TW201908343A - Tandemly repeated antibody-binding protein and its applications - Google Patents

Abstract

The invention provides a tandemly repeated protein comprising at least two repeats of an amino acid sequence of an antibody binding protein or a fragment thereof and a cell having the repeats expressed on the membrane thereof, which can be used in immunoassay to improve detection sensitivity and detection limit.

Description

Tandem repetitive antibody binding protein and application thereof

The present invention relates to the field of immunoassays. In particular, the present invention relates to tandem repetitive proteins and their use in detecting analytes of interest.

Immunoassays are used to quantify the biomolecule of interest based on the specificity and selectivity of the generated antibody reagent. Immunoassays are a set of sensitive assays that use specific antibody/antigen complexes to generate signals that can be measured and correlated with the concentration of a target analyte in solution, including immunosorbent assay (ELISA). Western blot, flow cytometry and immunohistochemistry (IHC). Antibody-based immunoassays utilize immunological reactants (antigens or antibodies) that bind to an immunosorbent (an antigen or antibody that binds to a solid support) by hydrophobic, hydrophilic or electro-bonding. However, antibody-based immunoassays have the following problems: (i) the amount of capture antibody loaded on the surface of the solid support is limited; (ii) the direction in which the capture antibody binds to the surface of the solid support is different, since the antigen-binding region is located in the solid support The bottom, which results in a decrease in sensitivity; (iii) since other proteins can compete with the capture antibody for binding sites on the surface of the solid support, which results in a reduced loading of the capture antibody on the surface of the solid support, purified antibodies must be used. US 20110312104 provides a method for the release of immunoglobulin G bound to the surface by protein A from a substrate surface, which is not only an antigen having a high molecular weight but also an antigen having a low molecular weight. However, this reference still does not completely avoid the problems mentioned above. Therefore, there is still a need for improved immunoassay methods for better sensitivity and specificity.

The present invention provides a tandem repetitive protein comprising at least two repeats of an amino acid sequence of an antibody IgG binding protein or a fragment thereof and one or more linkers that are ligated to each other. In some embodiments, the antibody IgG binding protein is a protein A, protein G, protein A/G, protein L or Fc receptor or a fragment, combination or fragment thereof. In some embodiments, the tandem repetitive protein comprises 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 of the amino acid sequence of the antibody IgG binding protein or fragment thereof. Up to 20, 9 to 20, 10 to 20, 11 to 20, 12 to 20, 13 to 20, 14 to 20, 15 to 20, 16 to 20, 2 to 16, 3 to 16, 4 to 16, 5 to 16 , 6 to 16, 7 to 16, 8 to 16, 9 to 16, 10 to 16, 11 to 16, 12 to 16, 2 to 14, 3 to 14, 5 to 14, 6 to 14, 7 to 14, 8 To 14, 9 to 14, 10 to 14, 2 to 12, 3 to 12, 4 to 12, 5 to 12, 6 to 12, 7 to 12, 8 to 12, 9 to 12, 2 to 10, 3 to 10 4 to 10, 5 to 10, 6 to 10, 7 to 10, 2 to 9, 3 to 9, 4 to 9, 5 to 9 or 6 to 9 repeats. In another embodiment, the tandem repetitive protein comprises 8 repeats of an amino acid sequence of an antibody IgG binding protein or fragment thereof. Illustrative examples of amino acid sequences include, but are not limited to, the sequence of the C1, C2 or C3 fragment of protein G. A preferred embodiment of the amino acid sequence of the C2 fragment of protein G is SEQ ID NO: 1. The linkers used in the tandem repetitive proteins may be the same or different. Exemplary embodiments of linkers include, but are not limited to, amino acid sequences of GGGSG (SEQ ID NO: 2) or GGGGSGGGGSV (SEQ ID NO: 3). Exemplary embodiments of the tandem repetitive protein also include, but are not limited to, 8 repeats of the amino acid sequence of protein G or a fragment thereof and an amino acid sequence comprising SEQ ID NO: 2 or SEQ ID NO: 3. a linker; 8 repeats of the amino acid sequence of SEQ ID NO: 1, and a linker comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, which are ligated to each other; and SEQ ID NO : a tandem repetitive protein of the amino acid sequence of 4. The present invention provides a cell comprising a tandem repetitive protein expressed on a cell membrane, wherein the tandem repetitive protein is fused to a transmembrane protein of a cell. The invention also provides antibody-repetitive protein complexes comprising a plurality of antibodies or fragments thereof that bind to a tandem repetitive protein of the invention or to a cell of the invention. In one embodiment, the anti-system detects the antibody or captures the antibody. The invention also provides a method for detecting an analyte in a sample comprising using the tandem repetitive protein, cell and antibody-repetitive protein complex of the invention to capture a sample in an immunoassay or antibody-coated immunoassay Analyte in the analyte and qualitatively or quantitatively detect the analyte. In one embodiment of Western blotting and ELISA, the method of detecting an analyte in a sample comprises the steps of: providing a solid support coated with an analyte as appropriate; and binding a plurality of detection antibodies to the tandem repeat of the present invention a protein or a cell of the invention to form a detection antibody complex; binding the detection antibody complex to an analyte coated in a solid support; and qualitatively or quantitatively detecting the analyte. In one embodiment of the sandwich ELISA, the method of detecting an analyte in a sample comprises the steps of: providing a solid support; immobilizing the capture antibody on a solid support; capturing the analyte in the sample by capture of the antibody; The detection antibody binds to the tandem repetitive protein of the invention or the cells of the invention to form a detection antibody complex; the detection antibody complex is added for binding to the analyte; and the analyte is detected qualitatively or quantitatively. In one embodiment of the sandwich ELISA, the method of detecting an analyte in a sample comprises the steps of: providing a solid support; immobilizing the tandem repetitive protein of the invention or the cell of the invention on a solid support; The capture antibody binds to the tandem repetitive protein of the invention or the cells of the invention; captures the analyte in the sample by capture of the antibody complex; adds the detection antibody to bind to the analyte; and qualitatively or quantitatively detects the analyte. In one embodiment of the competition ELISA, the method of detecting an analyte in a sample comprises the steps of: providing a solid support; immobilizing the tandem repetitive protein of the invention or the cell of the invention on a solid support; An antibody binds to a tandem repetitive protein of the invention or a cell of the invention to form a capture antibody complex; a signaled analyte having a predetermined concentration is mixed with an analyte in the sample to form a mixture; by capture The antibody complex captures the analyte of the mixture; and the analyte is qualitatively or quantitatively detected. The invention also provides a kit for detecting an analyte in a sample comprising a solid support optionally coated with an antigen, an analyte or a capture antibody; and a tandem repetitive protein of the invention or a cell of the invention. In one embodiment, the kit further comprises a detection antibody or a capture antibody. In one embodiment, the detection antibody can be further labeled. The invention further provides a kit for detecting an analyte in a sample using a Western blot or ELISA comprising a solid support coated with an analyte as appropriate and a tandem repetitive protein of the invention or a cell of the invention. In one embodiment, the invention provides a kit for detecting an analyte in a sample using a sandwich ELISA comprising a solid support and a tandem repeat protein of the invention or a cell of the invention. In one embodiment, the invention provides a kit for detecting an analyte in a sample using a competition ELISA comprising a solid carrier coated with a tandem repetitive protein of the invention or a cell of the invention and a tandem repeat of the invention A protein or a cell of the invention. In another embodiment, the detection antibodies described herein can be further labeled.

