KR101311736B1 - Sandwich immunoassay Biochip and immunoassay method using the same - Google Patents

Abstract

The present invention provides a sandwich immunoassay biochip and an immunoassay using the same, specifically, a first protein which binds to an Fc region of an antibody bound to a solid-supported functional group whose surface is modified with a functional group; A second protein that binds to the Fc region of the antibody bound to the functional group of the polystyrene beads whose surface is modified with the capture antibody and the functional group bound to the first protein; It provides a biochip comprising a detection antibody bound to the second protein and an immunoassay using the same. According to the present invention, there is an advantage in that pathogenic bacteria can be detected accurately and quickly even in the presence of trace amounts of pathogenic microorganisms.

Description

Sandwich immunoassay biochip and immunoassay using the same {Sandwich immunoassay Biochip and immunoassay method using the same}

The present invention relates to a sandwich-immunoassay biochip and an immunoassay using the same, wherein the present invention can accurately and quickly detect trace amounts of pathogenic bacteria present in a sample.

Biochip is an analytical instrument that measures the state and concentration of substances by using the functions of living organisms, and can detect small targets, and has the advantages of high specificity, sensitivity, simplicity, and detection in a short time. Among the biochips, a biochip using an antigen antibody reaction is called an immuno-biochip.

In biochip detection systems, the use of fine particles with a large surface area per volume allows immobilization of a large amount of antibodies, increasing sensitivity. Recently, studies on solid-state immunoassay systems using polystyrene latex fine particle-protein bonds with constant dispersion and easy surface treatment (Gozalez et al., J. Mater. Sci .: Mater. Med. 19: 777, 1994) . Immobilization of antibodies on the surface of polystyrene particles is accomplished by intermolecular bonds such as electrostatic attraction, hydrogen bonding, and van der Waals forces (D. song and S. Forciniti, J. colloid Interface Sci. 221: 25, 2000).

However, immobilization of antibodies using polystyrene microparticles allows immobilization of a large amount of antibodies, but does not impart proper orientation of the antibody which is very important for specific binding to antigens, resulting in loss of biological activity (Buij et al., Colloids Surf, B. B5 (1/2): 11-23, 1995).

Therefore, in the field of biochips, there is a need for a technique capable of immobilizing a large amount of antibodies and imparting proper orientation to the immobilized antibodies.

Escherichia coli (E. coli O157: H7), on the other hand, is a gut parasitic bacterium due to the ingestion of altered food. Escherichia coli is a bacterium that occurs in many countries, such as Canada, Europe, and Japan. Examples of the symptoms of the E. coli infection include diarrhea, renal failure, seizures, colitis, hemolytic uremic syndrome, and the like. In addition, according to the release of the US Center for Disease Control and Prevention, 61 of 75,000 people infected with E. coli die in the United States each year (Su and Brandt; 1995, Mead et al., 1999).

For this reason, research is being conducted on methods and devices for rapidly detecting E. coli in food, beverages or the body. In the past, the detection method of microorganisms was obtained by collecting microorganisms from contaminated samples, culturing them in a liquid medium, and taking a single colony grown in a solid medium, and proceeding the analysis, which takes two to three days. There was.

Accordingly, a recent study on the technique using the immune biochip for the detection of pathogenic microorganisms, such as E. coli, there was a problem that can not accurately detect the microorganisms when a small amount of microorganisms in the sample.

Therefore, the development of an immuno-biochip that can not only dramatically reduce the detection time of microorganisms present in a sample, but also accurately detect even microorganisms present in the sample, is urgently needed.

Accordingly, the present inventors have made intensive efforts to develop an immunoassay that can accurately and quickly detect trace pathogens using immuno-biochips. A sandwich immunoassay system using detection antibodies immobilized on polystyrene beads and capture antibodies immobilized on the solid-support of the biochip via protein G or protein A can quickly and accurately detect trace amounts of E. coli O157: H7 present in the sample. It confirmed that it exists and completed this invention.

