. 70/30 concentrated H was prepared in the beaker₂SO₄And H₂O₂The solution (Piranha) was cooled to room temperature. The Piranha solution was poured into a glass staining bath to cover the slides with liquid. It was covered and left to stand at room temperature for 12 hours. They were then removed and thoroughly rinsed with deionized water. The slides were dried by centrifugation.

The bath was heated to 80 ℃. The glass slides were immersed in 3-glycidoxypropyltrimethoxysilane and placed in a water bath for 5-6 hours with gentle shaking. The slides were cooled and then immersed 2 times with DMF. The slides were again spin dried and stored under argon for use in the next step.

The temperature of the water bath was maintained at 80 ℃. Submerging the glass slides in 2L of poly (ethylene glycol) and 8mLH₂SO₄And placed in a water bath. The water bath was stirred at moderate to low speed for 6 hours. The slides were cooled and then washed with DI water while stirring for about 1 hour. The slides were again spin dried and stored under argon for use in the next step.

PMPI was measured and dissolved in anhydrous DMSO to make a 50mM solution. The slide was then placed on the PMPI reaction vessel with the scored side facing upward. PMPI was injected under the slide to completely fill the container and some excess flowed into the reservoir. All air bubbles on the downward surface of the slide are removed. The reaction vessel was stirred on a shaker under argon overnight. Slides were removed from the reaction vessel and washed in DMSO for 1 hour. This step is repeated. The column was washed with DMF for another 1 hour. The slides were again spin dried and stored under argon for use in the next step.

Hybridization protocol: microarray slides were covered with hybridization cassettes and equilibrated with 1XTBST (50mM Tris, pH 8.0, 150mM NaCl, 0.1% Tween20) for 15 minutes. 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 about 1ml serum (20. mu.g/ml) at 4 ℃ for 16 hours and gently shaken. In an alternative method, slides were blocked with 1ml of E.coli lysate (1.5mg/ml) for 1 hour. Coli lysates were removed and incubated with 1ml of serum (15. mu.g/ml) in E.coli lysates for 18 h at 4 ℃ with gentle shaking. The microarray was then washed 3 times with 1 XTSST and hybridized with Alexa-647 labeled anti-IgG antibody (5. mu.g/ml) for 2 hours on an orbital shaker at 4 ℃. Remove microarray slide from cassette and wash with 1X TBST (3X15 min); then washed with 0.1X TBST (1X 10). The slides were dried by centrifugation (1500rpm, 5 minutes) and scanned on a microarray scanner (Gene Pix Autoloader4200) using a 635-nm laser at 100% power and a photomultiplier tube at 600 or 650 for magnification. All scan images were analyzed using Gene Pix Pro6.0 software and Genespring software.

Example 2: sarcosine scanning of ADP1-3

The effect of the sarcosine scan was to sequentially replace each side chain with a methyl group instead of hydrogen, leaving the tertiary amide bond, but removing the side chain moiety. Peptoids ADP1-3 each contained 8 positions that were altered in the initial library synthesis (fig. 14). To determine which of the side chains at these 8 positions would be important for binding AD specific antibodies, 24 derivatives were synthesized, in which each side chain was in turn substituted with a methyl group. Each compound may be formed from a solid phase "sub-monomeric" chemical structure in which bromine is replaced with methylamine at the desired substitution position. To test the antibody binding properties of each derivative containing a methyl side chain, all 24 compounds as well as ADP1-3 peptoids were printed on a microarray. Post mortem confirmed AD patient sera were hybridized to these arrays. After washing, a fluorescently labeled secondary antibody is applied to the array. After the washing again, the fluorescence intensity of each position was measured with a fluorescence scanner. The intensity is shown in fig. 14. When a significant decrease in fluorescence is observed at a given point associated with the parent compound (indicating less antibody captured), this is interpreted as indicating that the side chain present at that position of the parent compound is important for binding to AD-specific antibodies.

Example 3 materials and methods

Examples of materials methods used for these studies include the following.

And (4) synthesizing and purifying peptoids. Peptoids can be synthesized on Rink amide resin according to the submonomer method using an ABI 433A peptide synthesizer or using a parallel synthesis machine. (see, e.g., Zuckermann, R.N., Kerr, J.M., Kent, S.B.H.,&moos, W.H, (1992) j.am. chem. soc., 114, 10646-_N2Substitution to form side chains. After synthesis, the peptoid can be cleaved and deprotected in trifluoroacetic acid (TFA), triisopropylsilane, water (volume ratio 95: 2.5). Compounds can be purified by RP-HPLC on a C18 column with a linear acetonitrile/water gradient. The molecular weight of the purified product can be determined by mass spectrometry.

