WO2008122241A1 - Rapid protein analyses and the device thereof - Google Patents

Abstract

The present invention discloses methods of making rapid, definitive identification of different protein profiles by array analyses using a lateral flow-through process and the devices thereof. Dot-blot, slot-blot or reverse dot-blot arrays for high throughput screening are provided. The present invention also provides methods and devices comprising sets of biomarkers for cancers diagnosis.

Description

RAPID PROTEIN ANALYSES AND THE DEVICE THEREOF

[0001] This application claims the benefit of priority of U.S. Serial No. 60/910,208, filed April 4, 2007, the content of which is hereby incorporated in its entirety by reference into this application.

[0002] Throughout this application, various publications are referenced. Disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

FIELD OF INVENTION

[0003] The present invention relates to methods and devices for making rapid, definitive identification of different protein profiles by arrays analyses using a flow-through process.

BACKGROUND OF THE INVENTION

[0004] Accurate genotyping by DNA analysis such as HLA typing is essential for matching donor and recipient in organ or marrow transplantation (Thomas, 1983) to prevent the development of acute graft-versus-host disease (GVHD) . Recent studies have demonstrated that DNA genotyping can provide more accurate and definitive result (Kaneshige et . al . , 1993; Chow and Tonai, 2003; Mach et al . , 2004). Results of HLA-A, B, DQ, DR and DP genotyping provided data for accurate matching which is necessary in selecting potential organ donors (Tarn, 1998; 2004; 2005; 2006; 2007) . [0005] However, in the cases of extremely high heterogeneities of HLA complex, the number of polymorphic SNP (single nucleotide polymorphism) needed to achieve comprehensive differentiation is very high by DNA method because many SNPs in which mutation (s) do not result in polymorphic protein. Hence the number of HLA proteins expressed will be much less than the number of SNPs. If a full set of antibodies can be made, a set of Antibody Array and/or HLA antigen array is most appropriate for HLA protein typing to generate full HLA profile for an individual. Nowadays, these sets of antibodies or proteins can be made available easily by reverse genetic engineering and monoclonal antibody expression and screening. Depending on the number of antibodies/antigens used, the typing classification can be set from low (degenerate) to complete differentiation. In fact standard serological typing (Kaneshige et . al . , 1993; Chow and Tonai, 2003; Mach et al . , 2004) of HLA has been done for many years. The present invention provides improved methods and devices for rapid and cost efficient process to conduct protein analysis/typing using a flow-through protein array format. In addition to HLA typing, dot-blot, reverse dot-blot or slot blot can be used for other protein systems for rapid analysis described in this invention.

SUMMARY OF THE INVENTION

[0006] In one embodiment, the present invention provides a lateral flow-through device for protein analysis, comprising: (a) one or more reaction chambers, each of which comprises a membrane for immobilizing capture molecules capable of capturing target analysts; and (b) liquid delivery elements capable of accepting and removing solution comprising the target analysts to and from the reaction chambers, wherein the solution is maintained in a lateral flow direction that allows the solution flows through the membranes in cross-section so that the sensitivity of detection is dependent on the ratio of the width of an area comprising the capture molecule to the width of the membrane.

[0007] In another embodiment, the present invention provides a lateral flow-through device for protein analysis, comprising: (a) one or more membrane cassette assemblies, each of which comprises a plurality of wells, wherein each of the wells comprise a membrane for immobilizing capture molecules capable of capturing target analysts; and (b) liquid delivery elements capable of accepting and removing solution comprising the target analysts to and from the membrane cassette assemblies, wherein the solution is maintained in a lateral flow direction that allows the solution flows through the membranes in cross-section so that the sensitivity of detection is dependent on the ratio of the width of an area comprising the capture molecule to the width of the membrane .

[0008] In one embodiment, the devices described above further comprise (a) controlling elements that can be regulated to maintain the reaction chamber or membrane cassette assembly in controlled conditions; and (b) connecting elements for connection to a power supply and control unit that can regulate and maintain the controlled conditions.

[0009] The present invention also provides a lateral flow- through protein analysis system comprising a pluraity of the devices described above, wherein the devices are connected to a power supply and control unit capable of supplying energy and providing regulatory control to the devices.

[0010] The present invention also provides a method of performing rapid protein detection, comprising the steps of: (a) immobilizing capture molecules on membranes placed in any one of the devices described above, wherein the capture molecules are capable of capturing one or more target protein molecules on the membranes; (b) applying to the membranes a sample comprising the target protein molecules, wherein the sample is maintained in a lateral flow direction that allows the sample flows through the membranes in cross-section so that the sensitivity of detection is dependent on the ratio of the width of an area comprising a capture molecule to the width of the membrane; and (c) detecting the captured target protein molecules on the membranes .