It is an object of the present invention to capture an antibody bound to a Fc region of an antibody bound to a solid-supported functional group whose surface is modified with a functional group and an Fc region of an antibody bound to a functional group of a polystyrene bead surface-modified with a functional group. It is to provide a sandwich immunoassay biochip comprising a detection antibody bound to a protein that binds to.

Another object of the present invention is to use the biochip, (a) contacting a sample containing an antigen to the biochip; (b) specifically binding the antigen to the capture antibody; (c) binding the detection antibody to another epitope that is not bound to the capture antibody of the antigen; And (d) detecting the specific binding of the antigen-antibody.

In order to achieve the above object, in one embodiment the present invention provides a protein comprising: a first protein that binds to an Fc region of an antibody bound to a solid-supported functional group whose surface is modified with a functional group; A second protein that binds to the Fc region of the antibody bound to the functional group of the polystyrene beads whose surface is modified with the capture antibody and the functional group bound to the first protein; It provides a sandwich immunoassay biochip comprising a detection antibody bound to the second protein.

Biochip is a machine for investigating the properties of substances by using functions of living organisms, and there are biochips used for enzyme analysis and immunoassay, optical biochips and electrochemical biochips, and the biochips of the present invention are specific for antigen-antibodies. Biochips used in immunoassays for detecting antigens through binding, and in particular, the biochips of the present invention can specifically and specifically capture antigens and capture antibodies and antigens so as to quickly and accurately detect traces of microorganisms present in the sample. It relates to a sandwich immunoassay biochip detecting the.

In the present invention, the functional group is a common atomic bond form that causes each property in a group of organic compounds having common chemical properties, and the surface of the solid-supported phase and polystyrene beads of the present invention is bound to the Fc region of the antibody. The surface is modified with functional groups to allow the proteins to bind.

The functional group may be, but is not limited to, a hydroxyl group, an amino group, a carboxyl group, a nitro group, a phenyl group, an aldehyde group, a hydroxyl group, a sulfate group, a thiol group, preferably the solid-supported functional group is an amine group, and a polystyrene bead The functional group of may be a carboxyl group.

There is no limit to the method that can be used to modify the surface of the solid-supported phase and polystyrene beads with a functional group, and for modification methods, such as organic solvents, which can be performed by those skilled in the art to which the present invention pertains. By surface modification, surface modification using atmospheric pressure plasma, and the like.

In the present invention, the solid-supporting phase is a medium that is located inside the biochip and serves as a support for fixing the antibody, and the present invention provides an appropriate orientation in which the activation site that binds the antigen of the antibody is directed in the direction of the solution containing the antigen. The surface of the solid-supported phase is modified with a functional group to impart a protein, and the protein that binds to the FC site of the antibody and the functional groups protruding from the surface of the solid-supported phase are covalently bound.

The solid-supported phase may be all or part of polymethylmethacrylate, polycarbonate, polystyrene, cyclic olefin copolymer, polynorbornene, styrene-butadiene copolymer, acrylonitrile butadiene styrene, glass, silicone, hydrogel, silicone , Metal, ceramic or porous membrane, preferably glass, polystyrene or metal, more preferably glass.

In the present invention, the polystyrene beads (polysytrene bead) is a polystyrene microparticles, the surface area per volume can be immobilized a large number of detection antibodies to play a role of enhancing the sensitivity of the immunoassay. In the case of conventional biochips using polystyrene beads, a detection antibody binding to polystyrene beads binds without orientation and has a disadvantage in that the sensitivity of antigen binding is low. Accordingly, the present invention provides a biochip with a component that can be oriented by binding a protein capable of binding to an Fc region of an antibody such as protein G or protein A to polystyrene beads for the first time, thereby dramatically increasing antigen detection ability. You can.

