WO2009079192A1 - Universal tandem solid-phases based immunoassay - Google Patents

Abstract

Universal tandem solid-phases based immunoassay (UTSIA) is a sandwich-ELISA equivalent assay that overcomes limitations of sandwich-ELISA (antibody inactivation by solid phase and strict requirement of a pair of primary and secondary antibodies) by using an affinity binding solid phase to capture antigen specifically from a fluid sample, sequentially dissociating the antigen from the solid phase, transferring and coating the antigen to a non-affinity binding solid phase for specific detection. Cell-based UTSIA is a cell-based ELISA equivalent assay that overcomes limitations of cell-based ELISA for quantitatively detecting an antigen in the cells or tissue immobilized on a solid phase by dissociating and transferring the detection antibody bound on the antigen of the cells or tissue immobilized on the solid phase to another solid phase and coating the antibody there for specific detection of the antigen via the detection of the antibody.

Description

UNIVERSAL TANDEM SOLID-PHASES BASED IMMUNOASSAY

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to US Provisional Application No. 61014426, filed on December 17, 2007, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to immunoassay methods for detecting and/or quantitating antigen(s) in a fluid sample and detecting and/or quantitating antigen(s) in cells or tissue immobilized on solid support(s).

BACKGROUND OF THE INVENTION

In this post-genomics era, it is critically important to develop a convenient and robust method for detection and quantification of low abundance proteins for biological and biomedical researches, clinical diagnostics, environmental and food monitoring, and biotech- pharmaceutical industries (Duncan et al 2005, Srinivas et al 2002, and Albala 2001).

Traditionally sandwich-ELISA is a popular immunoassay to detect and quantitate antigen in biological and non-biological samples (Zangar et al, 2006, Zhou et al 2005). Its applications in clinical diagnostics, bio-medical research, food and environmental monitoring, and biotech and pharmaceutical industries are depended on its advantages: simplicity, sensitivity, and specificity. However, setting up a sandwich-ELISA for most of analytes is a challenge until current invention.

Sandwich-ELISA uses specific antigen-antibody binding reaction to detect its antigen (Voller et al 1978, Lequin 2005, and Zangar et al 2006). The basic requirements for the method to detect antigen include having an antigen with at least two binding sites for specific antigen antibody binding reactions, having a pair of matched capture and detection antibodies which can be used to form a sandwich-like complex with an antigen for the antigen's capture and detection, and having a capture antibody with capacity to maintain its antigen binding activity after immobilized on a solid phase. The general procedure for antigen detection includes that the antigen first reacts with excess solid-phase antibody (the capture antibody) immobilized on a solid phase, after incubation and followed by washing, the bound antigen on the solid phase is reacted with excess labeled antibody (the detection antibody). After further washing, the label is measured, and the signal of the label is directly correlated with the amount of antigen present. A modification of the sandwich-ELISA is the double sandwich-ELISA, which involves a third antibody. The third antibody carries a label and reacts with an unlabeled detection antibody already bound to the antigen. Then the signal of the label is measured, and like before, the signal of the bound label is directly correlated with the amount of the antigen present. The advantages of sandwich-ELISA are that, by using a capture antibody on a solid phase, the method can enrich and purify an analyte from a fluid sample containing complicated components (this is especially important for the detection of a low abundance protein), and that, by using a pair of capture antibody and detection antibody, the double affinity bindings significantly increase the specificity of the detection. However, as mentioned above, in this post-genomics era, to develop a sandwich- ELISA is still a big challenge for scientists. Why is it so difficult to develop a sandwich- ELISA? Because it is hard to find a capture antibody which maintains the antigen binding capacity after its coating on a solid phase, as about 80% of antibodies are denatured after coating on the solid phase; it is even more hard to find a pair of matched capture antibody and detection antibody which form a sandwich complex with an antigen for the successful antigen capture and detection. All of these problems of sandwich-ELISA lead to the assay only can be used in limited number of proteins discovered.

Since Engvall and Perlmann published their first paper on ELISA in 1971 (Lequin 2005), there are many inventions and literatures in the field of immunoassay. However, most of these researches are focused on improvements of sandwich-ELISA, not worked on inventing an alternate convenient and robust immunoassay which not only overcomes the technical difficulties of sandwich-ELISA but also preserves the advantages of sandwich- ELISA. For example, US5236849 worked on reducing the label signal background and increasing sensitivity of sandwich-ELISA by adding additional steps of dissociating the antigen- antibody complex from the solid phase and then rebinding the complex to a new affinity solid phase which attached with a reactive group which is capable of specifically binding with the complex; although US5236849 used two solid phases and a dissociating step in its patent claims, the invention does not solve the technical limitations of sandwich-ELISA by following facts: US5236849 uses sandwich complex mechanism of sandwich-ELISA for antigen or antibody's capture and detection; and in dissociating step of US5236849 the complex of antigen and antibody is dissociated from the solid phase in a form of the immuno-complex, not breaking the complex to elute un-complexed antigens into the liquid phase of the dissociating buffer; in addition, both solid phases of US5236849 used are affinity binding solid phases (in which there is at least an affinity binding component immobilized), which means the method is not an universal method, as the setting of two different affinity binding solid phases for each analyte analysis is very difficult; finally, the detection step of US5236849 focuses on the detection of complex of antigen and antibody, not on the single antigen or single antibody, that is "assaying for the complex, bound to the second solid carrier. "(Copied from claim 1C of US5236849). Another example is the patent of US5236830; the inventor also used two solid phases and a dissociating step for antigen assay. But the goal of the invention is to increase the sensitivity of the antigen detection, and the mechanism of the antigen's capture and detection is sandwich-ELISA, and the two solid phases used are all affinity binding solid phases to increase detection sensitivity, and the antigen is required to be modified first before be captured by the first solid phase. Therefore, both patents US5236830 and US5236849 are not an invention which solves the technical challenges of sandwich-ELISA. In contradictory, they are a more complicated and two affinity solid phases based sandwich-ELISA. Similarly, all other disclosed patents and literatures in the field of immunoassay before current invention have not solved the technical limitations of sandwich-ELISA successfully; specifically, they continue using sandwich- ELISA mechanism, the formation of antibody- antigen- antibody complex, to capture and detect antigen (analyte) of interest specifically from a complicated fluid sample. If the sandwich-ELISA for the antigen is not available, then alternate way for the determination of the antigen from the sample comprises at least two methods: first using immunoprecipitation or affinity chromatography to isolate the antigen from the fluid sample, then using western blot, mas s- spectrometry, or ELISA to detect the antigen isolated. As these methods are not integrated together in a single assay format as sandwich-ELISA, they are not efficient and 90 very time consuming. Thus, there is a need to invent a convenient and robust method to replace sandwich-ELISA with a novel alternate mechanism other than sandwich complex for the antigen capture and detection.

This invention discloses a novel immunoassay method to replace sandwich-ELISA for protein or other analytes' detection and quantification. The basic idea of the invention is 95 that by separating the process of antigen capture and detection of sandwich-ELISA in a solid phase into two solid phases-based processes (one affinity binding solid phase for specific antigen capture and one non-affinity binding solid phase for specific antigen detection) to break the strict requirement of a perfect matched two antibodies required for formation of sandwich antibody-antigen-antibody complex in sandwich-ELISA. The first solid phase (the

100 affinity binding solid phase) of present invention is for the capture of the analyte by affinity binding, and the second solid phase (non-affinity binding solid phase) of present invention is for immobilizing the analyte for further specific detection. As the invention need two solid phases and can be used to set up immunoassays for unlimited number of proteins, and is capable to up scale to formats of microarray for multiple analytes detection and

105 quantification, the invention is named as universal tandem solid-phases based immunoassay (UTSIA). The application of UTSIA comprises antigen determination, especially for low abundance antigen determination.

Another important immunoassay is immunohistochemistry (IHC) and immunocytochemistry (ICC). IHC is a technique developed by Coons six decades ago

110 (Coons AH CH et al., 1941). Today IHC has a central role in the field of diagnostic medicine pathology. IHCs variation ICC also has important role in biomedical research, clinical diagnostics, and drug discovery. Both techniques of IHC and ICC are using a detection antibody to detect an antigen immobilized on a solid phase. The major difference is that the antigen detected by IHC is located in tissue immobilized on a solid phase, while the antigen

115 detected by ICC is located in cells fixed on a solid phase.

The major challenge of ICC and IHC is that it is difficult to set up quantitative ICC and IHC. Traditionally ICC and IHC are qualitative methods or semiquantitative methods (Kaczmarek E et al 2004, Lang et al 2006, Dodson 2002, Ramos-Vara 2005, Guardigli et al 2005). The presence and amount of the antigen on the tissue or cells immobilized on a solid

120 phase is judged by assigning scores by an skilled observer such as pathologist by using a proper microscope based on certain criteria, or by cellular or tissue imaging method that involves image capture and image data analysis. It is well known that the scores method is not objective; the results from the scores method for quantification can be varied significantly with different observers. The imaging method also is not an ideal quantitative method as it is

125 not a direct measurement of antigen quantity, needs expensive equipment, consists of complicated manipulation procedures, is very time consuming, and produces data with big variation and the data is hard to replicate. Thus, there is an urgent need to develop better method for quantitative ICC or quantitative IHC.

The invention discloses an important variant of UTSIA, cell-based UTSIA, for

130 quantitative IHC and ICC. It is a cell-based ELISA equivalent immunoassay which is used to detect and quantitate antigen(s) of interest in cells or tissue immobilized on solid phase(s). The mechanism of the cell-based UTSIA is that dissociating, transferring, and coating the specific antibody that bound on the antigen of interest on the cells or tissues immobilized on a solid phase to another solid phase for specific detection. The antibody on the late solid

135 phase is directly correlated with the amount of the antigen on the first solid phase. The advantages of the cell-based UTSIA comparing with cell-based ELISA include it has less strict antibody requirement to set up assay, it can be used either in cell-based antigen or tissue-based antigen determination, it avoids complicated background (such as background enzyme activity) of tissue or cells on the detection steps, and it also can be used for detection

140 of low expression protein in cells or tissue by changing the relative sizes of the solid phase for cell or tissue immobilization and the solid phase for antibody detection. The applications of the cell-based UTSIA may comprise cell based or tissue based biomarker evaluation, detection, and quantification for biomedical research, clinical diagnosis, and drug discovery.

145 THE DETAIL DESCRIPTION OF THE INVENTION

The following definitions, description of figures, general descriptions, steps, figures, preferred embodiments, and prototype example are offered by way of illustration of present invention, and not by way of limitation of present invention. 150

DEFINITIONS:

Adaptor molecule: as used herein, is a molecule which can be immobilized on a solid phase (the first solid phase) to be part of a capture system, wherein the adaptor molecule

155 immobilized on the solid phase is capable of binding a capture molecule and is capable of preventing the inactivation of the capture molecule's analyte binding ability by the solid phase; the adaptor molecule is selected from a group comprising Protein G, Protein A, Protein A/G, Protein L, antibodies, proteins, recombinant proteins, recombinant antibodies, small organic molecules, poly-peptide, or nucleic acid aptamers; adaptor molecule comprises

160 site(s) for attaching or coating to a solid phase and site(s) for binding of a capture molecule. Affinity binding: as used herein, is a binding reaction same as or similar to the specific binding interactions between an antigen and its antibody or between a ligand and its receptor; the affinity bindings usually can be disrupted by the buffer comprising extreme pH.

Analyte: as used herein, is a substance of interest in a fluid sample or a sample; it may

165 have same meaning of target analyte, analyte of interest, antigen, or antigen of interest; the analyte comprises proteins or other components which have binding site(s) for a capture molecule and a detection molecule as well as have hydrophobic structures that can be used to coating the analyte on a detection solid phase; if the capture molecule or the detection molecule is an antibody, then the analyte is named as antigen.

170 Antibody: as used herein, the antibody is a protein encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically bind and recognize an antigen; the antibody comprises the intact antibody or the fragment antibody produced by animal, tissue culture, and recombinant DNA technology; if the adaptor molecule is an Fc- domain binding protein (such as Protein G), then the capture antibody is an antibody

175 comprising Fc-domain which binds with the Fc-domain binding protein specifically.

Antigen: as used herein, is an analyte that can be captured by a capture antibody and/or that can be detected by a detection antibody.

Aptamer: as used herein, is a polymer molecule that has high specificity and affinity for its target molecule; it may replace the antibody for UTSIA or UTSIA based assays; it

180 comprises nucleic acid polymer.