The present invention provides at least two repetitive longitudinal repetitive proteins comprising an amino acid sequence of an antibody IgG-binding protein or a fragment thereof, and cells having a repeat expressed on the membrane thereof, which can be used in an immunoassay to improve detection sensitivity And detection limits. Except as defined below, the terms used in the claims and the terms used in the specification should be understood in accordance with their ordinary meaning as understood by those skilled in the art. The singular forms "a", "the" and "the" are used in the <RTI ID=0.0> </ RTI> </ RTI> </ RTI> <RTIgt; As used herein, the use of "or" means "and/or" unless otherwise indicated. In the case of multiple sub-items, use "or" to re-reference one or more of the aforementioned separate items or sub-items in an alternative manner. The terms "analyte" or "target analyte" as used herein are used interchangeably and generally refer to a substance or group of substances that are detected and/or measured in a sample. The term "binding molecule" as used herein refers to a molecule that is capable of binding another molecule of interest. The term "analyte binding" as used herein refers to any molecule (eg, an antibody) that is capable of participating in a specific binding reaction with an analyte molecule. As used herein, the term "detecting or detecting" is used to obtain the absolute value of the amount or concentration of the analyte present in the sample and to obtain an index, ratio, percentage, visual or other value indicative of the amount of analyte in the sample. Above, it is intended to include both quantitative and qualitative measurements. The evaluation may be direct or indirect, and the chemical or biochemical substance actually detected need not of course be the analyte itself, but may be, for example, a derivative thereof. The term "antibody" as used herein generally includes both single and multiple antibodies and binding fragments thereof, particularly Fc fragments and so-called "single chain antibodies", chimeric, humanized, in particular CDR-grafted antibodies and bivalent or tetravalent antibodies. Immunoglobulin-like proteins are also included which are selected by techniques including, for example, phage display to specifically bind to the molecule of interest contained in the sample. In this case, the terms "specificity" and "specific binding" refer to antibodies or fragments thereof produced by the molecule of interest. The term "fixed" as used herein refers to the immobilization of an agent to a solid surface. When the reagent is immobilized to a solid surface, it is non-covalently bound or covalently bound to the surface. In one aspect, the invention provides a tandem repetitive protein comprising at least two repeats of an amino acid sequence of an antibody IgG-binding protein or a fragment thereof and one or more linkers that are ligated to each other. In some embodiments, the antibody IgG binding protein is a protein A, protein G, protein A/G, protein L or Fc receptor or a fragment, combination or fragment thereof. Antibody IgG binding proteins are commercially available (e.g., sold by Thermo Fisher Scientific Inc.) and are known in the art. Protein A, Protein G, Protein A/G, and Protein L are natural and recombinant proteins that bind to microbial sources of mammalian immunoglobulin molecules. The proteins can be obtained in purified, salt-free, lyophilized form, and coated in microplates and covalently immobilized to a variety of solid carriers. Protein A is originally in Staphylococcus aureus (Staphylococcus aureus The 42 kDa surface protein found in the cell wall. Its use in biochemical research has been found due to its ability to bind immunoglobulins. It consists of five homologous Ig binding domains folded into a triple helix bundle. Each domain is capable of binding proteins from many mammalian species, most primarily IgG. A protein A/G recombinant fusion protein comprising an IgG binding domain of both protein A and protein G. Thus, protein A/G can be used to bind a broad range of IgG subclasses from rabbit, mouse, human, and other mammalian samples. Protein L binds to certain immunoglobulin kappa light chains. Since the kappa light chain is present in members of all classes of immunoglobulins (ie, IgG, IgM, IgA, IgE, and IgD), protein L can purify these different classes of antibodies. However, only antibodies within each of the classes with the appropriate kappa light chain will bind. Typically, empirical testing is required to determine if protein L is effective in purifying a