The polystyrene beads are modified with a functional group on the surface of the beads so that the protein binding to the Fc region of the antibody to impart the proper orientation of the antibody to the surface of the beads, wherein the polystyrene beads are functional and solid-supported The functional groups may or may not be identical.

The diameter of the polystyrene beads may be, but is not limited to, 200 nm to 300 nm, preferably 220 nm to 260 nm, and more preferably 250 nm. In addition, the polystyrene bead may be derived from the functional group on the surface by the emulsion polymerization method, preferably, the carboxyl group may be derived on the surface by the emulsion polymerization method.

In the present invention, the protein that binds to the Fc region of the antibody is a protein having activity that binds to the Fc region of the antibody, proteins and peptides having such activity can be used without limitation.

Preferably, the protein that binds to the Fc region of the antibody may be Protein A or Protein G, more preferably, Protein G of SEQ ID NO: 1 or Protein A of SEQ ID NO: 2.

Protein G: SEQ ID NO: 1

LKGETTTEAVDAATAEKVFKQ YANDNGVDGEWTYDDATKTFT VTEKPEVIDASELTPAVTTYK LVINGKTLKGETTTEAVDAAT AEKVFKQYANDNGVDGEWTYD DATKTFTVTEKPEVIDASELT PAVTTYKLVINGKTLKGETTT KAVDAETAEKAFKQYANDNGV DGVWTYDDATKTFTVTELEHH HHHH

Protein A: SEQ ID NO: 2

AQHDEAQQNAFYQVLNMPNLN ADQRNGFIQSLKDDPSQSANV LGEAQKLNDSQAPKADAQQNN FNKDQQSAFYEILNMPNLNEA QRNGFIQSLKDDPSQSTNVLG EAKKLNESQAPKADNNFNKEQ QNAFYEILNMPNLNEEQRNGF IQSLKDDPSQSANLLSEAKKL NESQAPKADNKFNKEQQNAFY EILHLPNLNEEQRNGFIQSLK DDPSVSKEILAEAKKLNDAQA PKEEDNKKPGKEDGNKPGKED GNKPGKEDNKKPGKEDGNKPG KEDNNKPGKEDGNKPGKEDNN KPGKEDGNKPGKEDGNKPGKE DGNGVHVVKPGDTVNDIAKAN GTTADKIAADNKLADKNMIKP GQELVVDKKQPANHADANKAQ ALPETGEENPFIGTTVFGGLS LALGAALLAGRRRELLEHHHH HH

Protein G is a bacterial cell wall protein isolated from the group G streptococci and is known to bind to the Fc and Fab sites of mammalian antibodies (J. Immunol. Methods 1988, 112, 113-120) . However, it is known that the binding power of the protein G to the Fc region of the antibody is about 10 times higher than that of the Fab region.

Protein A is a cell surface protein found in Staphylococcus aureus. It has the property of binding to the Fc region of mammalian antibodies, in particular IgG class antibodies. Within antibodies of a given class, the affinity varies little with respect to species origin and antibody subclass or allotype (Surolia, A. et al., 1982 Protein A: Nature's universal, antibody, TIBS 7, 74). -76; Langone et al., 1982, Protein A of staphylococcus aureus and related immunoglobulin receptors, Advances in Immunology 32: 157-252). Protein A can be isolated directly from the culture of Staphylococcus aureus secreting Protein A and conveniently obtained by recombinant expression in E. coli.

In the present invention, protein A and protein G are not particularly limited in origin, and a protein in which amino acids are deleted, added, or substituted in native protein A or protein G, as long as it retains the binding force to the Fc region of the antibody. A or protein G derivatives may also be used for the purposes of the present invention.

In the present invention, the detection antibody and the capture antibody are used in the sandwich immunoassay of the present invention to perform specific binding with the antigen administered in the biochip, thereby serving to quickly and accurately detect a small amount of antigen.