The growing peptoid polymer monomer can be assembled in two steps using two already obtained subunit. Rink amide resin was bromoacetylated with diisopropylcarbodiimide activated bromoacetic acid. The bromoacetylated resin is then subjected to S of primary amine to bromine_N2Instead, this introduces the desired side chain. Hundreds of useful amine subunits and corresponding side chains are commercially available or synthesized. The synthesis of peptoids using a sub-monomer procedure provides for easy access to sequences with greater diversity in chemical structure than can be obtained directly via the monomer approach, and simply by sequence order, length, and/or N-pendant side chain structure is sufficient to provide the desired activity.

More specifically, Rink amide resin (4- (2 ', 4' -dimethoxyphenyl- (9-fluorenylmethyloxycarbonyl) -aminomethyl) -phenoxy resin, 0.25 mmol; Novabiochem) in CH may be initially prepared₂Cl₂Medium swelling for 30 min. After resin swelling, the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group was removed by treatment with 20% N-hexane in 1-methyl-2-pyrrolidone (NMP). The resin bound deprotected amine was then acetylated by reaction with 4.2ml of a 1.2M solution of bromoacetic acid (50mmol) in N, N-methylformamide (DMF) and 1.0ml (11mmol) of solvent-free N, N' -Diisopropylcarbodiimide (DIC) for 60 minutes at room temperature with constant stirring. Subsequently, the resin was washed with DMF (3 × 10 ml). NMP or CH with 6ml 1M Primary amine (sub-monomer)₂Cl₂The solution (6mmol) reacted with the resin bound bromoacetyl moieties to replace the bromine. Protected subunits are synthesized to yield N- (4-aminobutyl) glycine residues (Nlys). Then a further dip with NMP (3X10ml) followed by a dip with DMF (3 times 10 ml). The product of these two reactions produces peptoid "residues" whose structure depends on the subunit amine employed. The peptoid is extended by this submonomer method until the desired chain length is reached.

After synthesis, the peptoid oligomer can be cleaved from the resin simultaneously by treatment with 2, 2, 2-trifluoroacetic acid (TFA)/triisopropylsilane/H₂O (volume ratio 95: 2.5) treatment removed the tert-butyloxycarbonyl (Boc) protecting group from the Nlys residue. After cleavage, the peptoid can be purified to > 97% homogeneity by preparative scale reverse phase HPLC. The specific gradient employed for HPLC depends on the peptoid structure and hydrophobicity.

These syntheses and characterizations are also described in U.S. patent No.6,887,845, which is incorporated herein by reference. As described therein and as will be understood by those skilled in the art in light of the present disclosure, the present N-substituted glycine residues and resulting peptoid compounds are limited only by the corresponding amine reagents, either synthetic or commercially available.

Printing a microarray. An 8-mer peptoid library with 1 constant residue (C-terminal cysteine) was synthesized on 500 μm polystyrene beads using a conventional split-and-pool method. 7 different amines were employed using conventional split-pool and microwave-assisted protocols. Beads were placed in 96-well plates, one bead per well. The peptoid was then cleaved from the beads with a cleavage mixture of 95% TFA, 2.5% water, and 2.5% triisopropylsilane. The liquid contents of 4 96-well plates were then transferred to 384-well plates using a Tecan Genesis workstation. After evaporation of the liquid from the Master plate, DMSO (dimethyl sulfoxide) was added to each well using the Tecan Genesis workstation. The plates were stored in a 4 degree refrigerator ready for printing of microarrays. The contents of the 384 well plates were then printed onto maleimide coated glass slides using NanoPrintLM 360. The complex array was printed with all 48 pins and 410 μm sized dots using a 10 by 10 dot matrix. After printing, the slides were placed for 12 hours and then scanned with a GenePix Autoloader4200 AL scanner. The slide was scanned with 500PMT to confirm the position and morphology of the spots.