Assaying: as used herein, refers to qualitative or quantitative analysis of a substance. Blocking buffer: as used herein, is a buffer with blocking components which blocks a solid phase's non-specific adsorption to reduce the non-specific signals; the blocking components comprise BSA, Tween-20, and the like; usually the blocking buffer is used after

185 the immobilization of the adaptor molecule and/or the capture molecule on a capture solid phase, or the immobilization of analyte of interest on a detection solid phase; the blocking buffer is also used in steps of affinity binding such as the bindings between adaptor molecule (Protein G) and the capture molecule (antibody), the capture antibody and the antigen, the antigen and the detection antibody, the detection antibody and the labeled third anti-

190 detection- antibody, and the like; the blocking buffer is capable to block nonspecific binding reaction of solid phases to reduce background noise signals; the blocking buffer can't be used before and during the steps of physical adsorption on a solid phase, because the blocking buffer will block or reduce the adsorption of the molecule (adaptor molecule, capture molecule, or analyte) onto the solid phase; if the adaptor molecule immobilized on a

195 capture solid phase is Protein G or Protein A, then after binding with a capture antibody, the un-occupied Fc -binding sites of Protein G or Protein A is preferred to be blocked by a blocking buffer comprising serum, Fc-domains, or immunoglobulins.

Capture molecule: as used herein, is a molecule used to capture an analyte being assayed via a specific affinity binding reaction between the capture molecule and the analyte

200 in a liquid phase or on a solid phase; the capture molecule comprises antibody, recombinant antibody, protein, recombinant proteins, small or big organic molecules, and peptide or nucleic acid aptamers; if the capture molecule is an antibody, then it is named as capture antibody.

Capture system and capture solid phase: Capture system is the system of UTSIA to

205 capture analyte(s) from a fluid sample and the system comprises a capture solid phase which comprises a first solid phase; the capture solid phase refers to the solid phase immobilized with affinity binding molecule(s) for specific analyte capturing; the affinity binding molecules comprise adaptor molecule and / or capture molecule.

Cell-based ELISA: as used herein, is an immunoassay for detecting an antigen of

210 interest on cells immobilized on a solid phase; its major steps comprise growing the cells on a solid phase, then fixing the cells on the solid phase, penetrating the cells to expose antigen of interest, binding with a labeled detection antibody or an unlabeled detection antibody which recognizes the antigen of interest on the solid phase, finally detecting the antigen of interest on the solid phase by detecting the signal of the label on the detection antibody

215 bound on the solid phase; or detecting the unlabeled detection antibody by adding a labeled antibody against the unlabeled detection antibody and then detecting the signal from the label of the labeled antibody bound; the label comprises an enzyme. Cell-based UTSIA: as used herein, is a cell-based or tissue-based immunoassay for detecting an antigen of interest on cells or tissues immobilized on a solid phase; its function 220 is equivalent to the cell-based ELISA, but with less strict antibody requirement and has less problem of background noise signals, so it is much easier to set up than the cell-based ELISA.

Chromogen: as used herein, is a substance that lacks definite color but may be transformed into a pigment.

Coating: as used herein, is a physical adsorption process to immobilize substance on a 225 solid phase; in contrast to the affinity binding, the substance immobilized on a solid phase or a solid support by the coating or the physical adsorption process is nearly not dissociable from the solid phase by buffer with extreme pH.

Detection molecule: as used herein, is a molecule used to detect an analyte on a detection solid phase via a specific affinity binding reaction between a detection molecule 230 and the analyte; the detection molecule comprises antibody, recombinant antibody, protein, recombinant proteins, small or big organic molecules, and peptide or nucleic acid aptamers; if the detection molecule is an antibody, then it is named as detection antibody and its target analyte is an antigen.

Detection solid phase: as used herein, is the solid phase used for immobilizing analyte(s) 235 or antigen(s) dissociated from a capture solid phase; the detection solid phase comprises of hydrophobic surface such as polystyrene plate; the detection solid phase is also named as second solid phase.

Determining and determination: as used herein, determining refers to qualitative, quantitative, or qualitative and quantitative analysis of a substance; determination has same 240 meaning of the determining; assaying also has same meaning of the determining.

Dissociating buffer: as used herein, is a buffer comprising extreme low or high pH (e.g., less than 2.5), which is capable of eluting or dissociating analyte(s) bound on a capture solid phase into liquid phase of the dissociating buffer; the dissociating buffer can't contain blocking components which might interfere with the analyte' s coating process onto a 245 detection solid phase.

ELISA: as used herein, is an enzyme linked immuno-sorbent assay. Fc-domain: as used herein, is the protein or fragment of protein which can bind with Fc -binding protein by affinity binding.

Fc -binding protein: as used herein, is a protein which is capable of binding Fc-domain 250 protein, such as an antibody with a Fc-domain; the Fc-binding protein comprises protein-A, Protein G, protein or protein fragment derived from protein-A and/or Protein G, antibodies specifically targeting on Fc-domain of an antibody, and any other proteins or other molecules which can specifically bind with Fc-domain of a protein or an antibody; Fc-binding protein can be used as adaptor molecules in UTSIA if the capture molecule is a molecule with Fc- 255 domain.

First solid phase: as used herein, refers to the solid phase for making capture solid phase in UTSIA's capture system, which is usually immobilized with affinity molecule(s) for capturing an analyte or antigen from a fluid sample; in case of cell-based UTSIA it is the first solid phase for immobilizing the cells or tissues containing an analyte or antigen of interest, 260 but the solid phase does not capture an antigen from the sample (cells or tissue). Fluid sample: as used herein, fluid sample is a fluid that might contain an analyte (antigen), analytes (antigens), or analyte-analyte complex to be analyzed, comprising biological fluids, extracts or lysates of cells or tissues, biological samples such as blood, serum, plasma, cerebrospinal fluid, lymphatic fluid, semen, urine, sputum, synovial fluid,

265 saliva, lacrimal tears, nipple aspirate, and eye fluid; fluid samples may be of animal, plant, bacterial, viral, prion, or other life species; in this invention there are many descriptions such as the fluid sample, target sample, sample of interest, and the sample; all of them have same or similar meaning as the fluid sample; for analysis, if a sample is not a fluid, such as the tissue and the organ, then the sample can be changed to fluid sample by adding suitable

270 buffer and elements such as proteinase inhibitors and by processing with suitable procedures such as vortex and ultrasonic disruption under proper temperature and other proper conditions; the control sample is that does not contain the analyte of interest, it is equal to the negative control sample; for UTSIA, side by side to run a sample and a control sample can make sure that the detection of an analyte is not a false positive result.

275 Hole punch: as used herein, is a cutting instrument to make sections with defined area and defined shape.

HRP: as used herein, is horseradish peroxidase; it is tagged on an antibody for signal production and detection; it is an enzyme that needs a substrate such as TMB to produce a colorful product for the signal detection.

280 IHC: as used herein, is immunohistochemistry.

ICC: as used herein, is immunocytochemistry.

Immuno-complex: as used herein, is the complex of antigen and antibody formed by specific antigen antibody affinity binding.

Immobilize, immobilizing, and immobilization: as used herein, is a process or an

285 action of fixing a substance onto a part of solid phase which comprising surface of the solid phase; the process comprises physical adsorption (coating), affinity binding, or chemical covalent attachment.

IP: as used herein, is a method of using a solid phase immobilized with an Fc -binding protein to precipitate or capture an immuno-complex of antibody and antigen from a fluid

290 sample; the antigen obtained from the IP is usually further processed and detected by SDS- PAGE and Western Blot; IP is the abbreviation of immunoprecipitation.

Separated defined region: as used herein, refers to a region with border, so different regions are separated with each other; for example, the well in 96 well polystyrene plate for ELISA is a separated defined region.

295 Label: as used herein, is a molecule with a physical property or biochemical activity that is analyzable by a detector via the label's physical property or the label's catalyzed activity; it is usually tagged on a detection molecule or a third antibody against an unlabeled detection molecule for an analyte detection and quantification; the label comprises dye, fluorescent tag, radioactive isotope, or enzyme; the signal produced from the label itself or

300 mediated by itself, can be fluorescent, chemiluminescent, light- scatting, nano-crystalline, colorimetric, or radioactive, or any combination thereof; in special case, an unlabeled detection molecule bound on an analyte on a detection solid phase can be detected by an instrument, and the unlabeled detection molecule is acted as a detection molecule and a label molecule simultaneously. 305 Labeled: as used herein, it refers that a substance is tagged with a label.

Liquid phase: as used herein, comprises the phase of a fluid sample, a buffer, or a solution; it has same meaning of fluid, solution, and fluid phase; and the components of liquid phase comprises water.

Microarray or array: as used herein, is a solid phase containing multiple numbers of 310 immobilized probe elements, in a linear or two-dimensional array of discrete regions, each is in a finite area; this may also be referred to as antibody array or microarray, protein-array or microarray, antibody chip, or protein chip; the goal of antibody microarray is to detect multiple analytes in a fluid sample in a same time manner.

Neutralizing buffer: as used herein, is a buffer with opposite extreme high or low pH of 315 a dissociating buffer; the neutralizing buffer neutralizes the dissociating buffer's extreme pH and brings back the pH to a level that is suitable for an analyte' s adsorption on a detection solid phase; the neutralizing buffer can't contain blocking components which might interfere with the analyte' s coating process on the detection solid phase.

Physical adsorption and coating process: as used herein, is a physical process of 320 immobilizing a molecule on a solid phase without involving chemical covalent coupling reactions; usually the solid phase used for coating or physical adsorption is a hydrophobic surface such as polystyrene plate, and the molecule for coating is a macromolecule containing hydrophobic residues; in the buffer condition without blocking elements such as BSA, tween-20, or serum, the macromolecule is adsorbed onto the surface of the 325 hydrophobic solid phase; as it is more convenient to immobilize the adaptor molecule (e.g., Protein G) or capture molecules (e.g., antibody) on the first solid phase by simple physical adsorption, and more convenient to immobilize the analyte on the detection solid phase by simple physical adsorption, it prefers that there are hydrophobic residues in the molecules of adaptor molecule, capture molecule, and analyte; usually the coated molecules (such as 330 Protein G, antibody, or analyte) on the hydrophobic solid phase are not dissociated from the solid phase during the assay process, or the dissociation is negligible experimentally.

Primary antibody and secondary antibody: as used herein, primary antibody is the antibody used directly to bind an antigen being assayed, it is same as the capture antibody; secondary antibody is the antibody used to bind an antigen for its detection, it is same as the 335 detection antibody.

Protein or polypeptides or peptides: as used herein, are molecules which comprise multiple amino acid residues linked via peptide bonds.

Quantitate: as used herein, is to determine the amount(s) or the concentration(s) of analyte(s) of interest in a fluid sample; it has same meaning of quantify; its noun is 340 quantitation.

Quantification: as used herein, is the noun of measuring the amount or concentration of a substance; it has same meaning of quantitation.

(s): as used herein, refers single or multiple; for example, molecule(s) refer to a molecule or many molecules, antigen(s) refer to an antigen or many antigens, analyte(s) 345 refers to an analyte or many analytes, site(s) refer to one site or many sites, biomarker(s) refer to a biomarker or many biomarkers, phase(s) refer to a phase or many phases, compound(s) refer to a compound or many compounds, pool(s) refer to a pool or many pools, library(s) refer to a library or many libraries, and candidate(s) refer to a candidate or many candidates, proteins refer to one protein or many proteins.

350 Sample: for cell-based UTSIA, the sample is cells or tissue; it may have other meanings that are decided by the paragraphs that use the word of sample.

Sandwich-ELISA: as used herein, is an ELISA method in which both a capture antibody and a detection antibody bind to an antigen being assayed to form a sandwich-like complex on a solid phase for the antigen analysis; the sandwich-ELISA is especially useful in 355 the detection and quantization of the low abundance proteins in a fluid sample.

Second solid phase: as used herein, is the detection solid phase in UTSIA for coating the analyte(s) of interest; but for cell-based UTSIA, the second solid phase is the solid phase for coating detection antibody dissociated in the liquid phase of the dissociating buffer.

Section: as used herein, is a solid support immobilized with a slice of tissue; the solid 360 support comprises polystyrene sheet as it is easy to be cut into small pierces with defined area for further tissue biomarker detections.

Sheet: as used herein, is 2-dimensional solid phase comprising membrane, leaf, surface, pane, or slide; wherein the 2-dimensional solid phase is suitable for immobilizing cells or tissue for IHC or ICC.

365 Solid phase: as used herein, the solid phase comprises plastic, glass, metal, or other insoluble materials containing a number of regions wherein adaptor molecule, capture molecule, analyte, detection molecule, or sample (antigen, protein, or other macromolecule having hydrophobic structure) can be immobilized on its surface, and the solid phase can be used to carry out the steps of UTSIA or cell-based UTSIA; the solid phase may have same 370 meaning as the solid surface and solid support.

Signal generating system: as used herein, is a system for detecting a substance of interest bound on second solid phase (it is also named as detection solid phase); the system comprises a labeled detection molecule against the substance, or an unlabeled detection molecule against the substance and a labeled molecule against the unlabeled detection 375 molecule.