The detection antibody and the capture antibody bind to the protein binding to the Fc region of the antibody bound to the polystyrene beads and the solid-supporting functional groups, respectively, so that the activation site for binding the antigen is directed in the direction of the solution. That is, the biochip of the present invention is a biochip capable of sandwich immunoassay using a capture antibody and a detection antibody, and the detection antibody is used to maintain an appropriate orientation in which the Fab region, which is an activation site that binds to the antigen of the antibody, is directed in the direction of the solution. And the capture antibody is respectively bound to the surface of the support via a protein that binds to the Fc region of the antibody.

Due to the orientation of the capture antibody and the detection antibody, the biochip of the present invention can efficiently react with the antigen by the activation site that binds to the antigen of the antibody, through which a small amount of pathogenic bacteria causing food poisoning, such as E. coli and Salmonella Even if it has the advantage of detecting pathogens quickly and accurately.

In the biochip of the present invention, for the optimal frequency of reaction between the capture antibody and the antigen, preferably, the immobilization concentration of the capture antibody may be 100 μg / ml to 250 μg / ml, and more preferably 180 μg / ml to 220 μg / ml And even more preferably 200 μg / ml.

However, it will be appreciated that the concentration of such antibodies can be freely determined depending on the size of the antibody against the pathogen to be detected. That is, if the concentration is too high according to the size of the antibody, the reaction frequency may be rather lower due to the space disturbance on the slide chip, the concentration may be freely determined according to the size of the antibody used.

In one embodiment of the present invention, E. coli 0157: H7 is reacted on an amine slide glass with various concentrations of capture antibodies to determine an optimal capture antibody immobilization concentration, and polystyrene beads labeled with FITC-Protein G- anti-E. After reacting with the coli antibody complex (detection antibody), fluorescence intensity was measured at 516 nm using a confocal microscope. As a result, when the capture antibody immobilization concentration is 200μg / ml showed the highest fluorescence signal, it was found that the immobilization concentration of the capture antibody for the optimal frequency of reaction with the antigen is 200μg / ml (Example 3 and Figure 6) .

In another aspect, the present invention provides a sandwich immunoassay using the biochip.

In the present invention, the immunoassay comprises (a) contacting a sample containing an antigen with a biochip; (b) specifically binding the antigen to the capture antibody; (c) binding the detection antibody to another epitope that is not bound to the capture antibody of the antigen; And (d) detecting specific binding of the antigen-antibody.

In the biochip of the present invention, since the capture antibody and the detection antibody are respectively bound on the support, an antigen specifically binding to the antibody can be detected. Such a detection method may be referred to as a sandwich immunoassay.

In the present invention, the term "sample" includes, but is not limited to, tissues, cells, serum, plasma, saliva, cerebrospinal fluid, or samples, such as urine, containing antigens which are target substances to be detected.

An antigen is a substance that specifically binds to an antibody, and the antigen may be a pathogenic bacterium, yeast, or a fungus, since the present invention has the purpose of quickly and accurately detecting a small amount of pathogenic bacteria.

The pathogenic bacteria are all microorganisms that cause disease or harm while invading animals and animals, including Gram-positive bacteria and Gram-negative bacteria, preferably Escherichia coli O157: H7, Salmonella choleraesuis, Salmonella bongori, Salmonella typhimurium), Staphylococcus aureus, Listeria monocytogenes, Listeria denitrificans, Listeria grayi, Listeria murrayi, Cholera, Helicobacter, Pertussis, Diphtheria, Typhoid, Pest, Hemolytic Streptococcus or Staphylococcus aureus It may be more preferably E. coli O157: H7, Salmonella choleraesuis, Salmonella bongori, Salmonella typhimurium, Listeria monocytogenes, Listeria denitrificans, Listeria grayi, Listeria murrayi.

The pathogenic yeasts and fungi are pathogenic yeasts and fungi, but are not limited thereto, for example, Candida albicans, Aspergillus humigatus, Saccharomyces cerevisiae ( Saccharomyces cerevisiae) and Cryptococcus neoformans.