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 shows an exploded view of a lateral flow- through device of the present invention: Figure IA shows a device containing a reaction chamber where the membrane is placed directly onto the thermal plate which consists of the heating unit and the sensor regulator and electrical leads to the central power unit [P] as indicated in the Figure IB. Figure IB shows an arrangement of multiple lateral flow-through detection devices (I), (II) and (III) etc. which can be regulated when connected to a central control unit [P] . The sample and reagent solutions shall be delivered from the top cover openings and flow across the membrane for target molecules capture and development or additional assembly for solution removal i.e. at the drainage outlets .

[0012] Figure 1C shows a detailed view of another embodiment of a flow-through reaction chamber assembly. This embodiment is designed for use in the direct flow-through device of the prior art. The sample as well as reagent solutions flow through the incubation well [I] along the direction of the arrow → through the reaction chamber [R] . Reaction solution is drawn out by the suction (drainage system) provided by the main device unit, followed by signal development and interpretation.

[0013] Figure 2 shows another embodiment of a lateral flow reaction chamber assembly as described herein.

[0010] Figure 3 shows cancer biomarkers commonly used for clinical diagnosis. Quantitative assays are usually done with ELISA separately for each of these markers. Proteomic markers are identified and determined either by mass spectrometry (MS) and/or 2 dimensional gel electrophoresis as described in the given literatures .

[0011] Figure 3A shows plasma/serum concentration ranges of cancer biomarkers and their cut-off values among normal individuals reported from various studies.

[0012] Figure 4 shows examples of array images of cancer biomarkers. Panels A-D are image profiles obtained from some cancer samples; panel E is data from a normal sample.

[0013] Figure 5 shows example of concentration gradient and the detection limit of the present flow-through method for protein array detection. PVDF membrane was used for such experiment. The membrane was equilibrated with IX transfer buffer and blot dry. 0.4 ul human gamma-globulins with different concentrations, as shown in the figure insert, were dotted on the pre-wet PVDF membrane, air dry for 1 hour. The membrane was mounted on a R2M flow-through DNA analyzer, washed with ImI IX TBST 2 times (pump rate = low, wash time about 15sec) . The membrane was incubated with 0.5ml anti-human IgG AP-conjugate (1:10000) in S-AP for 15 min. Wash 3 times with ImI IX TBST followed by color development with 0.5ml NBP/BCIP substrate for 3 min, wash with TBST. Rinse with distill water 3 times and inspect signal. The total time require for the whole process was 25 minutes. This was at least 5 to 10 times faster than conventional Western analysis. Evidently as shown in Figure 5, this rapid process can readily achieve detection limit of fetal mole ranges with color development and the sensitivity is at least equal or better compared to conventional Western Blot tests. If chemiluminescence substrate is used the sensitivity shall be many folds better. Thus the feasibility of using the present lateral flow-through process for protein detection was indeed proven. This is the first application data demonstration using the flow-through technique of the present invention for faster, more efficient and more sensitive protein analysis .

[0014] Figure 6 shows a typical protein array profile (relative concentrations of different biomarkers) and biomarkers used for liver cancer diagnosis.

[0015] Figure 7 shows dot-blot analyses in a lateral flow- through manner of several lysates in identifying the culture conditions that generate positive signals (Figure 7A) as a mean for rapid screening for protein expression studies followed by Western Blot for detailed protein identification (Figure 7B) . PC 12 cells were stimulated with NGF for 0'; 15'; 30'; lhr; 3hrs; 5hrs and 24 hrs . Lysates were screened by dot blot assays with anti-ERK MAP kinase with the flow-through device as shown in Figure 7A. To confirm the feasibility of screening, parts of the same lysates were subjected to 10% PAGE, followed by conventional blotting procedures and flow-through procedures. Figure 7B shows the Western Blot analyses of lysates for the corresponding culture conditions. Evidently the results from rapid screening correlate well with results obtained by Western in positive signal for final identification of the protein (s) induced in given conditions. The dot-blot screening time was 30-40 minutes whereas the total Western Blotting assay required a two-days process that include protein separation and transfer etc. Figure 7B also showed that the results obtained by conventional process (which requires over two hours) compared well with the results obtained from flow- through process in 30 minutes at much reduced reagents used.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides methods and devices for rapid protein detection. In one embodiment, there is provided a lateral flow-through device for protein analysis, comprising: (a) one or more reaction chambers, each of which comprises a membrane for immobilizing capture molecules capable of capturing target analysts; and (b) liquid delivery elements capable of accepting and removing solution comprising the target analysts to and from the reaction chambers, wherein the solution is maintained in a lateral flow direction that allows the solution flows through the membranes in cross-section so that the sensitivity of detection is dependent on the ratio of the width of an area comprising the capture molecule to the width of the membrane. In one embodiment, the reaction chambers are disposable.