Hybridization protocol. Microarray slides were covered with hybridization cassettes and equilibrated with 1XTBST (50mM Tris, pH 8.0, 150mM NaCl, 0.1% Tween20) for 15 minutes. Then blocked with 1ml of blocking buffer at 4 ℃ for 1 hour. The blocking buffer was removed and the slides were incubated with about 1ml of serum at 4 ℃ for 16 hours and shaken gently. The microarray was then washed 3 times with 1XTBST and hybridized with Alexa-647 labeled goat anti-mouse antibody for 2 hours on an orbital shaker at 4 ℃. The microarray slide was removed from the cassette and washed with 1X TBST. Slides were then spin dried and scanned on a microarray scanner using a 100% power 635-nm laser and 600 photomultiplier tube magnification. All scan images were analyzed using Gene Pix Pro6.0 software and Genespring software.

Peptoid variants were designed. The effect of the sarcosine scan is to sequentially replace each side chain of the peptoid with a methyl group instead of hydrogen, leaving the tertiary amide bond, but removing the side chain moiety. The peptoid of interest can be altered at one or more positions in the peptoid to determine which side chain position affects peptoid binding. Each peptoid may be formed from a solid phase "sub-monomeric" chemical structure (as described above) in which bromine is replaced with methylamine at the desired substitution position. To test the binding properties of these peptoid variants, each peptoid variant can be printed on a microarray or some other scanning platform along with appropriate controls. Samples containing peptoid targets were hybridized to these arrays. After washing, a second antibody or other detection method is applied to the array and the fluorescence intensity at each position is measured. When a significant decrease in fluorescence is observed at a given point associated with the parent compound (indicating a weaker binding affinity), this is interpreted as indicating that the side chain present at that position of the parent compound affects binding.

Variant peptoids incorporating one or more of the amine or R groups described herein are then incorporated, and variants are identified using similar synthesis and scanning methods.

***************

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

VII reference

The following references are specifically incorporated by reference herein to the extent that they provide exemplary method or other details supplementary to those set forth herein.

U.S. Pat. No. 3,817,837

U.S. Pat. No. 3,850,752

U.S. Pat. No. 3,939,350

U.S. Pat. No. 3,996,345

U.S. Pat. No. 4,275,149

U.S. Pat. No. 4,277,437

U.S. Pat. No. 4,366,241

De Jager et al.，Semin.Nucl.Med.，23(2)：165-179，1993.

Doolittle and Ben-Zeev，Methods Mol Biol，109：215-237，1999.

Gulbis and Galand，Hum.Pathol.，24(12)：1271-1285，1993.

Nakamura et al.，In：Handbook of Experimental Immunology(4th Ed.)，Weir et al.(Eds)，1：27，Blackwell Scientific Publ.，Oxford，1987.

Claims (23)

1. Use of a support having a peptoid attached thereto for the preparation of a composition for diagnosing alzheimer's disease by detecting antibodies in a sample comprising antibodies, said peptoid having:

(i) formula (1A):

wherein R is¹And R⁴Independently selected from hydrogen; an alkyl group; alkene(s)Propyl; n-butylamine; an aryl group; a heteroaryl group; a cycloalkyl group; an alkenyl group; a cycloalkenyl group; a methoxyethyl group; (R) -methylbenzyl; is covered with NH₂OH or SH substituted C_1-6Alkyl radicals or unsubstituted or substituted by NH₂OH or SH substituted C_2-6An alkynyl group; lysyl groups; a carboxyl group; and a hydroxyl group; and

R²、R³and R⁵Independently selected from C_0-6An alkylaryl group; c_0-6An alkyl heteroaryl group; c substituted by_1-6Alkyl groups: OH, SH, halogen, OR¹⁵、COOR¹⁵、NR¹⁵(wherein R is¹⁵Is H, C_1-6Alkyl or C_1-6Alkynyl) or R¹⁶(wherein R is¹⁶Is 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_2-6An alkynyl group;

(ii) formula (1B):

wherein R is₆Selected from hydrogen; an alkyl group; an allyl group; n-butylamine; an aryl group; a heteroaryl group; a cycloalkyl group; an alkenyl group; a cycloalkenyl group; a methoxyethyl group; (R) -methylbenzyl; is covered with NH₂OH or SH substituted C_1-6Alkyl radicals or unsubstituted or substituted by NH₂OH or SH substituted C_2-6An alkynyl group; lysyl groups; a carboxyl group; and a hydroxyl group; and

R⁷and R⁸Independently selected from C_0-6An alkylaryl group; c_0-6An alkyl heteroaryl group; c substituted by_1-6Alkyl groups: OH, SH, halogen, OR¹⁵、COOR¹⁵、NR¹⁵(wherein R is¹⁵Is H, C_1-6Alkyl or C_1-6Alkynyl) or R¹⁶(wherein R is¹⁶Is 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_2-6An alkynyl group;