In the immunoassay by the biochip of the present invention, after the antigen is specifically bound to the capture antibody bound to the solid-supporting phase, the detection antibody is specifically bound to another epitope of the antigen not bound to the capture antibody. And by detecting specific binding of the capture antibody and the detection antibody to the antigen.

Detecting specific binding of the capture antibody and the detection antibody to the antigen can be performed by visually, optically, or electrochemically. Particularly, as an example of the visible method, the antibody is fluorescence. Surface plasmon resonance (SPR) technology is an example of a technique for measuring fluorescence intensity by labeling and confocal microscopy and an optical method.

In one embodiment of the present invention, anti-E. The coli antibody was immobilized and reacted with various concentrations of E. coli O157: H7, and polystyrene bead-protein G-anti-E labeled with the detection antibody FITC. After reacting with the coli antibody complex, fluorescence intensity was measured at 516 nm using a confocal microscope. As a result, it was confirmed that pathogens of 10 ² cell / ml can be detected by immunoassay using the biochip of the present invention (Example 4 and FIG. 7).

Thus, in the case of using the present invention, even when a small amount of pathogenic bacteria causing food poisoning or the like exists in the sample by providing proper orientation to the antibody and performing sandwich immunoassay using the detection antibody and the capture antibody to which the orientation has been given Accurate and rapid pathogenic bacteria can be detected.

According to the present invention, the detection sensitivity can be improved by fixing a large number of detection antibodies to polystyrene beads having a large surface area per volume. In addition, the antibody binds to the support through a protein that binds to the Fc region of the antibody to impart proper orientation, and trace pathogenic microorganisms causing food poisoning and the like in the sample through sandwich immunoassay using the capture antibody and detection antibody with the orientation Even if present, there is an advantage that can detect the pathogenic bacteria accurately and quickly.

1 is a diagram showing a PS bead-protein G- IgG complex according to an embodiment of the present invention.
Figure 2 is a photograph showing the fluorescence image of the complex conjugated IgG of various concentrations on the PS bead.
3 is a schematic diagram of antibody immobilization on amine slide glass.
4 is a photograph showing a confocal microscope fluorescence image showing the immobilization of the antibody on the amine slide glass.
Figure 5 is a schematic diagram showing the detection of E. coli O157: H7 by the detection antibody and capture antibody.
6 is a graph showing the fluorescence intensity according to the concentration of the antibody immobilized on the amine slide glass.
Figure 7 is a graph showing the fluorescence intensity to determine the sensitivity for E. coli 0157: H7 detection.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example  1: polystyrene Polystyrene bead phase  Antibody Immobilization

1-1: Polystyrene Bead  Protein G IgG  Complex formation

The antibody was immobilized on polystyrene (PS) beads (250 nm diameter) in which carboxyl groups were derived from the surface by emulsion polymerization. Synthesis of polystyrene beads with carboxyl groups derived from the surface occurs when a polymer of a hydrophilic comonomer is attached to the surface of the polymer when the syrene monomer is polymerized by an initiator. do. Polymerization is induced by adsorption of the radicals of the initiator onto the surface of the polymer, where the radicals of the copolymer are also stabilized by adsorption on the surface of the polymer and then chemically anchored to the surface of the polymer particles. Through this process, a polystyrene bead from which a carboxyl group having a surface hydrophilicity was produced.