[0015] In another embodiment, the present invention provides a lateral flow-through device for protein analysis, comprising: (a) one or more membrane cassette assemblies, each of which comprises a plurality of wells, wherein each of the wells comprise a membrane for immobilizing capture molecules capable of capturing target analysts; and (b) liquid delivery elements capable of accepting and removing solution comprising the target analysts to and from the membrane cassette assemblies, wherein the solution is maintained in a lateral flow direction that allows the solution flows through the membranes in cross-section so that the sensitivity of detection is dependent on the ratio of the width of an area comprising the capture molecule to the width of the membrane .

[0016] In general, one of ordinary skill in the art would employ a variety of liquid delivery elements capable of accepting and removing solution comprising the target analysts to and from the reaction chambers or membrane cassette assemblies. For example, for the miniature device (meant to be used for very small volume) showed in Figure IA, the solution is drawn across the membrane by absorbance wicks on one side. Alternatively, one can use forced injection. In another embodiment, accepting and removing solution to and from the reaction chamber or membrane cassette assembly can be done by regulated liquid pumping. In one embodiment, the liquid pumping is used to recirculate the solution comprising the target analysts through the membrane.

[0017] In another embodiment, the devices described above further comprise (a) controlling elements that can be regulated to maintain the reaction chamber or membrane cassette assembly in controlled conditions; and (b) connecting elements for connection to a power supply and control unit that can regulate and maintain the controlled conditions. In general, the power supply and control unit is capable of supplying energy and providing regulatory control to maintain the reaction chambers in the controlled conditions. In one embodiment, the controlling elements are heating or cooling elements. [0016] In one embodiment, the membrane can be made of nitrocellulose, nylon, Nytron, Biodyne, Porex or any kind of porous matrix material support, and the capture molecules can be arranged in an array on the membrane .

[0017] The present invention also provides a lateral flow- through protein analysis system comprising more than one of the devices described above, wherein the devices are connected to a power supply and control unit capable of supplying energy and providing regulatory control to the devices. In one embodiment, each of the devices is controlled independently by the power supply and control unit so that each device can perform different analysis under different conditions.

[0018] The present invention also provides a method of performing rapid protein detection, comprising the steps of: (a) immobilizing capture molecules on membranes placed in any one of the devices described above, wherein the capture molecules are capable of capturing one or more target protein molecules on the membranes; (b) applying to the membranes a sample comprising the target protein molecules, wherein the sample is maintained in a lateral flow direction that allows the sample flows through the membranes in cross-section so that the sensitivity of detection is dependent on the ratio of the width of an area comprising a capture molecule to the width of the membrane; and (c) detecting the captured target protein molecules on the membranes. In one embodiment, the target protein molecules are of human, bacterial, or viral origin. In another embodiment, the human is having or is suspected of having cancer.

[0019] In general, the captured target protein molecules are detected by a method such as fluorescence tags, quantum dot labeling, colloidal gold particle labeling, magnetic particle labeling, or enzyme-linked substrate assay. In one embodiment, the target protein molecules are mixed with a signal generating agent before being applied to the membranes for capture detection.

[0020] In one embodiment, the target protein molecules are biomarkers for cancer, therefore detection of the target protein molecules would provide diagnostic information for cancer. For example, the method described herein can be used to provide diagnostic information for liver cancer when the capture molecules are capable of binding to target protein molecules comprising AFP, AFP-L3, CA19.9, CA125, CA15.3 , TGF-Bl, HS-GGT, ALF-161, des-gamma carboxyprothrombin, ferritin, HbsAg, and anti-HCV antibody. In one embodiment, diagnosis or prognosis of liver cancer is indicated by an increase in expression levels as compared to normal cut-off values for two or more target proteins selected from the group consisting of AFP, AFP-L3, HS-GGT, TGF-Bl and DCP. In another embodiment, the increased expression levels are detected in a sample obtained from a human infected with hepatitis B virus, hepatitis C virus, or hepatitis B and C viruses.

[0021] In another embodiment, the method described herein can be used to provide diagnostic information for a specific cancer when the capture molecules are capable of binding to two or more of the biomarkers as listed in Figure 3. In one embodiment, diagnosis or prognosis of cancer is indicated by an increase in expression levels as compared to normal cut-off values for two or more of the biomarkers as listed in Figure 3.