(iii) formula (1C):

wherein R is⁹、R¹¹And R¹³Independently selected from hydrogen; an alkyl group; an allyl group; n-butylamine; an aryl group; a heteroaryl group; a cycloalkyl group; an alkenyl group; a cycloalkenyl group; a methoxyethyl group; (R) -methylbenzyl; is covered with NH₂OH or SH substituted C_1-6Alkyl radicals or unsubstituted or substituted by NH₂OH or SH substituted C_2-6An alkynyl group; lysyl groups; a carboxyl group; and a hydroxyl group; and

R¹⁰、R¹²and R¹⁴Independently selected from C_0-6An alkylaryl group; c_0-6An alkyl heteroaryl group; c substituted by_1-6Alkyl groups: OH, SH, halogen, OR¹⁵、COOR¹⁵、NR¹⁵(wherein R is¹⁵Is H, C_1-6Alkyl or C_1-6Alkynyl) or R¹⁶(wherein R is¹⁶Is 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_2-6An alkynyl group;

(iv) formula (1D):

wherein R is¹⁷-R²⁰And R²³Independently selected from hydrogen; an alkyl group; an allyl group; n-butylamine; an aryl group; a heteroaryl group; a cycloalkyl group; an alkenyl group; a cycloalkenyl group; a methoxyethyl group; (R) -methylbenzyl; is covered with NH₂OH or SH substituted C_1-6Alkyl radicals or unsubstituted or substituted by NH₂OH or SH substituted C_2-6An alkynyl group; lysyl groups; a carboxyl group; and a hydroxyl group; and

R²¹、R²²and R²⁴Independently selected from C_0-6An alkylaryl group; c_0-6An alkyl heteroaryl group; c substituted by_1-6Alkyl groups: OH, SH, halogen, OR¹⁵、COOR¹⁵、NR¹⁵(wherein R is¹⁵Is H or C_1-6Alkyl or C_1-6Alkynyl) or R¹⁶(wherein R is¹⁶Is 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 radical

(v) Formula (1D):

wherein R is¹⁷-R²⁰、R²³And R²⁴Independently selected from hydrogen; an alkyl group; an allyl group; n-butylamine; an aryl group; a heteroaryl group; a cycloalkyl group; an alkenyl group; a cycloalkenyl group; a methoxyethyl group; (R) -methylbenzyl; is covered with NH₂OH or SH substituted C_1-6Alkyl radicals or unsubstituted or substituted by NH₂OH or SH substituted C_2-6An alkynyl group; lysyl groups; a carboxyl group; and a hydroxyl group; and

R²¹and R²²Independently selected from C_0-6An alkylaryl group; c_0-6An alkyl heteroaryl group; c substituted by_1-6Alkyl groups: OH, SH, halogen, OR¹⁵、COOR¹⁵、NR¹⁵(wherein R is¹⁵Is H, C_1-6Alkyl or C_1-6Alkynyl) or R¹⁶(wherein R is¹⁶Is 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_2-6An alkynyl group; or

(vi) Formula (1D):

wherein R is¹⁷、R¹⁹、R²⁰And R²³Independently selected from hydrogen; an alkyl group; an allyl group; n-butylamine; an aryl group; a heteroaryl group; a cycloalkyl group; an alkenyl group; a cycloalkenyl group; a methoxyethyl group; (R) -methylbenzyl; is covered with NH₂OH or SH substituted C_1-6Alkyl radicals or unsubstituted or substituted by NH₂OH or SH substituted C_2-6An alkynyl group; lysyl groups; a carboxyl group; and a hydroxyl group; and

R¹⁸、R²¹and R²⁴Independently selected from C_0-6An alkylaryl group; c_0-6An alkyl heteroaryl group; c substituted by_1-6Alkyl groups: OH, SH, halogen, OR¹⁵、COOR¹⁵、NR¹⁵(wherein R is¹⁵Is H, C_1-6Alkyl or C_1-6Alkynyl) or R¹⁶(wherein R is¹⁶Is 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_2-6An alkynyl group; and

and

z is a coupling group; l is an optional linker moiety. M is a substrate or support; and m, n, o and/or p are each an integer of 0 to 6.

2. The use of claim 1, wherein R¹And R⁴Independently selected from methyl; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAn azole group; isoquinoline; a phenyl group; a pyridyl group.

3. The use of claim 1, wherein R₆Is selected from methyl; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAn azole group; isoquinoline; a phenyl group; a pyridyl group.