particular antibody. Protein G is in group C and group G streptococci (Streptococcal An immunoglobulin binding protein expressed in , and has been found to be useful in the purification of antibodies by virtue of its binding to the Fab and Fc regions. The C-terminus of protein G can bind to an antibody. In the amino acid sequence of protein G, amino acid 306-331 is a C1 fragment, amino acid 347-402 is a C2 fragment and amino acid 418-473 is a C3 fragment. In the fragment, C1 and C3 can bind to the Fc fragment and Fab fragment of the antibody, and C2 can bind to the Fc fragment of the antibody. In some embodiments, the tandem repetitive protein comprises 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 of the amino acid sequence of the antibody IgG binding protein or fragment thereof. Up to 20, 9 to 20, 10 to 20, 11 to 20, 12 to 20, 13 to 20, 14 to 20, 15 to 20, 16 to 20, 2 to 16, 3 to 16, 4 to 16, 5 to 16 , 6 to 16, 7 to 16, 8 to 16, 9 to 16, 10 to 16, 11 to 16, 12 to 16, 2 to 14, 3 to 14, 5 to 14, 6 to 14, 7 to 14, 8 To 14, 9 to 14, 10 to 14, 2 to 12, 3 to 12, 4 to 12, 5 to 12, 6 to 12, 7 to 12, 8 to 12, 9 to 12, 2 to 10, 3 to 10 4 to 10, 5 to 10, 6 to 10, 7 to 10, 2 to 9, 3 to 9, 4 to 9, 5 to 9 or 6 to 9 repeats. In another embodiment, the tandem repetitive protein comprises 8 repeats of an amino acid sequence of an antibody IgG binding protein or fragment thereof. In some embodiments, the antibody IgG binding protein or fragment thereof is a protein A, protein G, protein A/G, protein L or Fc receptor or fragment thereof. In some embodiments, the antibody IgG binding protein is a protein A, protein G, protein A/G, protein L or Fc receptor or a fragment, combination or fragment thereof. In some embodiments, the amino acid sequence of an antibody IgG binding protein or fragment thereof comprises the sequence of a C1, C2 or C3 fragment of protein G. In another embodiment, the amino acid sequence comprises the sequence of the C2 fragment of protein G. In one embodiment, the amino acid sequence of the C2 fragment of Protein G is as follows. TYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (SEQ ID NO: 1) In some embodiments, the linkers used in the tandem repetitive proteins may be the same or different. In some embodiments, the linker comprises an amino acid sequence of GGGSG (SEQ ID NO: 2) or GGGGSGGGGSV (SEQ ID NO: 3). In some embodiments, the tandem repetitive protein comprises 8 repeats of the amino acid sequence of protein G or a fragment thereof and a linker comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In one embodiment, the tandem repetitive protein comprises 8 repeats of the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence comprising SEQ ID NO: 2 or SEQ ID NO: 3 which is ligated to each other. Connector. In another embodiment, the tandem repetitive protein comprises the amino acid sequence of SEQ ID NO:4. TYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGGGGSGGGGSVETYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGGGGSGGGGSVETYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGGGGSGGGGSVETYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGGGGSGGGGSVETYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGGGGSGGGGSVETYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGGGGSGGGGSVETYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGGGGSGGGGSVETYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEGGGGSGGGGSV (SEQ ID NO: 4) In another aspect, the present invention provides a cell comprising a longitudinal row on the cell membrane performance of repetitive protein, wherein the transmembrane protein tandem repetitive fusion protein of the cell. In another aspect, the invention provides an antibody-repetitive protein complex comprising a plurality of antibodies or fragments thereof that bind to a tandem repetitive protein of the invention or to a cell of the invention. In one embodiment, the anti-system detects the antibody or captures the antibody. The tandem repetitive protein, cell and antibody-repetitive protein complexes of the invention can be used in immunoassays; in particular, in immunoassays for ELISA and antibody coating. The tandem repetitive protein, cell and antibody-repetitive protein complex of the present invention provides high antibody binding ability, increases the amount of detection antibody accumulated in a solid vector, and can be used in an immunoassay using an unpurified detection antibody or Capture antibodies. Accordingly, the present invention provides kits and methods for using the tandem repetitive protein, cell and/or antibody-repetitive