[0022] The present invention also provides a method of concentrating target molecules from large volume and detect low concentration of target molecules, comprising the steps: (a) the sample is first incubated with secondary affinity molecules such as substrates for enzymes; ligands for receptors; secondary antibodies for antibodies etc. immobilized solid matrix; (b) separate matrix and remove sample solution; (c) re-dissolve target molecule from matrix follow by (d) flow-through detection with reduction of non-specific binding for increase in signal to noise for increased sensitivity.

[0023] The present invention also provide a method for rapid and high throughput screening of protein expression profile followed by Western Blotting identification, comprising (a) antibodies array screening membrane; and (b) applying in the flow- through manner multiple lysates of cell cultures having different conditions of inductions for either up-regulated or down regulated expression in identifying component (s) in regulatory factor for biochemical pathways; (c) detecting and identifying changes of expression and identifying the culture conditions responsible for the changes which enables the investigator to have tremendous increase in speed and efficiency to explore the complex biochemical world and (d) follow the conventional time consuming Western Blotting experiments for molecular identification only a limited fraction of experiments. Thus the method provided by this invention would be of utmost importance for a saving of many folds in time and material cost for research.

EXAMPLE 1 Flow-Through Array System

[0024] Certain embodiments of a lateral flow-through detection device have been described (see Tarn, 1998; 2004; 2005; 2006). Figure 1 shows an exploded view of a lateral flow-through detection device of the present invention, and possible arrangement of multiple lateral flow-through detection devices connected to a central control unit.

[0025] In one embodiment, the device comprises a central controlling unit connected to one or more lateral flow-through devices. The central controlling unit provides power to and controls the lateral flow device where protein binding reactions and developing procedures are carried out. Several reactions (or several samples and/or analytes) can be tested simultaneously in a single lateral flow device, or in several devices controlled individually at different conditions.

[0026] The lateral flow device can accommodate an array in a format of n x m dot matrix (array) or in the form of linear arrays (as shown in Figure 1) . During the reaction process, a test solution flows from one end of the array to the other end of the array (i.e., in an east to west, or in a north to south direction), hence the sensitivity of detection is increased substantially. The extent of increase in sensitivity depends on the ratio of the total area of the array/membrane to the area of the dot or line containing the capturing probes. For example, assuming the total area of an array/membrane is 100mm square, and the dot size is lmm square. In a direct flow-through process (i.e., the solution flows from top surface through the membrane down to the other side of the membrane as in a conventional flow-through process) , only 1/100 of the total test solution used will flow through the dot, the location where the target molecule will bind to the probe (s) immobilized on the membrane.

[0027] However, if a lateral flow-through process is used, the sensitivity is only dependent on the ratio of the width of the dot to the width of the membrane (i.e., the cross section of the membrane) . For instance, in a lateral flow-through process, the total amount of solution that will pass through a 1 mm dot provided on a 10mm x 10mm membrane will be about 1/10, which represents a 10-fold increase in sensitivity using the same amount of test solution containing the target molecules. When a line array format is used in the lateral flow-through process, the sensitivity will also be increased since all the target molecules will pass through the line extending across the strip (or membrane) . The lateral flow-through process allows quantitative measurements to be taken during the reaction process because the flow of the analyst is more uniform.

[0028] One embodiment for the lateral flow-through device is shown in Figure IA. This embodiment can be made as a new device that comprises (i) an electronic control unit for operations; (ii) an optional temperature block; (iii) a reaction chamber; and (iv) a liquid delivery system. Temperature control may not be essential for antibody-antigen reaction as generally done for Western Blot analyses. However, some proteins may be less stable, others may react better at certain temperature higher or lower than room temperature, therefore accurate controlled conditions including temperature is one embodiment. On the other hand, if less stringent condition is favorable for certain analytes detection, a simpler device is in order for a low cost alternative device .

[0029] In one embodiment, the reaction chamber where the membrane is located can be designed as a new separate and/or disposable unit. The flow direction, the speed of flow and the sequence of solution reagents flow can be controlled accurately by the controller. This disposable reaction chamber can be made as single or multiple cassettes for separate or parallel reactions to provide increase in throughput. Evidently this embodiment can provide optimal conditions for effective detection with increase in sensitivity and specificity. The disposable as well as the totally enclosed setting in this embodiment can prevent any possibility of cross contamination.