4. The use of claim 1, wherein R⁹、R¹¹And R¹³Independently selected from methyl; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAn azole group; isoquinoline; a phenyl group; a pyridyl group.

5. The use of claim 1, wherein R¹⁷-R²⁰And R²³Independently selected from methyl; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAn azole group; isoquinoline; a phenyl group; a pyridyl group.

6. The use of claim 1, wherein R¹⁷-R²⁰、R²³And R²⁴Independently selected from methyl; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAn azole group; isoquinoline; a phenyl group; a pyridyl group.

7. The use of claim 1, wherein R¹⁷、R¹⁹、R²⁰And R²³Independently selected from methyl; an ethyl group; n-propyl; isopropyl group; n-butyl; an isobutyl group; sec-butyl; a tertiary butyl group; a pentyl group; hexyl; isoamyl; a furyl group; an indolyl group; a thienyl group; a thiazolyl group; an imidazolyl group; different from each otherAn azole group;an azole group; piperonyl radical; a pyrazolyl group; a pyrrolyl group; a pyrazinyl group; a pyridyl group; a pyrimidinyl group; a pyrimidine base; a purine group; a cinnolinyl group; a benzofuranyl group; benzothienyl; a benzotriazole group; benzo (b) isAn azole group; quinoline; different from each otherAn azole group; isoquinoline; a phenyl group; a pyridyl group.

8. The use of claim 1, wherein the peptoid is selected from the group consisting of:

wherein Z is a functional group capable of coupling to a linker, substrate or label.

9. The use of claim 1, wherein the sample is contacted with more than one species of peptide of any of formulae (i) to (vi) of claim 1.

10. The use of claim 9, wherein the sample is contacted with the peptoids AD1, AD2, and AD 3.

11. The use of claim 1, wherein the support is a bead, plate, dip stick, filter, membrane, needle or well, or wherein the sample is blood, serum, saliva or CSF.

12. The use of claim 1, wherein detecting comprises using RIA, FIA, ELISA, Western blot, flow cytometry, FRET or surface plasmon resonance.

13. The use of claim 1, wherein

The sample comprising the antibodies is a serum,

exposing said serum to a plurality of peptoids of any one of formulae (i) to (vi) as defined in claim 1, and

the diagnosis is performed in a patient suspected of having AD,

the antibodies include AD-associated antibody 1 bound to peptoid a and optionally AD-associated antibody 2 bound to peptoid B.

14.A kit for the detection of an antibody indicative of a neurodegenerative disease, the kit comprising one or more of the peptoids of any of formulae (1A) to (1D), wherein all variables are the same as defined in claim 1, optionally coupled to a substrate.

15. The kit of claim 14, wherein the one or more peptoids are selected from AD1, AD2, AD3 of any of the definitions in claim 8, and wherein Z is also the same as defined in claim 8.

16. A composition comprising antibodies isolated from serum of a subject suffering from a neurodegenerative disease, wherein the antibodies specifically bind to AD1, AD2, AD3 of any defined in claim 8, and wherein Z is also the same as defined in claim 8.

17. A kit for the detection of AD-associated antigens, said kit comprising an antibody as defined in claim 16.

18. An antibody isolated by the steps comprising:

(a) contacting a sample comprising an antibody indicative of a neurodegenerative disease with an immunoaffinity matrix comprising the peptoid of claim 1 that binds to the neurodegenerative disease indicator antibody;

(b) eluting the bound antibody from the affinity matrix.

19. A composition comprising a peptoid having any of formulae (i) to (vi) as defined in claim 1.

20. A peptoid composition comprising AD1, AD2, AD3 of any of the definitions in claim 8, and wherein Z is also the same as defined in claim 8.

21. The composition of claim 19 or 20, further comprising an antibody indicative of a neurodegenerative disease.

22. An isolated peptoid having any of formulae (i) to (vi) as defined in claim 1, wherein the peptoid is any of AD1, AD2, and AD3 as defined in claim 8, and wherein Z is also the same as defined in claim 8.

23. The use of claim 1, wherein detecting antibodies in the sample comprising antibodies comprises the steps of:

(a) incubating the peptoid array with a blocking buffer comprising bacterial lysate at a concentration of 0.5 to 2 mg/ml;

(b) removing the blocking buffer;

(c) incubating the peptoid array with a sample solution comprising a bacterial lysate; and

(d) detecting antibodies bound to the peptoid array.