protein complexes of the present invention in immunoassays. In another aspect, the invention provides a method of detecting an analyte in a sample comprising using the tandem repetitive protein, cell and/or antibody-repetition of the invention in immunoassay or antibody-coated immunoassay The protein complex is used to capture the analyte in the sample and to detect the analyte qualitatively or quantitatively. In one embodiment, the method of detecting an analyte in a sample comprises the steps of: providing a solid support coated with an analyte as appropriate; binding a plurality of detection antibodies to the tandem repetitive protein of the invention or the invention Cells to form a detection antibody complex; binding the detection antibody complex to an analyte coated in a solid support; and qualitatively or quantitatively detecting the analyte. The examples mentioned above are applicable to Western blots and ELISA. In one embodiment, the method of detecting an analyte in a sample comprises the steps of: providing a solid support; immobilizing the capture antibody on a solid support; capturing the analyte in the sample by capturing the antibody; and binding the plurality of detection antibodies To the tandem repetitive protein of the invention or the cells of the invention to form a detection antibody complex; the detection antibody complex is added for binding to the analyte; and the analyte is detected qualitatively or quantitatively. The examples mentioned above are applicable to sandwich ELISA. In one embodiment, the method of detecting an analyte in a sample comprises the steps of: providing a solid support; immobilizing the tandem repetitive protein of the invention or the cell of the invention on a solid support; and binding the plurality of capture antibodies to A tandem repetitive protein of the invention or a cell of the invention; captures an analyte in a sample by capture of the antibody complex; adds a detection antibody to bind to the analyte; and qualitatively or quantitatively detects the analyte. The examples mentioned above are applicable to sandwich ELISA. In one embodiment, the method of detecting an analyte in a sample comprises the steps of: providing a solid support; immobilizing the tandem repetitive protein of the invention or the cell of the invention on a solid support; and binding the plurality of capture antibodies to a longitudinally repetitive protein of the invention or a cell of the invention to form a capture antibody complex; a signaled analyte having a predetermined concentration is mixed with an analyte in the sample to form a mixture; by capturing the antibody complex Capturing the analyte in the mixture; and qualitatively or quantitatively detecting the analyte. The examples mentioned above are applicable to competitive ELISA. In another aspect, the invention provides a kit for detecting an analyte in a sample comprising a solid support optionally coated with an antigen, an analyte or a capture antibody; and a tandem repetitive protein of the invention Or a cell of the invention. In one embodiment, the kit further comprises a detection antibody or a capture antibody. In one embodiment, the detection antibody can be further labeled. Whether or which antigen, analyte or capture antibody is optionally coated on a solid support depends on the type of immunoassay. In one embodiment, the invention provides a kit for detecting an analyte in a sample using Western blots or ELISA, comprising a solid support coated with an analyte as appropriate, and a tandem repetitive protein of the invention or the invention The cells. In one embodiment, the invention provides a kit for detecting an analyte in a sample using a sandwich ELISA comprising a solid support and a tandem repeat protein of the invention or a cell of the invention. In one embodiment, the invention provides a kit for detecting an analyte in a sample using a competition ELISA comprising a solid carrier coated with a tandem repetitive protein of the invention or a cell of the invention and a tandem repeat of the invention A protein or a cell of the invention. A solid support for immobilizing a tandem repetitive protein or antibody-repetitive protein complex can be any inert carrier or carrier that is substantially water insoluble and useful in immunoassays, including, for example, flat surfaces, particles, A carrier in the form of a porous substrate or the like. Examples of commonly used carriers include pellets, beads and assay plates or test tubes made from polyethylene, polypropylene, polystyrene, and the like, including 96-well microtiter