[0030] Alternative embodiment for the lateral flow-through device shown in Figure 1C is modified assembly of the reaction chamber which enables one to adopt using the Direct Flow-through Device described in the prior invention [U.S. patent No. 6,020,187] . The assembly comprises a plurality of wells, each of which further comprises a membrane. The cassette like membrane assembly by which the sample and subsequent reagent solutions can be applied on top of one side of the array membrane where the solution will flow across the membrane and drained from the other side of the membrane into the waste. Recirculation is also possible provided re-circulating pumping mechanism is added.

EXAMPLE 2 Procedures For The Flow-Through Process

[0031] The flow-through array system described herein can be use for dot-blot, reversed dot-blot or slot blot analysis, where multiple array assays can be done simultaneously.

Dot-Blot

[0032] In one embodiment of dot-blot assay, target samples to be tested are dotted onto the membrane as arrays in each well

(separated from the other wells) for which the number of well depend upon the number of antibodies (for antigen screening) or antigens (for antibody screening) . The following is exemplified for antigens screening:

[0033] Procedures:

[0034] 1. Immobilized a set of samples onto a number of wells or on the membrane as an array. A membrane refers to any porous matrix materials capable of binding the target antigens for detection.

[0035] 2. Block the membrane to prevent non-specific binding. [0036] 3. Flow-through solution containing antibody molecule to be targeted for detection; wash followed by signal detection (no further step is needed if signal generating dye has been labeled onto the antibody) .

[0037] 4. Develop color according to standard assay procedures (see e.g. Tarn et . al . , 1988).

Reverse Dot-Blot

[0038] In one embodiment of reverse dot-blot, a different type of antibodies (or antigens) are being dotted in array format for screening their complement molecules in the target sample solution:

[0039] Procedures:

[0040] 1. Immobilized a set of antibodies (for capturing antigenic proteins or antigens) or antigens (for capturing antibodies) onto a membrane or any matrix materials for detecting target molecules

[0041] 2. Block the membrane to prevent non-specific binding. This step can be deleted if the membrane has been pretreated with blocking reagents .

[0042] 3. Flow-through the solution containing target molecules; wash followed by signal detection (no further step is needed if labeled detection second antibody is added into the target solution in appropriate conditions) .

[0043] 4. Develop color according to standard assay procedures (see e.g. Tarn et . al . , 1988).

[0044] Slot Blots can be done either as Dot-blot or Slot-Blot as described above.

EXAMPLE 3 Detection of Concentration Limit Without Amplification

[0045] To determine the limits of detection by the lateral flow-through technique, a set of biotin labeled at various concentrations can be immobilized onto the membrane. Color development is done according to established procedures with avidin-alkaline phosphatase system. The lowest detection limit of the immobilized antigen is used as the lowest limit of detection for the present embodiment of the flow-through device. Next the flow-through reaction is determined by immobilizing various antibodies forming the specific array as capture molecules. Various concentrations of present gradient of target molecules are used to test the feasibility and effectiveness of quantitative capturing process by the immobilized antibodies for their corresponding target molecules.

EXAMPLE 4 Western Blot Assays by Flow Through Procedures

[0046] Western Blot has been a useful technique for the analysis of target protein (s) in a solution in question for definitive identification. The general procedures are: (1) separate the protein molecules as far as possible in a mixture of proteins by either conventional SDS electrophoresis or isoelectric focusing (IEF) in order to generate a clean and observable bands; (2) transfer the protein onto a membrane; and (3) assayed by antibody binding (by affinity) followed by (4) color development. This is, however, a very time consuming process requiring days or hours to perform. Hence improved protocols such as device and procedures which can be adopted for the Western Blotting studies are in great demand.

[0047] The present invention provides a rapid platform (the lateral flow-through device's reaction chamber) for carrying out all the steps of Western Blot after the proteins have been transferred onto a membrane. The lateral flow-through process by itself is suitable for such improvement by reducing the time and reagent cost by several folds simply by performing steps (3) and

(4) described above using the reaction chamber described herein. After protein transfer onto the membrane is completed, the membrane is placed onto the reaction chamber of the present lateral flow-through device, and start with the normal conventional procedures: (i) apply blocking solution and allow to flow into the membrane preventing non-specific binding; (ii) add antibody conjugate, add washing solution to remove unbound conjugates; and (iii) add substrate for signal development and analyze data. The platform of the present invention will provide rapid immuno-reaction between target molecules and their reactants

(e.g. antibodies or antigens).