TMB solution: as used herein, is the chromogenic reagent for peroxidase, designed for ELISA and UTSIA; it contains 3,3',5,5'-tetramethylbenzidine (TMB), hydrogen peroxide (H₂O₂), and other stabilizing agents.

Un-complexed: as used herein, is equal to the meaning of not binding with each other 380 by affinity binding( s) .

UTSIA: as used herein, is the abbreviation of universal tandem solid-phases based immunoassay; UTSIA is a novel two solid-phases based analyte or antigen capture and detection system to replace sandwich-ELISA' s sandwich complex based antigen capture and detection; UTSIA can be used in detection and quantification of low abundance proteins, 385 analysis of protein-protein interactions, cell-based or tissue-based antigen determination, and many other applications. UTSIA is sound like "You Cheer!" or "Cheer!" DESCRIPTION OF THE FIGURES

Fig. IA. Analyte Capture and Detection Mechanism in UTSIA: Ia, incubating a fluid

395 sample with a capture solid phase (the solid phase immobilized with a capture molecule) to immobilize an analyte on the capture solid phase; Ib, pre-incubating a fluid sample with a capture molecule to form a complex of the analyte and the capture molecule, then contacting the complex with a capture solid phase (the solid phase immobilized with an adaptor molecule) to immobilize the analyte; Ic, incubating a fluid sample with a capture solid phase

400 pre-immobilized with the complex of an adaptor molecule and a capture molecule to immobilize the analyte on the capture solid phase; 2a, adding a dissociating buffer to the capture solid phase to release the analyte to liquid phase and then transferring the liquid phase containing the analyte to a detection solid phase; 2b, adding a dissociating buffer to the capture solid phase to release the analyte to liquid phase and then transferring the liquid

405 phase containing the analyte to a detection solid phase; 3, immobilizing the analyte to the detection solid phase by physical adsorption; 4a, adding a labeled detection molecule to recognize the analyte coated on the detection solid phase for specific detection and quantification of the analyte; 4b, adding a unlabeled detection molecule to recognize the analyte coated on the detection solid phase; 5, then adding a labeled molecule against the

410 unlabeled detection molecule for the detection and quantification of the analyte. Fig. IB: Keys for fig. IA.

Fig. 2. Specific Detection and Quantification of Target Antigen (rabbit IgG) by Universal Tandem Solid-phases Based Immunoassay: a prototype example of UTSIA, which demonstrates that the UTSIA specifically detects and quantitates a target analyte (rabbit IgG),

415 but not a control analyte (mouse IgG), in concentration dependent manner.

Fig. 3 A. Antigen Detecting Mechanism of Cell-based UTSIA: 1. a first solid phase immobilized with cells or tissue is incubated with a detection antibody against an antigen of interest to bind the detection antibody on the first solid phase; 2. then the unbound detection antibody is removed and the first solid phase is incubated with a dissociating buffer (with

420 extreme pH) to release the detection antibody bound on the solid phase into a liquid phase of the dissociating buffer; then the liquid phase containing the detection antibody dissociated from the first solid phase is transferred to a second solid phase which may pre-contain a neutralizing buffer (with opposite pH of the dissociating buffer, the mixture of the dissociating buffer and the neutralizing buffer will get a pH optimal for coating the detection

425 antibody on the second solid phase) to coat the detection antibody on the second solid phase; 3. finally the determination of the antigen of interest in the cell or tissue immobilized on the first solid phase is decided by detecting the detection antibody coated on the second solid phase via using a labeled antibody against the detection body; the labeled antibody is incubated with the second solid phase for proper time, then the unbound is removed, and then

430 the bound labeled antibody is used for signal detection; the signal from the labeled antibody on the second solid phase is correlated with the presence and the amount of the antigen of interest in the cells or tissue immobilized on the first solid phase. Please note: if the detection antibody is labeled with a detectable tag, then it can be detected directly on the second solid phase without the step 3 of fig. 3A.

435 Fig. 3B. Keys for Fig. 3A. DETAIL DESCRIPTION OF THE INVENTION

(I) General Description

440 Immunoprecipitation (IP) is a simple method used to isolate antigen in protein chemistry (Qoronfleh et al 2003), Its principle is that, an antibody (capture antibody) against an antigen is allowed to form an immuno-complex with the antigen in a fluid sample, such as cell lysate. The immuno-complex is then captured on a solid phase to which either Protein A or Protein G has been immobilized. This process of capturing the immuno-complex to the

445 solid phase from a solution is referred as precipitation. Any proteins or substances that do not precipitated by Protein G or Protein A on the solid phase are washed away. Finally the antigen of the bound immuno-complex are eluted from the solid phase and analyzed by SDS- PAGE as well as western blot technology.

Obviously IP and sandwich-ELISA have similarity, that is, both methods use a solid

450 phase to capture an antigen from a fluid sample. But, people familiar with the art of sandwich-ELISA and IP know that IP has better antigen capture system, because the solid phase of IP does not directly coated with the capture antibody (the capture molecule), but instead, using an adaptor molecule Protein G or Protein A immobilized on the solid phase to bind the capture antibody (the capture molecule). As Protein G or Protein A only binds to

455 the Fc-domain of the antibody, it does not interfere with the antigen binding domain, the Fab domain, of the antibody. Due to this special design, the solid phase of IP acts as not only an affinity binding solid phase to capture the antigen of interest but also a solid phase preventing the inactivation of the capture antibody's antigen binding capacity. That's why IP can be used for nearly all proteins' isolation if the proteins have specific antibodies which can be

460 used to pull down the proteins by a solid phase immobilized with Protein G or Protein A or other similar adaptor molecules.

Therefore, it's good idea to use IP's antigen capture system to replace sandwich- ELISA's antigen capture system. But in reality it is a difficult challenge, as Protein G or Protein A immobilized on a solid phase binds not only the capture antibody but also the

465 detection antibody. Thus, IP's antigen capturing system does not work in the method of sandwich-ELISA, as it produces false positive results. That is why sandwich-ELISA doesn't use IP's antigen capture system. Please note, in sandwich-ELISA, the capture antibody is physically adsorbed onto the solid phase (this is the coating process or non-affinity binding process). It is convenient to use physical adsorption (coating) to immobilize a capture

470 molecule (e.g., antibody) or an analyte (e.g., antigen) on the solid phase. The greatest advantage of the physical adsorption of antibody or antigen to a solid phase is that the coated molecules such as antibody and analyte on the solid phase are nearly not dissociable (US 5009998) and the process of the coating is very convenient. However, as the physical adsorption is a random immobilization process, most of the antibodies usually loss most of

475 their antigen binding capacities (78-82%) after coated on the solid phase (Subramanian, A. and Velander, W.H. 1996). In another experiment, Lu (1996) reported that the immobilization of antibody using Protein A resulted in increasing sensitivity 10 times higher than that of random immobilization. Why antibodies inactivated after adsorption on the solid phase? My opinion is that most of the antibodies used today are not screened and selected 480 from the antibodies that already adsorbed on the solid phase. In contradictory, the antibodies are usually screened from the plate coated with antigens. The three dimensional structures of these antibodies will change to a structures not optimal for antigen binding after coating on a solid phase and may lead to loss of antigen binding activity. Therefore, it is relatively easy to find antibodies that bind to an antigen coated on a solid phase, but it is difficult to find

485 antibodies that maintain the antigen binding activity after directly coated on a solid phase, In consequence, immobilizing an adaptor molecule such as Protein A or Protein G on a solid phase first, then using the adaptor molecule bound to bind a capture antibody in a liquid phase overcomes the technical challenge of solid phase inactivation of the capture antibody, and naturally this leads to find more antibodies available for capture of antigens from their

490 fluid samples to a solid phase.

Another technology to review is indirect ELISA, an immunoassay specific for an antibody detection (Voller et al 1978 and Lequin 2005). In this method, an antigen purified is directly immobilized onto a solid phase via physical adsorption. The target antibody in a fluid sample reacts with the antigen bound on the solid phase to form an immuno-complex,

495 and thus the target antibody is captured on the solid phase from a fluid sample. Then a labeled detection antibody with specificity for the target antibody is added and incubated. Following washing off unbound labeled detection antibody, the amount of the target antibody is measured by detecting the signal produced by the label of the bound detection antibody on the solid phase. Similar to sandwich-ELISA, indirect ELISA also has a useful modification,

500 the double indirect ELISA, which involves a third antibody. The third antibody carries a label and reacts with the unlabeled detection antibody already bound to the target antibody. After washing to remove unbound substances, the signal of the label is measured, and the signal of the label is directly correlated with the amount of the target antibody present in the fluid sample.

505 Unlike sandwich-ELISA, the indirect ELISA is not used to detect the presence of an antigen (Voller et al 1978). This is because that the antigen or protein in a sample such as cell lysate usually exists in a mixture of million of other proteins and other biomolecules such as DNA, RNA, lipids, and carbohydrates; in addition, the antigen usually exists in a very low concentration. Therefore, it is impossible to directly bind enough amount of the target

510 antigen in the sample onto the solid phase of indirect ELISA specifically for further detection. However, I contemplated that it is possible to detect the antigen of low abundance using indirect ELISA if the method is combining with the antigen capture steps of IP or IP-like method mentioned above. Actually and fortunately, the idea is really working. Specifically, the antigen separated from the IP or the IP like antigen capture system is dissociated and then

515 coated onto the solid phase of indirect ELISA for further antigen detection. As the antigen separated from the IP antigen capture system is enriched and is relative pure, and in a water based buffer system without blocking agents such as Tween-20 and BSA, it can be adsorbed to a hydrophobic solid phase of indirect ELISA for further specific detection, This novel combination or integration of IP-like antigen capture system with indirect ELISA antigen

520 detection system forms the core elements of present invention UTSIA. It must point out, even though previously there are people trying to run ELISA such as sandwich-ELISA after getting an antigen from immunoprecipitation or affinity chromatography, they never integrated the two immunoassays of immunoprecitation and ELISA into a novel immunoassay like present invention UTSIA.

525 Obviously, to make the invention works well, it needs to design an efficient analyte

(antigen) capture solid phase system to obtain an analyte (antigen) from a fluid sample, an simple analyte (antigen) dissociating system to release the analyte (antigen) from the capture solid phase, and a detection solid phase to immobilize the released analyte (antigen), and a signal generating system to detect the presence and the amount of the analyte (antigen) bound

530 on the detection solid phase.

To capture an analyte (antigen) from a fluid sample, the best way is to immobilize the analyte on a solid phase via specific affinity binding reaction between the analyte and affinity binding molecule(s) (adaptor molecule, capture molecule, or complex of adaptor and capture molecules) attached on the solid phase. Therefore, the choices of solid phase and the affinity

535 binding molecules are critical for setting up the analyte (antigen) capture solid phase system (capture system).

Now let me talk about the solid phase for the capture system of present invention. The solid phase is insoluble in water based buffer or solution or samples, and stable in extreme low or high pH conditions. Also the solid phase has capacity to attract and immobilize the

540 capture affinity binding molecule directly, has chemical functional groups which can be used to couple the affinity molecules on the solid phase, or has combinations of these properties. Alternately, the solid phase can attain additional coating materials which can be used to physically adsorb or chemically couple the affinity binding molecules to immobilize them on the solid phase. The solid phase comprises plastic, derivative plastic, magnetic or non- 545 magnetic materials, glass, or silicon materials. The solid phase can be in any suitable shape such as plate, membrane, sheet, dipstick, bead, well, chip, and any proper configurations that known to those of ordinary skill in the art.

A preferred embodiment of the solid phase for the capture solid phase comprises polystyrene 96 well plate. The polystyrene plate has hydrophobic surface which is ideal for

550 protein adsorption or coating. Usually the hydrophobic adsorption is the most convenient method to immobilize an affinity binding molecule on a solid phase comparing with other methods such as the covalent chemical coupling. Of course the hydrophobic surface like polystyrene can be further modified by radiation or other techniques that alter the chemistry of the surface, such as adding hydrophilic groups (carboxyl and amine groups) onto the

555 hydrophobic surface that may optimize the process of physical adsorption or coating of biomolecules such as protein onto the solid phase surface, or adding other reactive groups that can be used for the covalent immobilization of biomolecules onto the surface of the solid phase.

One embodiment of the affinity binding molecule on the capture solid phase comprises

560 a capture molecule or capture antibody. The capture molecule is coated on a hydrophobic polystyrene solid phase by physical adsorption or coating process. Before and during the coating process, the buffer containing the capture molecule should avoid blocking components such as Tween-20, BSA, and serum, because these blocking components will reduce or prevent the adsorption of the capture molecule on the solid phase. After the solid

565 phase immobilized with the capture molecule, it should be blocked by a buffer containing blocking components such as Tween-20 and BSA to reduce nonspecific binding in late experimental procedures. The advantage of coating the capture molecule on the solid phase is that it is the simplest analyte (antigen) capture system. The disadvantage of the coating is that it usually inactivates the capture molecule or capture antibody by the solid phase during

570 the adsorption or coating process.