After diluting the PS beads in 600 ml distilled water to be present in ¹¹ of 3.1X10, 200 mM of EDC and 100 mM of NHS were added to 600 µl of the beads solution to activate the carboxyl groups of the PS beads to react with the amine groups of the protein G. 50 μl of distilled water solution mixed with 1: 1 was added and reacted at room temperature for 15 minutes. After washing, 500 μl of distilled water was added and sonicated to disperse the PS beads. Then, 100 μl of distilled water (1 mg / ml) in which Protein G was dissolved was added and reacted at room temperature for 1 hour, followed by centrifugation for 40 minutes ( 17000 rpm, 4 ° C) to remove unreacted protein G. Thereafter, 500 µl of distilled water was added, and then, 100 µl of a 1% BSA solution was added to prevent non-specific reactions, and reacted at room temperature for 20 minutes, followed by washing. Next, 100 μl of various concentrations of IgG (immunoglobulin G; 10, 100, 200, 300 μg / ml) labeled with Fluorescein isothiocyanate (FITC) fluorescent substance and 100 μl of control BSA (1%) were added to each other. After reacting for a time, the supernatant was removed by centrifugation. Thereafter, 500 μl PBS was added and sonicated to obtain PS beads-protein G-IgG complexes having various concentrations of IgG bound to protein G bound to the surface of the PS beads (FIG. 1).

1-2: PS Bead  Protein G IgG  Check for complex formation

It was confirmed whether IgG was effectively immobilized on the PS bead-protein G-IgG complex obtained in Example 1-1. To this end, the various concentrations of IgG obtained in Example 1-1 were dispensed on 5 μl slide glass of PS beads-protein G-IgG complexes immobilized on the surface of PS beads, and then at a temperature of 37 ° C. After drying for a period of time, a fluorescence image was obtained at 516 nm using a Nikon C1si Laser Confocal Microscope (Tokyo, Japan) and is shown in FIG. 2.

As a result, the fluorescence intensity increased proportionally when the concentration of the antibody immobilized on the surface of the PS beads increased from 10 μg / ml to 300 μg / ml, but when reacted with the control BSA, the fluorescence image did not appear (FIG. 2). ). These results show that since the Fc portion of the IgG specifically binds to the protein G bound to the PS beads, the orientation of the antibody can be imparted, so that the PS beads can be effectively used for immunoassays.

Example  2: amine slide Glass top  Antibody Immobilization

As shown in the schematic diagram of FIG. 3, the possibility of immobilization of the antibody on the surface of the slide chip was confirmed. To this end, 5 μl of protein G (1 mg / ml) is dropped onto a slide glass (Matsunami, Osaka, Japan) surface-treated with an amine group, and left at room temperature for 3 hours. To immobilize.

Wash with PBS (pH 7.4) buffer to remove unbound Protein G. Next, 5 μl of 1% BSA was reacted at room temperature for 20 minutes to prevent nonspecific binding of the antibody. Then, after removing 1% BSA unbound with buffer, reacted with 5 μl of various concentrations of anti-IgG (10, 100, 200, 300 μg / ml) fluorescently labeled with FITC for 1 hour at room temperature, 37 ° After drying for one hour at a temperature of C, fluorescence images were acquired by confocal microscopy at 516 nm (FIG. 4).

As a result, the fluorescence image did not appear when reacted with the control 1% BSA, but when reacted with anti-IgG, the fluorescence intensity increased proportionally as the concentration of the antibody increased (Fig. 4). These results show that protein G successfully binds to the surface of the slide glass, thereby conferring orientation by the Fc portion of the antibody specifically binding to protein G.

Example  3: E. coli O157 : H7  Determination of Optimal Concentration of Antibodies for Detection

In order to determine the optimal antibody concentration capable of obtaining the highest antigen detection signal when detecting pathogenic bacteria using a sandwich immunoassay biochip, a representative pathogenic bacterium E. coli 0157: H7 was used.

To this end, in the same manner as in Example 2, various concentrations of anti-E. Coli O157: H7 antibodies (100, 200, 300, 400, 500 μg / ml) were immobilized on an amine slide glass to which protein G was bound. . In addition, the PS bead-protein G-anti-E.coli antibody complex was immobilized by fixing the anti-E. Coli antibody (300 µg / ml) fluorescently labeled with FITC on the surface of the PS beads in the same manner as in Example 1-1. Was formed.