EXAMPLE 5

Rapid Screening of Expression Profiles From Cell Culture Lysabes

By Flow-Through Antibody Array

[0048] In the post genomic era, protein expression profiling is now in the center stage for discovery of novel protein (s) or for drug developments using in vitro cell culture followed by analysis of protein expression profiles generated by 2 -dimensional gel electrophoresis. Western Blotting is the most frequent tool for novel protein identification and/or identification of regulatory element (s) of metabolic pathway (s). Unfortunately, 2-dimensional gel and Western Blot analysis is time consuming, labor intensive and therefore is the rate limiting step for these proteomic studies. The lateral flow-through method and device described herein can provide alternative analyses that can reduce the time and increase the efficiency of these proteomic studies.

[0049] Since antibodies production are very efficient nowadays and specific monoclonal molecules are readily available, one can use antibody array as a screening tool for rapid analysis of proteins in lysates obtained from cells cultured under different stimuli for up-regulated or down regulated protein expression. Instead of completing all detailed analysis of every lysate from cells cultured in different conditions by the time consuming Western and/or 2-dimensional gel analyses, the present invention provides an antibody array and the lateral flow-through system as a first tool for qualitative screening analyses. Detailed analyses will be done only on those lysates that are identified to have significant changes. For illustration purpose, the MAP kinase activation is used as an example. Antibody array was prepared by immobilizing individual antibodies onto the membrane. The time required for performing 15 wells reactions (i.e. 15 different membranes or antibody arrays used to analyze 15 different lysates from cultured cells) was 90 minutes (with 30 minutes incubation extension time for lysate-antibody reaction) , resulting in substantial reduction of more than 30-folds of technical time required by the complete set of Western Blot analysis. Hence the present lateral flow-through system can be used for rapid initial screening for the presence or absence of target proteins before performing the long electrophoresis separation and Western analyses. Using the new lateral flow-through system and dot -blot to screen many samples for target protein requires only 20-30 minutes without performing conventional electrophoresis separation, transfer and Western Blot analysis. Since dot-blot is a rapid and high throughput assay, the flow-through system of the present invention will save 10-100 folds in time and materials. Furthermore, using a recirculation flow-through system disclosed herein would even further increase the sensitivity of the assay.

EXAMPLE 6 Biomarkers Assay for Early Cancer Diagnosis

[0050] The Flow-through Array described herein can generate useful profiles by simultaneously assaying multiple biomarkers in a single assay. Figure 3 shows some of the biomarkers useful for cancer screening in clinical laboratories for the past decade or two as single marker assays. Although an individual marker may have some prognostic value, a single marker alone cannot provide significant sensitivity and specificity for the diagnosis of solid cancers. Quantitative profiles of a group of markers would be much more useful in delineating possible types of cancer and to get a more accurate early diagnosis. Figure 4 shows a typical set of profiles and their corresponding cancers suggest that such an approach is feasible.

[0051] The recent BIOMARKERS C-12 cancer screening kit has claimed an advantage of increased detection. Since most of those markers are non-specific for cancer, increase in detection may also be caused by non-cancerous diseases. For example, most liver cancer patients have abnormally high blood serum levels of alpha- fetal protein (AFP) , yet similar increase can also be caused by cirrhosis or chronic hepatitis. Hence further increase in sensitivity of detection will also lead to increase in false positive predictions. Indeed recent general screening data using the C-12 screening test have resulted in over 30% positive rate, which is 10 times the cancer prevalence rate of 3% from the China national statistic data, indicating that the false positive rate is much too high. Evidently the selection of such general markers per se is not the right approach despite the real advantages of using protein arrays as analytical tool. For this reason, microarrays capable of analyzing thousands of genes differentially expressed between cancerous and normal patients are used to identify biomarkers responsible for the set cancer as diagnostic tool (MacBeath G 2000; Nguyen et al . , USPTO No. 7049151; Diamond; Scott L, USPTO No. 7332286). Liew CC. et al . , (WO/2005/042725) claim a collective set of biomarkers which include amyloid beta precursor-like protein 2(APLP2); BCL2-related protein Al (BCL2A1) ; phosphoprotein regulated by mitogenic pathways (C8FW) ; complement Component 5 (C5) ; CD14 antigen (CD14) and many others including RAS viral gene sequences and expressed said markers for use as diagnostic tool for liver cancer. Evidently, product (s) in related pathways as well as tumorgenesis factors are more specific diagnostic tool for a given cancer. Nevertheless, regardless of the choice of biomarkers, the flow-through device of the present invention is an efficient tool for performing the protein arrays analysis which would provide more sensitive and specific results. With the right selection of biomarkers for cancer diagnosis, the array profiles and diagnosis can be done and further validated through large clinical trails. In another embodiment, the present invention can simultaneously detect multiple proteins of different organisms such as viruses and/or bacteria. For example, phenotyping of drug resistant proteins in human such as P450; mutant oncogenic proteins such as Ras, p53 mutated proteins or viral proteins of HIV, HBV, HCV can be conducted singularly or in combinations .