In another embodiment of the affinity binding molecule on the capture solid phase comprises an adaptor molecule, The adaptor molecule is immobilized on a solid phase by physical adsorption (coating process), or covalently coupling reaction. The function of the adaptor molecule is acting as an intermediate molecule between the solid phase and the

575 capture molecule to prevent the inactivation of the capture molecule by the solid phase.

Therefore, at least one part of the adaptor molecule is capable of immobilizing onto the solid phase by physical adsorption or by chemical coupling reactions, and at least part of the adaptor molecule can bind with the capture molecule by affinity binding reaction. The solid phase comprises hydrophobic solid support such as polystyrene plate. Before and during the

580 coating process to immobilize the adaptor molecule on the solid phase, the buffer containing the adaptor molecule should avoid blocking components such as Tween-20, BSA, and serum, because these blocking components reduce or prevent the adsorption of the adaptor molecule onto the solid phase. However, after the solid phase immobilized with the adaptor molecule, it should be blocked by a buffer containing blocking components such as Tween-20 and BSA

585 to reduce nonspecific binding in late experimental procedures. The advantage of coating the adaptor molecule on the solid phase is that it prevents the inactivation of the capture molecule on the solid phase by providing proper orientation of the capture molecule or capture antibody on the solid phase. The alternate method of physical adsorption to immobilize an adaptor molecule to a solid phase is covalent coupling reaction. The method

590 is generally more complicated, and therefore, is not preferred for current invention if the physical adsorption working well. There are many publications that describe the chemical coupling reactions to couple affinity binding molecules to a solid phase, the papers to begin with are by Nisnevitch et al 2001 and by Qoronnfleh et al 2003. Alternately, there are commercial solid phase immobilized with adaptor molecule such as Protein G or Protein A/G

595 available for ordering, which may cost more from your budget; examples of these commercial solid phases are Reacti-BindTM Protein G coated 96 well strip plate and Reacti- BindTM Protein A/G coated 96 well strip plate from Pierce Biotechnology, Inc, Rockford, IL, USA.

Even though the adaptor molecule is critical for present invention, there are only two

600 ways available to find the adaptor molecules. One is to screen naturally available proteins such as Protein A, Protein G, Protein A/G, and protein L on a solid phase and test whether they can protect the capture antibody from inactivation; another is to make the adaptor molecule by recombinant molecular cloning technology or other technologies. Tanaka G et al (2006) reported that a recombinant Fc -binding protein (E72G3) with self-adhering ability on

605 hydrophobic solid phase is produced by linking the Fc -binding domain of Protein G (G3) and hydrophobic domain of elastin (E72) by a recombinant molecular cloning technology; the E72G3 retains the antibody binding activity after coating on a hydrophobic solid phase. Therefore, the E72G3 and E72G3 like recombinant proteins can be tested to use as the adaptor molecule for current invention. 610 After immobilizing the adaptor molecule on the solid phase and blocking the solid phase with proper blocking buffer (i.e., if the adaptor molecule is a protein G, then the blocking buffer can't contain the substance with Fc domain such as serum.), there are three ways to capture an analyte (antigen) from a fluid sample. One way is that a capture molecule reacts with the analyte in the fluid sample first to form a complex of the capture molecule and

615 the analyte in liquid phase; then the liquid phase containing the complex is incubated with the solid phase immobilized with the adaptor molecule to bind the complex via affinity binding interaction between the adaptor molecule and the capture molecule of the complex. This way is especially useful when the fluid sample doesn't contain substances that may interfere with the affinity binding reaction between the adaptor molecule and the capture molecule. For

620 example, if the adaptor molecule is Protein G and the capture molecule is a capture antibody, then a fluid sample such as cell lysate is mixed with the capture antibody first to form an immuno-complex of the antigen and the capture antibody. Because the cell lysate doesn't contain the antibody-like molecules that may directly binds with Protein G, the immuno- complex will efficiently bind to Protein G immobilized on the solid phase. As the interaction

625 between the immuno-complex and Protein G is an affinity binding, it's ideal to including blocking components Tween-20 and/or BSA in the affinity binding process to reduce nonspecific and non-affinity bindings. However, the blocking components such as serum can't be used in this step, because it contains immunoglobulin which will block the affinity binding between the immuno-complex and Protein G. The maximum amount of the capture molecule

630 added to the fluid sample is that the maximum amount that can be bound by the adaptor molecule immobilized on the solid phase. Another way is that a capture molecule is incubated with the solid phase immobilized with the adaptor molecule first to bind the capture molecule on the solid phase. After binding the capture molecule to the adaptor molecule immobilized on the solid phase, the solid phase is blocked with a blocking buffer

635 comprising components such as Tween-20 and BSA. If the adaptor molecule is Protein G and the capture molecule is a capture antibody, then the solid phase immobilized with the complex of Protein G and the capture antibody, the antibody immobilized is ready to capture the antigen in a fluid sample without the immunoglobulin-like molecules (the molecule containing Fc-domain) which will bind with Protein G. But if the antigen of interest is an

640 IgG, then the solid phase immobilized with the complex of Protein G and the capture antibody is not ready to capture the antigen yet. The solid phase must be further blocked with a blocking buffer which contains serum / immunoglobulins to completely block the unoccupied Fc -binding sites of Protein G on the solid phase to prevent un- wanted binding reactions. It is important to select blocking serum / immunoglobulins (for blocking the

645 capture solid phase) from an animal species that will not interfere with the late detection steps. For example, if the detection molecule is an unlabeled polyclonal antibody from rabbit, then the blocking serum / immunoglobulins should not get from rabbits, because it may interfere with the interaction between the anti-rabbit-HRP and the rabbit polyclonal detection antibody. At the moment, the solid phase immobilized with the complex of Protein G and

650 the capture antibody, after being blocked with proper serum/immunoglobulin, is ready to capture the antigen in the fluid samples, even though the antigen may be IgG like molecules. The third way is that a capture molecule, a fluid sample, and a solid phase immobilized with an adaptor molecule are mixed together to form a complex of analyte-capture molecule - adaptor molecule- solid phase to capture the analyte from the fluid sample. Similar to the first

655 way, the fluid sample should not contain substances that interfere with the affinity binding reaction between the adaptor molecule and the capture molecule. For example, if the adaptor molecule is Protein G and the capture molecule is a capture antibody, then a fluid sample (like cell lysate) is mixed with the capture antibody and the solid phase to form an immuno- complex of the antigen, the capture antibody, and Protein G on the solid phase, Because the

660 cell lysate doesn't contain the antibody-like molecules that may directly binds with Protein G, the complex of analyte-capture molecule- adaptor molecule will be efficiently formed on the solid phase. As the interactions among the components of the complex are affinity bindings, it's ideal to including blocking components Tween-20 and/or BSA to reduce non-specific and no-affinity bindings. However, the blocking component such as serum/immunoglobulins

665 can't be used in this step if the adaptor molecule is a Fc -binding protein such as Protein G, because it contains immunoglobulins which will block the affinity binding between the capture antibody and Protein G, thus, it inhibits the capture of the analyte from the fluid sample.

Now the analyte (antigen) is on the solid phase of the capture systems. As mentioned

670 previously, the direct detection of the analyte with a detection antibody on the capture solid phase is the technique of the sandwich-ELIS A, which is difficult due to the difficulty in finding a perfectly matched pair of the capture antibody and the detection antibody to form a sandwich complex. Therefore, I am not using the sandwich-ELIS A to continue the analyte detection; instead, I dissociate the analyte from the capture solid phase by using a

675 dissociating buffer to release the analyte bound, and then transfer the analyte and adsorb the analyte to another solid phase for further detection. The detail procedure is described further in following paragraphs.

After the analyte (antigen) is immobilized on the capture solid phase, the solid phase is washed by PBS or other water based buffers (the buffers do not contain blocking components

680 such as Tween-20, BSA, and serum) to remove unbound substances and to clean unbound blocking components such as BSA and Tween-20. Then the solid phase is incubated with a dissociating buffer to release the antigen into the liquid phase of the dissociating buffer from the solid phase. The dissociating buffer comprises a buffer with extreme pH (e.g., 0.2 M Glycine pH 2.5), which is capable of separating the analyte (antigen) bound on the solid

685 phase into the liquid phase of the dissociating buffer. The mechanism of the dissociation may be that the extreme pH will redistribute the charges of hydrophilic functional groups of amino acid residues in the analyte and in the capture molecule, and this may interrupt the affinity bindings between the capture molecule and the analyte; and therefore, the analyte is released into the liquid phase of the dissociating buffer. Please note, as the analyte

690 dissociated from the solid phase needs to be further coated onto the hydrophobic detection solid phase for further detection, the dissociating buffer can't contain the blocking components which might interfere with the antigen's coating process, and these blocking components comprise BSA, serum, tween-20, and the like. There may be other mechanisms besides extreme pH that may be helpful to release the analyte bound into the liquid phase,

695 these include salt concentrations, organic solvents, and detergents. Even though present inventor has not tested whether these mechanisms are compatible with present invention UTSIA, the obvious disadvantages of some of the mechanisms such as organic solvents and detergents are that they are not easy to be neutralized and, in addition, some of them may disrupt hydrophobic bindings (physical adsorption) which is critical for present invention.

700 Therefore, their applications in UTSIA should be further cautiously investigated.

Then the dissociating buffer containing the analyte (antigen) is transferred to a detection solid phase which contains a neutralizing buffer (e.g., IM Tris/HCl pH 9.0) with opposite extreme pH of the dissociating buffer. The mixing of the dissociating buffer and the neutralizing buffer neutralizes the dissociating buffer's extreme pH and brings the pH to a

705 level that is suitable for the antigen' s adsorption onto the detection solid phase, and the pH is suitable for the late binding steps for the detection of the analyte on the detection solid phase. This can be achieved by using different ratio of the dissociating buffer to the neutralizing buffer to see which combination results in maximum analyte (antigen) coating on the solid phase. The neutralizing buffer also can't contain blocking components which might interfere

710 with the antigen's coating process on the detection solid phase, and the blocking components comprise BSA, serum, tween-20, and the like. The detection solid phase comprises a solid phase for immobilizing the analyte (antigen) dissociated in the dissociating buffer, it comprises hydrophobic solid phase such as 96 well polystyrene plates (the hydrophobic surface like polystyrene can be further modified by radiation or other techniques that alter the

715 chemistry of the surface such as adding hydrophilic groups of carboxyl and amine groups onto the hydrophobic surface, that may optimize the process of physical adsorption or coating of biomolecules such as protein onto the detection solid phase surface). The detection solid phase does not require the affinity binding molecule(s) immobilized for the capture of the analyte from the liquid phase, so it is different from the capture solid phase, which

720 requires at least an affinity binding molecule (such as the adaptor molecule or the capture antibody) for the capture of the analyte. The detection solid phase immobilizes the analyte by a simple physical adsorption or coating process preferably. Before and during the adsorption or coating process, the detection solid phase can't be incubated with substances that have blocking capacity, such as Tween-20, BSA, and serum, as these substances block

725 the sites of the solid phase that are critical for the analyte' s adsorption or coating. As mentioned earlier, the dissociating buffer containing the analyte and the neutralizing buffer in the detection solid phase do not contain these blocking substances. In addition, pH of buffer may influence the coating of analyte such as protein on the detection solid phase significantly; it is reported that pH 9.6 of carbonate coating buffer is ideal for protein coating on the

730 radiation modified polystyrene plate, even though other pH ranges such as neutral pH (7-7.5) the protein also can be coated onto the solid phase. These special buffer conditions promote the physical adsorption of the analyte onto the detection solid phase. Therefore, after proper time of incubation in these conditions, the analyte dissociated in the liquid phase is immobilized onto the detection solid phase for further specific detection.

735 You may ask why the analyte dissociated in liquid phase does not coat back to the capture solid phase, but it is coated onto the detection solid phase? It is because that the capture solid phase was blocked previously and there is no way to coat the analyte back on the capture solid phase by physical adsorption, while the detection solid phase is not blocked and its hydrophobic surface is easily to be coated with analytes (like proteins) which also

740 have hydrophobic residues in their molecules. In addition to above reason, the capture solid phase immobilizes the analyte by affinity binding, but the detection solid phase immobilizes the analyte by physical adsorption (coating process); under extreme pH of the dissociating buffer, the analyte does not bind with the affinity molecule immobilized on the capture solid phase.

745 In case there are other molecules co-precipitated (co-capture) with the analyte by the capture system, then these molecules, if they are complexed with the analyte by affinity binding, they are also released to the liquid phase of the dissociating buffer with the analyte; and the molecules are also coated to the detection solid phase with the analyte. These molecules can be detected on the detection solid phase by their specific detection molecules.