Subsequently, E. coli O157: H7 (10 ⁷ cell / ml) was reacted on slides containing various concentrations of anti-E.coli antibodies, followed by PS bead-protein G-anti-E.coli O157: H7 antibody complex. Was continuously reacted for about 1 hour (FIG. 5). Then, washed with a buffer and then dried in an oven at 37 ° C, the fluorescence intensity was measured by confocal microscopy.

As a result, it showed the highest fluorescence signal at the concentration of anti-E. Coli 0157: H7 antibody of 200 ㎍ / ml, but the fluorescence signal began to decrease at a concentration higher than 200 ㎍ / ml (Fig. 6). These results indicate that when the concentration of antibody is higher than 200 ㎍ / ml, a steric hindrance occurs on the slide chip, and thus the frequency of reaction between E. coli O157: H7 and anti-E.coli O157: H7 antibody is reduced. Indicates. Therefore, it was found that the optimized immobilization concentration of the antibody for detecting the microorganisms present in the sample accurately and quickly and with high sensitivity was 200 µg / ml.

Example  4: E. coli O157 : H7  Sensitivity measurement for detection

Anti-E. Coli antibody (200 μg / ml) was immobilized on the bound protein G (1 mg / ml) on the amine slide glass, and various concentrations of E. coli O157: H7 (10 ^{1-10 7} cell / ml) After the reaction, the reaction was again performed with the PS bead-protein G-anti-E.coli 0157: H7 antibody complex for 1 hour at room temperature. After the reaction, washed with PBS and dried at a temperature of 37 ° C, and measured the fluorescence intensity at 516 nm using a confocal microscope. BSA and Salmonella typhimurium were used as controls.

As shown in Figure 7, the concentration of E. coli 0157: H7 of 10 ² ~ 10 ⁷ cells / ml increased the fluorescence signal than when reacted with the control group BSA and Salmonella typhimurium. However, at the concentration of 10 ¹ cell / ml E. coli O157: H7, the fluorescence intensity was not significantly higher than that of the control group, so the detection limit of E. coli O157: H7 was 10 ² cell / ml (Fig. 7).

Attach an electronic file to a sequence list

Claims (10)

(a) a solid-supported surface whose surface is modified with a functional group, (ii) a first protein bound to the functional group, and (iii) a capture antibody bound to the first protein; And
(b) polystyrene beads whose surface has been modified with a functional group, (ii) a second protein bound to the functional group, and (iii) a detection antibody bound to the second protein.
To include, wherein the first protein and the second protein will bind to the Fc region of the antibody, sandwich immunoassay biochip. The biochip of claim 1, wherein the functional group is a hydroxy group, an amine group, a carboxyl group, a nitro group, a phenyl group, an aldehyde group, a hydroxyl group, a sulfate group, a thiol group, a hydroxyl group, or a sulfate group.
The biochip of claim 2, wherein the solid-supported functional group is an amine group and the functional group of the polystyrene bead is a carboxyl group.
The biochip of claim 1, wherein the solid-supported phase is glass, polystyrene, or metal.
The biochip of claim 1, wherein the protein is Protein G or Protein A.
The biochip of claim 5, wherein the protein G has an amino acid sequence of SEQ ID NO: 1 and the protein A has an amino acid sequence of SEQ ID NO.
(a) contacting a sample comprising an antigen with the biochip of any one of claims 1 to 6; (b) specifically binding the antigen to the capture antibody; (c) binding the detection antibody to another epitope that is not bound to the capture antibody of the antigen; And (d) detecting specific binding between the antigen and the capture antibody and the antigen and the detection antibody. 8. The immunoassay according to claim 7, wherein the antigen is pathogenic bacteria, yeast or fungi.
The method of claim 8, wherein the pathogenic bacteria are Escherichia coli O157: H7, Salmonella, Staphylococcus aureus, Listeria spp., Cholera spp. At least one bacterium selected from the group consisting of leus.
The immunoassay according to claim 7, wherein the concentration of the capture antibody is 100 μg / ml to 250 μg / ml.

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