[0052] Liver cancer is one of the most prevalent cancer in China and Africa. Because it is difficult to detect at early stages, once diagnosed the 5-year survival rate is only 5-7%. Hence it is imperative to find more sensitive and specific assays for early diagnosis. Among the biomarkers, serum levels of alpha- fetal protein (AFP) are most frequently used as the prognostic marker because between 50-75% of HCC have abnormally high serum levels of AFP. Unfortunately it cannot be used as confirmational diagnosis because it can also be caused by many other diseases such as cirrhosis, chronic hepatitis and other infections. Recently liver specific HS-AFP or AFP-L3 (Li et al . , 2001; Wu W. et al., 2006), Midkine (USPTO No. 709083) and CRG-L2 (USPTO No. 7129328) have been suggested as better and more specific markers. It is well established that chronic hepatitis individuals infected by HBV and HCV have a much higher likelihood of developing liver cancer (Gao JD et al . , 2005) . Hence the presence of viral DNA and/or viral antigens can serve as positive association and differentiating factor for diagnosis when high levels of other biomarkers are tested positive. The present invention provides in this example a flow-through array comprising a set of biomarkers associated with tumorgenic release from the cancerous cells and this set of markers together with known cell specific mutagenic agents form an array that can serve as diagnostic assay for liver and other cancers. In one embodiment, the markers used for liver cancer diagnosis include AFP; AFP-L3; CA19.9; CA125; CA15.3; TGF- Bl; HS-GGT; ALF-161; DCP (des-gamma carboxyprothrombin) ; ferritin; HbsAg; and anti-HCV antibody. [0053] The disclosures in connection with preferred embodiments are not intent to limit the invention to the procedures and embodiments described. On the contrary, the intent is to cover all alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.

EQUIVALENTS

[0054] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

References

Bunce M et al . (1995) Tissue Antigens, 46, 355-367.

Chakraborty R, Stivers D N. Paternity exclusion by DNA markers: effects of paternal mutations. J Forensic Sci 1996 July; 41(4):

671-7. Chow R and Tonai R., "High throughput methods for HLA typing", US

Pat. No. 6,670,124, December 30, 2003. Gao JD et al . , Tight association of hepatocellular carcinoma with

HBV infection in North China. Hepatobiliary Pancreat Dis Int.

2005, 4: 46-9 Grondahl et . al . , Journal of Clinical Microbiology 1999 Jan; 37:

1-7 Kaneshige T et . al . , Rapid and Practical HLA Class II Genotyping by Reversed Dot Blotting, Transplantation Proceedings, 1993 Feb.;

25 (1) : 194-198.

Li D et . al . , A new generation of tumor marker for hepatocellular carcinoma, Clin Chim Acta. 2001, 313: 15-9.

MacBeath G. and Schreiber SL, Printing Proteins as Microarrays for High-Throughput Function Determination, Science 2000 September 8 ;

289(5485) : p. 1760-1763. Mach B et al . (1990) in Molecular Biology of HLA Class II Antigens, ed. Silver J (CRC, Boca Raton, FL), pp. 201-223. Mach et al . , "DNA sequences coding for the DR beta-chain locus of the human lymphocyte antigen complex and polypeptides, diagnostic typing processes and products related thereto" , US

Pat. No. 6,818,393, November 16, 2004.

Mackay et . al . Journal of Clinical Microbiology 2003 Jan; 100-105 Tarn, Flow Through Nucleic Acid Hybridization Device, U.S. Pat. No.

5, 741,647. Tarn, Flow Through Nucleic Acid Hybridization Device, U.S. Pat. No.

6, 020,187. Tarn, DNA Fingerprinting Using Allelic Specific Oligonucleotide

Reversed Dot Blot (ASO RDB) Flow Through Hybridization Process

And Device, U.S. Publication No. 2005/0079493, April 14, 2005. Tarn, SNP-BASED HLA-DP, DR And DQ Genotyping Analysis By Reverse

Dot Blot Flow Through Hybridization, U.S. Publication No.

2004/0209253, October 21, 2004. Tarn, Rapid Genotyping Analysis and the Device Thereof, U.S.

Publication No. 2006/0292601, December 28, 2006. Thomas ED (1983) J. Clinical Oncology 1, 517-531. Wu W et al . , Combined serum hepatoma-specific alpha-fetalprotein and circulating alpha-fetalprotein-mRNA in diagnosis of hepatocellular carcinoma. Hepatobiliary Pancreat Dis Int. 2006,

5:538-44.