750 If there are multiple molecules need to be coated on a detection solid phase, then a suitable area of the detection solid phase for proper adsorption need to be experimentally optimized. You can image, if the area is too narrow, it may be only part of the molecules can be coated on the solid phase; the coated part of the molecules may be not optimal enough for further detection.

755 After the immobilization of the analyte on the detection solid phase, the next step is to remove the unbound substances and then washing the detection solid phase with PBS or other water-based buffers, and to block the detection solid phase by using blocking buffer comprising blocking components such as Tween-20, BSA, and serum. It is important to select blocking serum from an animal species that will not interfere with the detection steps.

760 For example, if the detection molecule is an un-labeled antibody from rabbit, then the blocking serum / immunoglobulins should not get from the rabbit. After blocking, the analyte on the detection solid phase is detected by a signal generating system. The signal generating system is selected from the group comprising a labeled detection antibody or an unlabeled detection antibody and a labeled antibody against the unlabeled detection antibody.

765 If the signal generating system comprises a labeled detection antibody, the analyte immobilized on the detection solid phase is incubated with a solution containing the labeled detection antibody and the blocking components such as BSA. serum, and Tween-20, to let the labeled detection antibody specifically binds on the antigen coated on the detection solid phase. After removing and washing to remove unbound substances from the detection solid

770 phase, the signal from the label on the detection antibody bound on the detection solid phase is detected by an instrument or other suitable means. The label is selected from the group comprising enzyme, chromogens, luminescent compounds, chemiluminescent compounds, radioactive elements, and direct visual labels such as colored microparticles; the label is capable of producing a signal for detection either by itself or in conjunction with one or more

775 additional substances. For example, if the label is enzyme HRP, then the signal is produced by adding its substrate TMB and other substances required for the enzyme reaction under proper time and temperature to form a blue color product, and then the intensity of the color product (thus, the signal of the label) is measured by a spectrometry via detecting its absorbance at 650-655nm wavelength. The signal is correlated with the presence of the

780 analyte. To determine the amount or the concentration of the analyte, a standard curve of concentrations or amounts vs absorbances is produced by measuring absorbances produced from serial different concentrations or amounts of the analyte on the assay. Comparing with the standard curve, then the concentration or the amount of the analyte in a fluid sample is decided. If the signal generating system comprises an unlabeled detection antibody and a

785 labeled antibody against the unlabeled detection antibody, the analyte immobilized on the detection solid phase is incubated with a solution containing the unlabeled detection antibody and blocking components such as BSA and Tween-20, to let the unlabeled detection antibody specifically binding on the analyte coated on the detection solid phase. After removing and washing to remove unbound substance from the detection solid phase, the labeled antibody

790 against the unlabeled detection antibody is incubated with the solid phase in the presence of blocking components such as Tween-20, BSA, and serum to let the labeled antibody specifically binding on the unlabeled detection antibody bound on the detection solid phase. After removing and washing to remove unbound substance from the detection solid phase, the labeled antibody immobilized on the solid phase is detected by a detector or other means

795 via the label's physical property or the label's catalyzed activity. The label is selected from the group comprising enzyme, chromogen, luminescent compound, chemiluminescent compound, radioactive element, and direct visual label such as colored micro-particle; the label is capable of producing a signal for detection either by itself or in conjunction with one or more additional substances. To determine the amount or the concentration of the analyte,

800 a standard curve of concentrations or amounts vs absorbances is produced by measuring absorbances produced from serial different concentrations or amounts of the analyte on the assay. Comparing with the standard curve, then the concentration or the amount of the analyte in a fluid sample is decided. The advantage of the signal generating system comprising an unlabeled detection antibody and a labeled antibody against the unlabeled

805 detection antibody is that it provides a universal detecting platform for detection of multiple different analytes in multiplex immunoassay or in microarray immunoassay. For example, multiple different antigens immobilized on different spots of a detection solid phase can be detected by their corresponding different detection antibodies from a same animal species, and then the unlabeled detection antibodies are detected by a common labeled antibody

810 specifically against these unlabeled detection antibodies. For example, if these unlabeled detection antibodies are monoclonal mouse IgGs, then a common HRP-labeled anti-mouse IgG can be used to detect these monoclonal IgG antibodies.

In case the capture molecule is not very specific to the analyte that leads to the capture of un-wanted cross-reacting substances, the situation can be improved by additional loading

815 the neutralized dissociating buffer which contains the analyte and the unwanted cross- reacting substances to a new capture system which uses another capture molecule against the target analyte, then followed by dissociating, transferring, neutralizing, coating, and detecting. Repeating the capture steps may provide much clean enrichment of the target analyte from a fluid samples, that is, increasing specificity of the assay by repeating the capture steps.

820 In case the analyte level in the sample is very low, such as in pg level, its detection on the detection solid phase can be carried out by the technology of high sensitivity detection system which is disclosed in US patent 5731158. Basically, the HRP-detection antibody or HRP-antibody against the unlabeled detection antibody on the detection solid phase is reacted with H2O2 and biotinyl-tyramide to covalently attach many biotin residues on the

825 detection solid phase, and then using streptavidin-HRP to bind with the biotins on the solid phase, and then reacts with HRP substrate TMB to amplify the detection signal. Currently there is commercial high sensitivity detection kit available.

Now it is clear that UTSIA is a novel and convenient immunoassay which overcomes the technical limitations of sandwich-ELISA in the capture and detection of an analyte 830 (antigen) from a fluid sample. The UTSIA is not only a sandwich-ELISA equivalent immunoassay but also an immunoassay with more flexible powers and wider applications. You may wonder whether the UTSIA quantification can be used in the situation that the antigen of interest is located in the cells or tissue immobilized on a solid phase, such as IHC and ICC. The answer is yes. In IHC and ICC, there are many other substances immobilized

835 on the solid phase, these substances on the solid phase are not only interfering with the detection of the antigen of the interest, but also diluting the antigen of interest in a relative low density that is usually difficult to be directly measured by enzyme based assay such as cell-based ELISA. Therefore, currently routine quantitative method of the antigen in IHC and ICC is by imaging method (usually it is fluorescence based image method) that involving

840 the image capture and image analysis (Warford et al 2004 and Kaczmarek E et al 2004), As the imaging method is a fluorescent based method and not a direct antigen quantification method like cell-based ELISA, it is not robust, and the quantification data usually have big variations. Even though the cell-based ELISA is a very nice cells-based antigen quantification method, it is very difficult to set up for routine applications, as it needs a very

845 high quality antibody for a specific antigen and needs high expression of the antigen of interest in the cells (Versteeg et al 2000, Yang et al 2006). This bottleneck problem in quantitative IHC and ICC is solved by cell-based UTSIA, a variant technology of UTSIA. Specifically, first using an detection antibody binding on the antigen of interest in the cells or tissue immobilized on a first solid phase, then using a dissociating buffer to release the

850 detection antibody bound into a liquid phase of the dissociating buffer, then coating the detection antibody dissociated in the liquid phase onto a second solid phase; finally, an labeled antibody against the detection antibody is used to detect the detection antibody coated on the second solid phase. The presence and the amount of the antigen of interest on the first solid phase is directly correlated with the detection antibody coated on the second solid phase,

855 and thus directly correlated with the signal from the label of the labeled antibody bound on the second solid phase. Therefore, the antigen of interest on the cells or tissue immobilized on the first solid phase is detected by measuring the presence and the amount of the detection antibody coated on the second solid phase. The detail of the UTSIA based antigen detection and quantification in cells or tissue immobilized on a solid phase will be further described in

860 late paragraphs.

Followings are major applications of UTSIA in different fields. Please note, in following preferred embodiments of the invention, some detail steps or processes that may be missed in their descriptions, such as blocking, dissociating, neutralizing, transferring, coating, washing, or detecting, it should refer back to above paragraphs of general description and

865 refer forward to prototype example for experimental guidance. As indicated before, they are offered by way of illustration of present invention, and not by way of limitation of present invention.

(II) The Preferred Embodiments of the Invention 870 1. Detecting and quantitating an analyte in a fluid sample (Fig. IA, Fig. IB, and Fig 2):

Like sandwich-ELISA, UTSIA can be used to detect and quantitate an analyte of interest in a fluid sample. As there are three different capture solid phases, there are several ways to capture and detect an analyte from a fluid sample. A. using a first solid phase immobilized with a capture molecule: the fluid sample is first incubated with the first solid

875 phase to let the analyte immobilized on the first solid phase via the affinity binding between the analyte and the capture molecule immobilized on the first solid phase; after removing and washing to get rid of the unbound substances, the first solid phase is incubated with a dissociating buffer to release the analyte into the liquid phase of the dissociating buffer; then transferring the dissociating buffer containing the analyte to a detection solid phase (second

880 solid phase) which may be pre-incubated with a neutralizing buffer to coat the analyte onto the detection solid phase; after removing and washing to remove the unbound buffer mixture, the detection solid phase is blocked, and then incubated with either a labeled detection molecule to detect the analyte bound via the label of the labeled detection molecule in the presence of blocking components, or an unlabeled detection molecule and a labeled

885 molecule against the unlabeled detection molecule to detect the analyte bound via the label of the labeled molecule in the presence of blocking components; the signal strength of the detection is correlated with the amount or concentration of the analyte in the fluid sample; B. using a first solid phase immobilized with an adaptor molecule: the fluid sample is first incubated with a capture molecule to form a complex of the analyte and the capture molecule

890 in a liquid phase, and then the complex in the liquid phase is incubated with the first solid phase in the presence of blocking components to immobilize the complex via the affinity binding between the capture molecule of the complex and the adaptor molecule immobilized on the first solid phase; after removing and washing to get rid of the unbound substances, the first solid phase is incubated with a dissociating buffer to release the analyte into the liquid

895 phase; then transferring the liquid phase of the dissociating buffer containing the analyte to a detection solid phase (second solid phase) (which may be pre-incubated with a neutralizing buffer) to coat the analyte onto the detection solid phase; after removing and washing to remove the unbound buffer mixture, the detection solid phase is blocked, and then incubated with either a labeled detection molecule to detect the analyte bound via the label of the

900 labeled detection molecule in the presence of blocking components, or an unlabeled detection molecule and a labeled molecule against the unlabeled detection molecule to detect the analyte bound via the label of the labeled molecule in the presence of blocking components; the signal strength of the detection is correlated with the amount or concentration of the analyte in the fluid sample; C. using a first solid phase immobilized with an complex of an

905 adaptor molecule and a capture molecule (the capture molecule is binding with the adaptor molecule pre-immobilized on the first solid phase): the fluid sample is first incubated with the first solid phase to obtain the analyte from the fluid sample in the presence of blocking components, and then after removing and washing to get rid of the unbound substances, the first solid phase is incubated with a dissociating buffer to release the analyte into the liquid

910 phase; then transferring the liquid phase of dissociating buffer containing the analyte to a detection solid phase (second solid phase) (which may be pre-incubated with a neutralizing buffer) to coat the analyte onto the detection solid phase; after removing and washing to remove the unbound buffer mixture, the detection solid phase is blocked, and then incubated with either a labeled detection molecule to detect the analyte bound on the detection solid

915 phase via the label of the labeled detection molecule in the presence of blocking components, or an unlabeled detection molecule and a labeled molecule against the unlabeled detection molecule to detect the analyte bound on the detection solid phase via the label of the labeled molecule in the presence of blocking components; the signal strength of the detection is correlated with the amount or concentration of the analyte in the fluid sample. 920

2. Detecting multiple analytes (UTSIA based antibody microarray):

As mentioned in the background of the invention, the antibody based microarray is a new concept that intents to detect multiple proteins in a same assay. However, except in rare case such as cytokines, it is suggested that it is nearly impossible to setting up an antibody-

925 based microarray to detect hundreds of proteins in same time and in same assay with current methods (Zangar et al 2006). It is because every protein has its unique amino sequence and unique 3-dimensional structure, and thus, it is thought that every protein will need a special set of assay conditions such as antibodies, buffer and incubation time for its detection. Indeed, until today the availability of commercial antibody-based microarray is limited, only

930 sandwich-based antibody microarray for the detection of a few proteins such as cytokines available (please note, in this case, the microarray is developed from commercial available sandwich-ELISA for cytokines analysis). Therefore, to make the antibody-based microarray for multiple proteins detection possible, an alternate capture and detection mechanism other than the sandwich-ELISA is required.

935 UTSIA, different from the sandwich-ELISA, has many universal properties that make the setting up of antibody-based microarray for multiple proteins detection practical. One is that an adaptor molecule such as Protein G on a capture solid phase prevents the capture antibody's inactivation by the solid phase; because the adaptor molecule provides the antibody immobilized on the solid phase in an oriented manner. Another is that UTSIA uses

940 two solid phases, a capture solid phase and a detection solid phase, to replace sandwich-

ELISA's requirement of a perfect pair of the capture antibody and the detection antibody for sandwich complex formation; the third is that UTSIA uses a dissociating buffer with extreme pH to disrupt all of the affinity bindings between the capture antibodies and the analytes of interest on the capture solid phase; the dissociating buffer dissociates the analytes from the

945 capture solid phase into liquid phases of the dissociating buffer; and then the analytes dissociated are transferred to another solid phase and are coated on the solid phase, the detection solid phase, for specific detection by detection antibodies (detection molecules) which are from a same animal species. Finally, the detection antibodies (detection molecules) bound on the detection solid phase are detected by a common labeled third antibody against

950 the detection antibodies (detection molecules) for signal measurement. Therefore, these universal steps of capture, dissociating, coating, and detection make the UTSIA realistic for multiple analytes detections, such as scaled up to microarray format to detect and quantitate multiple analytes in a same time and in a same assay.

UTSIA based antibody microarray comprises a capture solid phase immobilized with

955 an adaptor molecule such as Protein G, wherein the adaptor molecule is coated on discrete separated regions on the solid phase. To capture multiple analytes from a fluid sample, the discrete separated regions of the solid phase pre-coated with the adaptor molecule are incubated with fluid mixtures of the fluid sample and the different capture antibodies which are from a same animal species targeting on different analytes in the presence of blocking

960 components, wherein each discrete separated region of the solid phase pre-coated with the adaptor molecule is incubated with a mixture of the fluid sample and a capture antibody. The multiple analytes can be immobilized on the discrete separated regions via the affinity binding interactions between the capture antibodies in the fluid mixture and the adaptor molecule Protein G immobilized on the solid phase. After washing the solid phase to remove

965 unbound substances, a dissociating buffer is added to these different regions on the solid phase to elute these analytes into liquid phases, with each liquid phase contains an analyte released from a discrete region of the capture solid phase; and then the analytes in the liquid phases are transferred and coated to discrete regions of a detection solid phase (which may pre-contain neutralizing buffer on these regions). After washing and blocking, the coated

970 analytes on the discrete regions of the detection solid phase are then detected by different detection antibodies (each analyte in each region incubated with its corresponding detection antibody) in the presence of blocking components. All detection antibodies are from a same animal species and are different from the animal species that making the capture antibodies to avoid cross immuno-reactions. Then, proper amounts of a common labeled third antibody

975 against these different detection antibodies (such as anti-mouse IgG-HRP if the detection antibodies are mouse monoclonal antibodies) are added to these regions of the detection solid phase for the analytes' detections in the presence of blocking components. The strengths of the signals of the label on the discrete regions of the detection solid phase are correlated with the amount of these different analytes in the fluid sample.

980

3. Cell-based UTSIA (Fig. 3A and Fig. 3B):

As mentioned in background, there is an urgent need to develop better method for quantitative ICC or quantitative IHC than the score method and the imaging method.

The cell-based UTSIA is a solution to the urgent need. Instead of taking images from

985 the labeled antibody bound on tissue or cells immobilized on the solid phase of IHC or ICC to quantify the antigen of interest, cell-based UTSIA uses a dissociating buffer to release the detection antibody bound on cells or tissue immobilized on the solid phase (first solid phase) to a liquid phase of the dissociating buffer, then transfers the liquid phase to a second solid phase (that may contain a neutralizing buffer) to coat the detection antibody dissociated in

990 the liquid phase on the second solid phase; this way, the detection antibody originally bound on the cells or tissue of the first solid phase is partially purified from the background cells or tissue on the first solid phase and then is coated to the second solid phase which has no background cells and tissue immobilized, and this will increase final detection specificity; since the detection of the detection antibody on the second solid phase avoids most of non- 995 specific bindings with cells or tissue immobilized on the first solid phase. In addition, if the area of the solid phase (the first solid phase immobilized with cells or tissue) is bigger than area of the second solid phase (the solid phase for coating the detection antibody), the detection antibody is also enriched on the second solid phase and this increases final detection sensitivity (since the bigger area of the first solid phase, it will immobilize more 1000 cells, so it will have more antigen of interest, thus it will bind with more detection antibody; when the detection antibody is coated to a smaller area of a second solid phase, it is enriched). If the detection antibody is labeled with a detectable tag, it can be directly determined on the second solid phase; if the detection antibody is unlabeled, it can be indirectly determined on the second solid phase by using a labeled antibody against the detection antibody. Finally the 1005 determining antigen of interest is via detecting the signal of the bound labeled secondary antibody against the detection antibody coated on the second solid phase, and the signal strength of the label is directly correlated with the amount of the antigen of interest on the cells or tissue immobilized on the first solid phase.

Please note, on the first solid phase, before and during the binding of the detection

1010 antibody to its antigen, the blocking components should be selected from the one that without significant cross-reaction with the detection antibody in late detection step on the second solid phase. For example, if the detection antibody is a mouse monoclonal antibody, then the blocking serum should not get from mouse or other animal species that may have cross- reactions with the mouse monoclonal antibody on the second solid phase when detected by a

1015 labeled antibody against the mouse monoclonal detection antibody.

Followings are further guidance of cell-based UTSIA for your reference which comprises: growing cells in a 96 well tissue culture plate (first plate or first solid phase) until the cells are near completely confluent; removing the medium from the well; washing with PBS; fixing the cell with 4% paraformaldehyde solution for proper time; removing the

1020 paraformaldehyde and washing the fixed cells with PBS; permeabilizing the fixed cells with cold methanol or PBS with 0.5% Triton X-100; blocking the cells with blocking buffer which comprises Tween-20, BSA, and serum; the serum is not from the species from which the detection antibody was made and the serum is preferred from the species from which the secondary antibody was taken; incubating the cells with an detection antibody against an

1025 antigen of interest in a blocking buffer comprises Tween-20 and BSA; removing the unbound detection antibody and washing the cells with PBS; adding a dissociating buffer with extreme pH to release the detection antibody bound on the antigen of interest on the cells fixed in the well of 96 well plate into the liquid phase of the dissociating buffer; transferring the liquid phase to a well of another 96 well polystyrene plate (second plate or

1030 second solid phase) which pre-contains a neutralizing buffer with opposite pH of the dissociating buffer to make buffer mixture with pH optimal for the detection antibody to be detected in late steps of the assay; gentle rotating the plate to coat the detection antibody on the well of the plate; removing the unbound buffer mixture from the well and washing with PBS; blocking the well with a blocking buffer with Tween-20, BSA, and serum which will

1035 not interfere with the detection of the detection antibody; incubating the well with an antibody-HRP against the detection antibody bound in the well of the plate in the blocking buffer comprising Tween-20 and BSA; removing unbound antibody-HRP and washing the well with PBS; adding TMB substrate solution to the well of the second plate and incubating without light for proper time; the signal of the antigen of interest in the cells fixed on the first

1040 plate is determined by detecting the absorbance of the second plate by reading the OD650nm.

There is alternate way to do cell-based UTSIA, which comprises: growing cells in a 96 well tissue culture plate (first plate or first solid phase) until the cells are near confluent; removing the medium from the well; washing with PBS; fixing the cells with 4% paraformaldehyde solution for proper time; removing the paraformaldehyde and washing the

1045 fixed well with PBS; permeabilizing the fixed cells with cold methanol or PBS with 0.5% Triton X-100; blocking the cells with blocking buffer which contains Tween-20, BSA, and serum (the serum should be from an animal species that is different from the animal species making the detection antibody, to avoid non-specific cross reactions); incubating the cells with an labeled detection antibody against an antigen of interest in the blocking buffer; 1050 removing the unbound labeled detection antibody and washing the cells with PBS; then adding a dissociating buffer with extreme pH to release the labeled detection antibody bound on the antigen of interest on the cells fixed in the well of 96 well plate into the liquid phase of the dissociating buffer; transferring the liquid phase to a well of another 96 well polystyrene plate (second plate or second solid phase) which pre-contains a neutralizing buffer with

1055 opposite pH of the dissociating buffer to make buffer mixture with pH optimal for the labeled detection antibody to be detected; gentle rotating the plate to coat the labeled detection antibody on the well of the plate; removing the buffer mixture from the well and then washing with PBS; the signal of the antigen of interest in the cells fixed on the first plate is determined by detecting the presence and the amount of the labeled detection antibody coated

1060 on the second plate (second solid phase), Please note, the success of the alternate way of cell- based UTSIA depends on whether the label of the labeled detection antibody maintains its activity after the antibody coated on the second plate. However, if the activity of the label is lost during the coating process, the labeled detection antibody coated on the second plate still can be detected by using a labeled antibody against the inactivated labeled detection antibody.

1065 For suspension cells, they can be coated in the wells of a poly-L-lysine-coated polystyrene plate or glass slide, then follow by fixing the cell with 4% paraformaldehyde solution for proper time; removing the paraformaldehyde and washing the fixed well with PBS; permeabilizing the fixed cells with cold methanol or PBS with 0.5% Triton X-100; blocking the cells with blocking buffer which contains Tween -20, BSA, and serum; the

1070 serum is not from the species from which the detection antibody was taken and the serum is preferred from the species from which the labeled secondary antibody was taken; incubating the cells with an labeled detection antibody or unlabeled detection antibody against an antigen of interest in a blocking buffer contains Tween-20 and BSA; removing the unbound labeled detection antibody and washing the cells with PBS; then adding a dissociating buffer

1075 with extreme pH to release the labeled detection antibody or unlabeled detection antibody bound on the antigen of interest on the cells fixed in the well of the poly-L-lysine-coated plate into the liquid phase of the dissociating buffer; transferring the liquid phase to a well of another 96 well polystyrene plate (second plate or second solid phase) which pre-contains a neutralizing buffer with opposite pH of the dissociating buffer; gentle rotating the plate to

1080 coat the labeled detection antibody or unlabeled detection antibody on the well of the plate; removing the buffer mixture from the well and washing with PBS; then blocking the plate and detecting the labeled detection antibody coated on the second plate directly, or detecting the unlabeled detection antibody coated on the second plate by adding and incubating a labeled secondary antibody against the unlabeled detection antibody to determine the

1085 presence and amount of the antigen of interest in the cells fixed on the first plate.

For frozen tissue, mounting the frozen tissue block on a cryostat holder and cutting the tissue to 5μm thick slice at minus 18 to minus 2O⁰C; loading the tissue slice to a surface of polystyrene sheet that may be coated with poly-L- Lysine or other materials which promote tissue adhesion on the surface; letting the section dry for at 5- 30 minutes at room

1090 temperature; then cutting the surface immobilized with tissue to a round small section with defined area (first solid phase) such as with diameter of 4.5mm, 9mm, or 13.5mm which is capable to be put into the well of 96 well plate, 48 well plate, or 24 well plate (the cutting section to round small pierce with defined area can be done with a hole punch); then putting the section with defined area to a well of one of the plates (such as 4.5mm diameter tissue

1095 section to well of 96 well plate, or 13.5mm diameter section to well of 24 well plate) to fix the tissue with 2-4% paraformaldehyde 10-15 min (or with acetone for 2 min) (if the antigen expression is weak in the tissue, it prefers to use larger section with bigger diameter), washing with PBS to remove paraformaldehyde or acetone; permeabilizing the fixed tissue with cold methanol or PBS with 0.5% Triton X-IOO; washing the tissue with PBS 2 times;

1100 blocking the section with blocking buffer comprising Tween-20, BSA, and serum (the serum should be from an animal species that is different from the animal species making the detection antibody, to avoid non-specific cross reactions), then incubating the section with an detection antibody diluted in PBST with 2.5% serum (the serum should be from an animal species that is different from the animal species making the detection antibody, to avoid non-

1105 specific cross reactions) 30 min to 1 hour; removing and washing the section with PBS to get rid of unbound detection antibody; adding a dissociating buffer with extreme pH to release the detection antibody bound on the antigen of interest on the fixed tissue on the section in the well of the plate; then transferring the liquid phase containing the dissociated detection antibody to a well of another polystyrene plate (second plate or second solid phase) which

1110 pre-contains a neutralizing buffer with opposite pH of the dissociating buffer to make buffer mixture with pH optimal for the detection antibody to be detected in late steps of the assay; gentle rotating the plate for proper time to coat the detection antibody on the well of the second plate; removing the buffer mixture from the well and washing with PBS; blocking the well with a blocking buffer with Tween-20, BSA, and serum; incubating the well with an

1115 labeled secondary antibody (such as antibody-HRP) against the detection antibody bound in the well of the second plate in the blocking buffer comprising Tween-20 and BSA; removing unbound labeled secondary antibody (antibody-HRP) and washing the well with PBS; adding TMB substrate solution to the well of the second plate and incubating without light for proper time if the labeled secondary antibody is the antibody-HRP; the signal of the antigen of

1120 interest in the tissue fixed on the first plate is determined by detecting the absorbance of the second plate by reading the OD650nm.