Claims

What is claimed is :

1. A lateral flow-through device for protein analysis, comprising:

(a) one or more reaction chambers, each of which comprises a membrane for immobilizing capture molecules capable of capturing target analysts,- and

(b) liquid delivery elements capable of accepting and removing solution comprising the target analysts to and from the reaction chambers, wherein the solution is maintained in a lateral flow direction that allows the solution flows through the membranes in cross-section so that the sensitivity of detection is dependent on the ratio of the width of an area comprising the capture molecule to the width of the membrane .

2. A lateral flow-through device for protein analysis, comprising:

(a) one or more membrane cassette assemblies, each of which comprises a plurality of wells, wherein each of the wells comprise a membrane for immobilizing capture molecules capable of capturing target analysts; and

(b) liquid delivery elements capable of accepting and removing solution comprising the target analysts to and from the membrane cassette assemblies, wherein the solution is maintained in a lateral flow direction that allows the solution flows through the membranes in cross-section so that the sensitivity of detection is dependent on the ratio of the width of an area comprising the capture molecule to the width of the membrane.

3. The device of claim 1 or 2 , further comprising

(a) controlling elements that can be regulated to maintain the reaction chamber or membrane cassette assembly in controlled conditions; and

(b) connecting elements for connection to a power supply and control unit that can regulate and maintain the controlled conditions.

4. The device of claim 3, wherein the controlling elements are heating or cooling elements.

5. The device of any one of claims 1-4, wherein the membrane comprises a material selected from the group consisting of nitrocellulose, nylon, Nytron, Biodyne, and Porex.

6. The device of any one of claims 1-5, wherein the capture molecules are arranged in an array on the membrane.

7. The device of any one of claims 1-6, wherein accepting and removing solution to and from the reaction chamber or membrane cassette assembly is by regulated liquid pumping .

8. The device of claim 7, wherein the liquid pumping is used to recirculate the solution comprising the target analysts through the membrane.

9. A lateral flow-through protein analysis system comprising a plurality of the device of any one of claims 1-8, wherein the devices are connected to a power supply and control unit capable of supplying energy and providing regulatory control to the devices .

10. The system of claim 9, wherein each of the devices is controlled independently so that each device can perform analysis under different conditions.

11. A method of performing rapid protein detection, comprising the steps of:

(a) immobilizing capture molecules on membranes placed in any one of the device of claims 1-10, wherein the capture molecules are capable of capturing one or more target protein molecules on the membranes;

(b) applying to the membranes a sample comprising the target protein molecules, wherein the sample is maintained in a lateral flow direction that allows the sample flows through the membranes in cross-section so that the sensitivity of detection is dependent on the ratio of the width of an area comprising a capture molecule to the width of the membrane ; and

(c) detecting the captured target protein molecules on the membranes .

12. The method of claim 11, wherein the captured target protein molecules are detected by a method selected from the group consisting of fluorescence tags, quantum dot labeling, colloidal gold particle labeling, magnetic particle labeling, and enzyme-linked substrate assay.

13. The method of claim 11 or 12, wherein the target protein molecules are mixed with a signal generating agent before being applied to the array.

14. The method of any one of claims 11-13, wherein the target protein molecules are of human, bacterial, or viral origin.

15. The method of any one of claims 11-14, wherein the sample is obtained from a human and the target protein molecules are biomarkers for cancer.

16. The method of any one of claims 11-15, wherein the capture molecules are capable of binding to target protein molecules comprising alpha-fetal protein (AFP) , lens culinaris agglutinin bound AFP (AFP-L3) , CA19.9, CA125, CA15.3, TGF-Bl, HS-GGT, ALF-161, des-gamma carboxyprothrombin (DCP) , ferritin, HbsAg, and anti -HCV antibody, wherein detection of the target protein molecules would provide diagnostic information for liver cancer .

17. The method of claim 16, wherein diagnosis or prognosis of liver cancer is indicated by an increase in expression levels as compared to normal cut-off values for two or more target proteins selected from the group consisting of AFP , AFP - L3 , HS - GGT , TGF - Bl and DCP .

18. The method of claim 17, wherein the increased expression levels are detected in a sample obtained from a human infected with hepatitis B virus, hepatitis C virus, or hepatitis B and C viruses.

19. The method of any one of claims 11-15, wherein the capture molecules are capable of binding to two or more of the biomarkers listed in Figure 3, wherein detection of the biomarkers would provide diagnostic information for cancer.

20. The method of claim 19, wherein diagnosis or prognosis of cancer is indicated by an increase in expression levels as compared to normal cut-off values for two or more of the biomarkers listed in Figure 3.