The formalin-fixed and paraffin-embedded tissues also can run cell-based UTSIA. After immobilizing the tissue on a glass slide, and followed by regular procedures of deparaffinizing, rehydrating, antigen retrieval, and blocking, the tissue on the glass slide is

1125 then incubating with an detection antibody diluted in PBST with 2.5% serum (the serum should be from an animal species that is different from the animal species making the detection antibody, to avoid non-specific cross reactions) 30 min to 1 hour; removing and washing the slide with PBS to get rid of unbound detection antibody; adding a dissociating buffer with extreme pH to release the detection antibody bound on the antigen of interest on

1130 the fixed tissue on the slide; then transferring the liquid phase containing the dissociated detection antibody to a well of a polystyrene plate (second plate or second solid phase) which pre-contains a neutralizing buffer with opposite pH of the dissociating buffer to make buffer mixture with pH optimal for the detection antibody to be coated and detected in late steps of the assay; gentle rotating the plate for proper time to coat the detection antibody on the well

1135 of the second plate; removing the buffer mixture from the well and washing with PBS; blocking the well with a blocking buffer with Tween-20, BSA, and serum; incubating the well with an labeled secondary antibody (such as antibody-HRP) against the detection antibody bound in the well of the second plate in the blocking buffer comprising Tween-20 and BSA; removing unbound labeled secondary antibody (antibody-HRP) and washing the

1140 well with PBS; adding TMB substrate solution to the well of the second plate and incubating without light for proper time if the labeled secondary antibody is the antibody-HRP; the signal of the antigen of interest in the tissue immobilized on the glass slide is determined by detecting the absorbance of the second plate by reading the OD650nm.

All above steps of cell-based UTSIA can be run on room temperature if the antigen fixed

1145 and antibodies used are stable in room temperature; you can optimize the protocols by modifying any steps of the protocols or by adding steps or by reducing steps that lead to better sensitivity and specificity to detect your antigen of interest. In summary, the detail steps and the experimental procedures for detection of each antigen of interest should be tested by experiments and referenced with the general guidance in this chapter. The

1150 dissociating buffer is 0.2 M Glycine pH 2.5, and neutralizing buffer is IM Tris/HCl pH 9.0; PBS is phosphate buffered saline pH 7.0, PBST is PBS with 0.05% Tween-20; blocking buffer is PBST with 1% BSA (bovine serum albumin), or blocking buffer is PBST with 10% goat serum if the detection antibody is not an antibody from a goat; and TMB solution (Sigma T8665) is the substrate for HRP tagged on an antibody. The first solid phase is the

1155 polystyrene plate for tissue culture or the polystyrene sheet such as Permanox and

Polystyrene Microscope Slides from Electron Microscopy Sciences for tissue immobilization, and the second solid phase is the polystyrene plates for ELISA (Sigma #6562, high protein binding ELISA plate). The polystyrene sheet can be treated with poly-L-lysine (Electron Microscopy Sciences catalog# 19320-A/19320-B) to promote slice of frozen tissue adhesion

1160 on the sheet if it is necessary.

Again, if the level of the antigen in the cells or tissue is very low, such as in pg level, its detection on the detection solid phase can be carried out by the technology of high sensitivity detection system which is disclosed in US patent 5731158. Basically, the HRP- detection antibody or HRP-antibody against the unlabeled detection antibody on the detection

1165 solid phase is reacted with H₂O₂ and biotinyl-tyramide to covalently attach many biotin residues on the detection solid phase, and then using streptavidin-HRP to bind with the biotins on the solid phase, and then reacts with HRP substrate TMB to amplify the detection signal.

After determining the presence and the amount of the antigen of interest on the first

1170 plate (first solid phase), it is now critical to get an internal normalization control data from a substance in the tissue or cells immobilized on the first solid phase that the substance is directly correlated with the presence and the amount of the cells immobilized on the first solid phase. The substance comprises proteins, carbohydrates, and DNA/RNA. There are many methods to detect the presence and the amount of these substances, one of the

1175 convenient methods is using Bicinchoninic Acid (BCA) protein assay (Stoscheck, CM. 1990) to detect the presence and the amount of the proteins on the cells or tissue immobilized on the first solid phase.

The detail steps of the BCA assay for cells or tissue immobilized on a solid phase (the solid phase is load in a well of 96 well plate) are described here: the cells/tissue immobilized

1180 on a solid phase (96 well tissue culture plate) is washed with PBS to clean the dissociating buffer left, then after removing PBS from the cells or the tissue immobilized, lOOul/well BCA mixtures (A:B=50:l) (PIERCE #23225 BCA Protein Assay kit) is added to the well immobilized with cells/tissue on the first plate; the plate is incubated at 37⁰C for proper time to develop purple product solution which the color density is directly correlated with the

1185 presence and the amount of the proteins, and thus correlated with the amount of cells/tissue immobilized on the first solid phase. The color product solution of the BCA Protein Assay on the first plate is detected by measuring absorbance at OD562nm.

Now we get the amount of proteins as the internal normalization control and the amount of the antigen of interest. Dividing the amount of the antigen by the amount of the proteins

1190 gets a number that can be used to compare with other data of the antigen got from other samples (if the protein amounts per cell from these samples are comparable), as the data are normalized amounts of the antigen of interest in the cells/tissues immobilized on the first plate.

Besides above internal normalization control, it may also need a negative control for

1195 cell-based UTSIA; this can be done by using a non-specific IgG which is from a same animal species of the detection antibody (IgG) to stain the tissue or cells, then follows above described steps to dissociate, transfer, coat the non-specific IgG on a second plate for determination of the non-specific IgG; this non-specific IgG level on the second plate acts as a negative control for the detection antibody.

1200 Cell-based UTSIA is especially useful for cell-based drug discovery, because the method is a simple and robust cell-based assay, and can be scaled up to medium or high through put by adapting to automation. For lead drug candidate screening, the compound is added in the cells growing on the 96 well plate for proper time, and then removing the treatment medium, and running the cell-based UTSIA as above instructed to detect the

1205 antigen of interest. The cells without treatment or only treated with the vehicle is also running cell-based UTSIA as instructed above as a negative control. If there is a significant signal difference between the control and the treatment cells on the antigen of interest, then the compound deserves further validations.

Similarly, cell-based UTSIA is also useful in animal-based drug discovery {in vivo

1210 drug discovery or pharmacodynamic (PD) analysis}, because the method is a simple and robust tissue-based assay, and has format of tissue-based multiplex antigen detection assay (see paragraph below). For lead drug candidate screening, the compound is dosed to animal for proper time with a proper schedule, then collecting tissue sample of interest and freeze the tissue to a frozen tissue block by liquid nitrogen and dry ice (the frozen tissue can be

1215 stored at -8O⁰C), and then running the cell-based UTSIA as above instructed in the paragraph starting with "For frozen tissue, mounting the frozen tissue block on a cryostat holder and cutting the tissue to 5μm thick slice at minus 18 to minus 2O⁰C" to detect the antigen of interest. The animal without treatment or only treated with the vehicle is also running the cell-based UTSIA as instructed above as a negative control. If there is a significant signal

1220 difference between the control and the treatment animals on the antigen of interest, then the compound deserves further validations. If the antigen responses well to the drug treatment in the animal experiment, it may be used as a biomarker for clinical treatment evaluation.

Cell-based UTSIA also can be used in detection and quantification of multiple clinical biomarkers. For example, a frozen tissue can be used to make multiple tissue slices which

1225 were fixed on many round polystyrene supports with defined area, such as the round sections with diameter 4.5mm; these many tissue sections from a same tissue block can be used to detect multiple antigens (or biomarkers) by using different detection antibodies which are made from a same animal species, and then a common labeled antibody against the detection antibodies is used to detect the detection antibodies by running cell-based UTSIA. This

1230 novel detection (quantification) of multiple antigens from a same frozen tissue sample will increase detection efficacy significantly. The detection and quantification of multiple antigens in a same tissue sample by cell-based UTSIA is an advance in microarray or multiplex methods. The detail procedure please refers to above paragraph of that start with "For frozen tissue, mounting the frozen tissue block on a cryostat holder and cutting the

1235 tissue to 5μm thick slice at minus 18 to minus 2O⁰C".

Similarly, the cell-based UTSIA can be used in multiple antigens determination in cell- based assay. For example, growing cells in a 96 well tissue culture plate (first plate or first solid phase) until the cells are near completely confluent; removing the medium from the wells; washing with PBS; fixing the cell with 4% paraformaldehyde solution for proper time;

1240 removing the paraformaldehyde and washing the fixed cells with PBS; permeabilizing the fixed cells with cold methanol or PBS with 0.5% Triton X-IOO; blocking the cells with blocking buffer which comprises Tween-20, BSA, and serum; the serum is not from the species from which the detection antibodies was made and the serum is preferred from the species from which the secondary antibody was taken; incubating the plate with different

1245 specific detection antibodies that from a same animal species, that cells in each well of the plate is incubated with a specific detection antibody against an antigen of interest in a blocking buffer comprises Tween-20 and BSA; removing the unbound detection antibodies and washing wells of the plate with PBS; adding a dissociating buffer with extreme pH to every well of the plate to release the bound every detection antibody in each well of the plate

1250 into the liquid phase of the dissociating buffer in each well; transferring the liquid phase of each well of the first plate to corresponding well of another 96 well polystyrene plate (second plate or second solid phase) which pre-contains a neutralizing buffer with opposite pH of the dissociating buffer to make pH optimal for the detection antibody to be coated on the well of the second plate; gentle rotating the plate to coat the detection antibodies on the wells of the

1255 plate; removing the unbound buffer mixture from the wells and washing with PBS; blocking the wells with a blocking buffer with Tween-20, BSA, and serum which will not interfere with the detection of the detection antibody; incubating the wells with an antibody-HRP against the detection antibodies bound in the wells of the plate in the blocking buffer comprising Tween-20 and BSA; removing unbound antibody-HRP and washing the wells

1260 with PBS; adding TMB substrate solution to the wells of the second plate and incubating without light for proper time; the signal of the antigens of interest in the cells fixed on the first plate are determined by detecting the absorbance of wells of the second plate by reading the OD650nm.

In this post-genomic era, there are many discussions that the future medicine will be a

1265 "personalized medicine". The main idea of "personalized medicine" is that the diagnosis, treatment, prognosis, and prevention will all depend on the exact target that causing the disease. That suggests that the detection and quantification of the target related biomarker(s) on cells/tissue will be critical for future medicine. As described above, the cell-based UTSIA has unique property that compliments with current routine IHC and ICC, specifically on the 1270 field of quantification, it will has critical roles in current and future medicine.

EXAMPLE OF THE INVENTION

UTSIA invention prototype example: to detect and quantitate an analyte in a fluid 1275 sample (Fig. 2) (Note: the prototype example's steps of dissociating, transferring, and coating also can be used in cell-based UTSIA to immobilize the detection antibody on the second or detection solid phase for specific detection):

I. Materials and agents:

The capture solid phase (first solid phase) and the detection solid phase are transparent 1280 96 well polystyrene plates for ELISA (Sigma #6562, high protein binding ELISA plate), the adaptor molecule is Protein G (Bio Vision #6510-5), dissociating buffer is 0.2 M Glycine pH 2.5, and neutralizing buffer is IM Tris/HCl pH 9.0, PBS is phosphate buffered saline pH 7.0, PBST is PBS with 0.05% Tween-20, 1^st blocking buffer is PBST with 1% BSA (bovine serum albumin), and 2^nd blocking buffer is PBST with 10% goat serum, and TMB solution 1285 (Sigma T8665) is the substrate for HRP tagged on the detection antibody. The fluid sample is PBS with 1%BSA containing an antigen rabbit IgG (Santa cruz #sc-577) and a background control antigen mouse IgG (Santa cruz #sc-46680). The capture molecule to the antigen rabbit IgG is anti-rabbit IgG from donkey (Chemicon #AP182). The labeled detection molecule is an anti-rabbit IgG-HRP from donkey (Amersham Biosciences #NA934V). The 1290 detection molecule to the control antigen mouse IgG is anti-mouse IgG-HRP from sheep (Amersham Biosciences #NA931V).

II. Procedures:

1. Immobilizing the capture molecule Protein G to the capture solid phase: In the well of 96 well polystyrene plates (first solid phase), 100 μl of (200 ng) Protein G per well in

1295 PBS is loaded and incubated for 1 hour at room temperature. Then the unbound fluid is washed away by two time 300 μl/well PBS washing, and then the solid phase is blocked by 1% BSA in PBS 200ul/well for 15 min.

2. Immobilizing a capture molecule to the capture solid phase coated with Protein G: A capture molecule, anti-rabbit IgG from donkey (Chemicon #AP182), is diluted 10

1300 times (1:10 dilution) with 1^st blocking buffer, then the diluted capture molecule solution lOOul/well is incubated in the capture solid phase immobilized with Protein G at room temperature for 30 min, then unbound is removed; then the solid phase is blocked with 10% goat serum PBST for 10 min in room temperature; and then the solid phase is washed by PBST 1 time;

1305 3. Capturing an antigen of interest from a fluid sample onto the capture solid phase: a fluid sample 100 μl/well containing an antigen rabbit IgG or containing a control antigen mouse IgG is added to the capture solid phase, and incubated for 30 min at room temperature. The unbound fluid sample is removed and washed by PBS 300μl/well for 2 times.

1310 4. Dissociating the antigen from the capture solid phase into the liquid phase: the dissociating buffer 110 μl/well is added to the capture solid phase immobilized with the antigen and incubated in room temperature for 10 min with gentle rotation to separate the bound antigen from the capture solid phase into the liquid phase of the dissociating buffer.

1315 5. Transferring the dissociated antigen and coating it to a detection solid phase: the dissociating buffer with the dissociated antigen is transferred to the detection solid phase having 20 μl of the neutralizing buffer, then the buffer mixture containing the dissociated antigen is mixed and incubated in the detection solid phase for 30 min at room temperature with gentle rotation to immobilize the antigen onto the detection

1320 solid phase. After removing the buffer mixture, the detection solid phase is blocked with 200 μl 2^nd blocking buffer each well for 15 min, then the blocking buffer was removed.

6. Detecting the antigen on the detection solid phase with a detection antibody against the antigen: a labeled detection antibody (anti-rabbit IgG-HRP) against the antigen

1325 rabbit IgG 1 :5000 at lOOμl/well in 2^nd blocking buffer, or a labeled detection antibody

(anti-mouse IgG-HRP) against the control antigen mouse IgG 1:5000 at lOOμl/well in 2^nd blocking buffer, is incubated with the detection solid phase immobilized with the antigen for 30 min. The unbound is removed by PBST 300 μl/well washing 3 times. Then the TMB solution 100 μl/well is added to the solid phase and is incubated

1330 without light for 30 min. The signal of the antigen on the detection solid phase is detected by reading the OD655nm.

III. Results (Fig. 2): The analyte of interest (rabbit IgG), but not control protein mouse IgG, is specifically detected and quantitated by UTSIA in a does-dependent manner.

1335

Claims

I claim:

1360 1. An universal tandem solid-phases based immunoassay (UTSIA) method for assaying an analyte in a fluid sample, comprising:

(a) providing: a capture system comprising (i) a first solid phase immobilized with an adaptor molecule and a liquid phase containing a capture molecule, wherein

1365 the adaptor molecule immobilized on the first solid phase is capable of binding with the capture molecule in the liquid phase, and the capture molecule is capable of binding with said analyte in said fluid sample, (ii) a first solid phase immobilized with a capture molecule, wherein the capture molecule immobilized on the first solid phase is capable of binding with said

1370 analyte in said fluid sample, or (iii) a first solid phase immobilized with a capture molecule via an adaptor molecule wherein the capture molecule is capable of binding with said analyte in said fluid sample; a dissociating buffer for dissociating analyte immobilized on the first solid phase into a liquid phase of the dissociating buffer;

1375 a second solid phase for immobilizing analyte dissociated in the liquid phase of the dissociating buffer; and a signal generating system for detecting analyte immobilized on the second solid phase, wherein the signal generating system comprising (a) a labeled detection molecule which is used for binding with analyte

1380 immobilized on the second solid phase, or (b) an unlabeled detection molecule which is used for binding with analyte immobilized on the second solid phase and a labeled molecule which is used for binding with the unlabeled detection molecule;

(b) contacting said fluid sample with said capture system for a time and under 1385 conditions sufficient for said analyte specifically to be immobilized on the first solid phase of the capture system;

(c) incubating the analyte immobilized on the first solid phase of the capture system with said dissociating buffer for a time and under conditions sufficient for the analyte immobilized on the first solid phase of the capture system to be dissociated into

1390 the liquid phase of the dissociating buffer;

(d) contacting the liquid phase of the dissociation buffer containing the analyte with said second solid phase for a time and under conditions sufficient for the analyte in the liquid phase to be immobilized onto the second solid phase;

(e) determining the presence of the analyte immobilized on the second solid phase 1395 by using said signal generating system specifically targeting on the analyte immobilized on the second solid phase; and

(f) determining the presence and the amount of the analyte in the fluid sample by relating the presence of the analyte immobilized on the second solid phase with proper standard. 1400

2. The method of claim 1, wherein said first solid phase and said second solid phase comprise polystyrene support, wherein the first solid phase and the second solid phase are either located in separated areas of a same polystyrene support or located in two polystyrene supports separately.

1405

3. The method of claim l(b), wherein said analyte specifically to be immobilized on the first solid phase of the capture system is an affinity binding process that comprises (a) incubating the capture molecule immobilized on the first solid phase with the analyte in the fluid sample, or (b) first incubating the capture molecule in the liquid phase with the analyte

1410 in the fluid sample to form a complex of the capture molecule and the analyte in the fluid mixture, and then incubating the adaptor molecule immobilized on the first solid phase with the complex of the capture molecule and the analyte in the fluid mixture;

4. The method of claim l(d), wherein said the analyte in the liquid phase to be 1415 immobilized onto the second solid phase comprises a physical adsorption process that the analyte in the liquid phase is coated onto the second solid phase.

5. The method of claim 1, wherein said analyte comprises protein.

1420 6. The method of claim l(a), wherein said adaptor molecule comprises Protein G.

7. The method of claim l(a), wherein said capture molecule comprises a capture antibody containing Fc-domain.

1425 8. The method of claim l(a), wherein said dissociating buffer comprises a buffer of

0.2 M Glycine pH 2.5.

9. The method of claim l(a), wherein said labeled detection molecule and said unlabeled detection molecule comprise antibodies, wherein the antibody for unlabeled

1430 detection molecule is derived from an animal species other than the one producing the capture antibody.

10. The method of claim l(a), said labeled is that the detection molecule or the molecule against the unlabeled detection molecule is tagged with a label molecule with a

1435 physical property or biochemical activity that is analyzable by a detector via the label molecule's physical property or the label molecule's catalyzed activity, wherein the label molecule is capable of producing a signal for detection by itself or in conjunction with one or more additional substances.

1440 11. The method of claim l(a), wherein the said labeled molecule against the unlabeled detection molecule comprises a labeled antibody.

12. A kit for determining an analyte in a fluid sample which comprises: (a) a capture system comprising (i) a first solid phase immobilized with an adaptor 1445 molecule and a liquid phase containing a capture molecule, (ii) a first solid phase immobilized with a capture molecule, or (iii) a first solid phase immobilized with a complex of an adaptor molecule and a capture molecule;

(b) a dissociating buffer for dissociating the analyte into a liquid phase from said first solid phase;

1450 (c) a second solid phase for immobilizing the analyte dissociated in the liquid phase; and

(d) a signal generating system for detecting the analyte immobilized on the second solid phase; wherein the signal generating system comprising (i) a labeled detection molecule, or (ii) an unlabeled detection molecule and a labeled molecule against the 1455 unlabeled detection molecule; wherein the first solid phase and the second solid phase may be located in different areas of a same solid support, or located in different solid supports.

13. A kit for determining multiple analytes in a fluid sample which comprises: 1460 (a) a capture system capturing said multiple analytes in said fluid sample comprising (i) a first solid phase containing multiple separated defined regions that each region immobilized with an adaptor molecule, and liquid phases containing capture molecules against said multiple analytes that each liquid phase contains one capture molecule against an analyte, or (ii) a first solid phase immobilized with many complexes 1465 of an adaptor molecule and multiple capture molecules against the multiple analytes in many separated defined regions of the first solid phase that each region immobilized with a complex of the adaptor molecule and a capture molecule against an analyte;

(b) a dissociating buffer for dissociating the analytes bound on said separated defined regions of the first solid phase into liquid phases that each liquid phase contains

1470 an analyte dissociated from a separated defined region of the first solid phase;

(c) a second solid phase with many separated defined regions for immobilizing multiple analytes dissociated in many liquid phases that each separated defined region of the second solid phase is for immobilizing an analyte dissociated in a liquid phase; and

(d) signal generating system(s) for detecting the analytes immobilized on many 1475 separated defined regions of the second solid phase; wherein the first solid phase and the second solid phase are either located in separated areas of a same solid support or located in different solid supports.

14. The method of claim 13(d), wherein said signal generating system(s)

1480 comprises (i) multiple unlabeled detection molecules against said multiple analytes that each unlabeled detection molecule against an analyte and a common labeled molecule against the multiple unlabeled detection molecules, or (ii) multiple labeled detection molecules against said multiple analytes that each labeled detection molecule against an analyte.

1485 15. An cell-based UTSIA for detecting and quantifying an analyte in a sample immobilized on a solid phase, comprising: (a) providing a first solid phase, a detection molecule or a labeled detection molecule against said analyte, a dissociating buffer, a second solid phase, and a labeled secondary molecule against the detection molecule; 1490 (b) immobilizing said sample on a defined area of said first solid phase;

(c) incubating the detection molecule or the labeled detection molecule against the analyte with the sample immobilized on the first solid phase to immobilize the detection molecule on the first solid phase;

(d) washing to remove the unbound detection molecule or labeled detection 1495 molecule from the sample immobilized on the first solid phase;

(e) incubating the sample bound with the detection molecule or the labeled detection molecule on the first solid phase with said dissociating buffer to dissociate the detection molecule or the labeled detection molecule into the liquid phase of the dissociating buffer from the first solid phase;

1500 (f) transferring and immobilizing the detection molecule or the labeled detection molecule dissociated in the liquid phase onto said second solid phase;

(g) determining the presence and the amount of the analyte in the sample immobilized on the first solid phase by detecting the presence and the amount of the detection molecule or the labeled detection molecule immobilized on the second solid

1505 phase, wherein the presence and amount of the detection molecule or the labeled detection molecule on the second solid phase is directly correlated with the presence and the amount of the analyte in the sample immobilized on the first solid phase; and

(h) making an internal normalization control for the presence and the amount of the analyte in sample immobilized on the first solid phase by determining the presence

1510 and the amount of a substance in the sample immobilized on the first solid phase which is directly correlated with the presence and the amount of the sample immobilized on the first solid phase.

16. The method of claim 15, wherein said sample comprises cells or tissue. 1515

17. The method of claim 15, wherein said analyte comprises antigen.

18. The method of claim 15(a), wherein said detection molecule and said labeled detection molecule comprise antibody.

1520

19. The method of claim 15(a), wherein said first solid phase and said second solid phase comprise polystyrene support.

20. The method of claim 15(g), wherein said detecting the presence and the amount of 1525 the detection molecule immobilized on the second solid phase comprises binding the detection molecule immobilized on the second solid phase with the labeled secondary molecule against the detection molecule.

21. The method of claim 15(a), wherein said labeled secondary molecule comprises a 1530 labeled antibody.

22. The method of claim 15(h), wherein said substance comprises proteins.

23. The method of claim 22, wherein said proteins are determined by method

1535 comprising Bicinchoninic Acid (BCA) protein assay to detect the presence and the amount of the protein(s) on the sample immobilized on the first solid phase.

24. The method of claim 15(f), wherein said immobilizing the detection molecule or the labeled detection molecule dissociated in the liquid phase onto said second solid phase

1540 comprises coating the detection molecule or the labeled detection molecule dissociated in the liquid phase onto the second solid phase,

25. A kit for determining an antigen in cells or tissue immobilized on a solid phase, which comprises:

1545 (a) a solid phase for cells or tissue immobilizing;

(b) a detection antibody against said antigen;

(c) a dissociating buffer for dissociating the detection antibody bound on tissue or cells on the first solid phase into liquid phase;

(d) a solid phase for coating the detection antibody dissociated in liquid phase; 1550 and

(e) a labeled secondary antibody against the detection antibody.

26. A kit for determining multiple antigens in cells or tissue immobilized on many spots with defined area and defined shape of a solid phase, which comprises:

1555 (a) a first solid phase for cells or tissue immobilization at said many spots;

(b) multiple detection antibodies from a same animal species against said multiple antigens respectively;

(c) a dissociating buffer for dissociating the detection antibodies bound on the cells or tissue immobilized on the spots of the first solid phase into liquid phases;

1560 (d) a second solid phase with multiple defined spots for coating the detection antibodies dissociated in liquid phases; wherein each spot is for coating a detection antibody dissociated; and

(e) a labeled secondary antibody against the multiple detection antibodies.

1565 * * * * *

END OF THE INVENTION

1570