plates and particulate materials such as filter paper, agarose, cross-linked polydextrose, and others. Polysaccharide. Alternatively, a reactive water insoluble substrate such as a cyanogen bromide activated carbohydrate and a reactive substrate can be used. In one embodiment, the immobilized capture reagent is coated onto a streptavidin coated 96-well microtiter plate. To facilitate immunoassay, the detection antibodies used in the kits and methods of the invention may also comprise a detectable label. The detectable label can be that does not interfere with binding of the analyte to the detection antibody and to any portion of the tandem repetitive protein of the invention or the cells of the invention. Numerous markers can be used in the methods and kits of the present invention. Examples of labels include, but are not limited to, radioisotopes, colloidal gold particles, fluorescent or chemiluminescent labels, and enzyme labels. Examples of radioisotopes include (but are not limited to)³⁵ S,¹⁴ C,¹²⁵ I,³ H and¹³¹ I. Antibodies can be labeled with radioisotopes using techniques known in the art, and radioactivity can be measured using scintillation counting. Other radioactive species include⁹⁹ Tc,⁹⁰ Y,¹¹¹ In,³² P,¹¹ C,¹⁵ O,¹³ N,¹⁸ F,⁵¹ Cr,⁵⁷ To,²²⁶ Ra,⁶⁰ Co,⁵⁹ Fe,⁵⁷ Se,¹⁵² Eu,⁶⁷ CU,²¹⁷ Ci and²¹² Pb. Examples of fluorescent or chemiluminescent labels include, but are not limited to, rare earth chelates (ruthenium chelates), fluorescent yellows and derivatives, rose bengal and its derivatives, isothiocyanates, phycoerythrins, algae Blue protein, allophycocyanin, o-phthalaldehyde, fluorescamine, sulfonamide, umbelliferone, luciferin, luminescent amine label, heteroluminescent amine label, aromatic acridinium ester label, imidazole Label, acridine salt label, oxalate label, aequor label, 2,3-dihydropyridazinedione, deuterated amine reactive N-hydroxy amber imidate label, Texas Red (Texas Red), sulforaphthalein, Lissamine, phycocrytherin or commercially available fluorophores. Fluorescent labels can be coupled to antibodies using techniques known in the art. Fluorescence can be quantified using fluorescence. Biotin labeling can be coupled to the antibody using techniques known in the art. Biotinylated antibodies can be detected by enzyme-conjugated avidin and streptavidin. Examples of enzyme labels include luciferase, malate dehydrogenase, urease, peroxidase (for example, horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucose Amylase, lysozyme, sugar oxidase (eg, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (eg, uricase and xanthine oxidase), milk peroxidation Enzymes, microperoxidases and the like. Techniques for coupling enzymes to antibodies are known in the art. Where the detection antibody is labeled, the kit and method can also include one or more reagents that produce a detectable signal when a sandwich is formed between the capture antibody, the analyte, and the detector binding molecule. For enzyme-labeled detector binding molecules, kits and methods can include the substrate and cofactor required for the enzyme, and for fluorophore labeling, the kit can include a dye precursor that produces a detectable chromophore. For biotinylated detector binding molecules, kits and methods can include enzymatically coupled avidin and streptavidin, the enzymes and cofactors required for the enzyme. If the detection antibody is not labeled, the kit and method may also comprise a detection member (eg, a labeled antibody that specifically binds to the detection antibody) and a step of using the detection member, respectively. The sample described herein may be any of the following various body fluids, including tissue, biopsy, biopsies, blood, serum, semen, breast exudate, saliva, sputum, urine, cytosol, plasma, ascites. , pleural effusion, amniotic fluid, bladder irrigation, bronchoalveolar lavage fluid and cerebrospinal fluid. In some embodiments, the sample is tissue, biopsy, blood, serum or plasma, and in a preferred embodiment, the sample is serum. Examples of immunoassays include, but are not limited to, ELISA, Western blot, flow cytometry, immunohistochemistry, radioimmunoassay, competitive immunoassay, magnetic immunoassay, memory lymphocyte immunostimulation assay (MELISA), side Flow-through immunochromatographic analysis, surround fiber immunoassay (SOFIA), and agglutination-PCR (ADAP). In immunoassays such as ELISA and Western blots, the tandem repetitive proteins or cells of the invention can bind multiple detection antibodies to form a complex, thereby increasing the accumulation of detection in the position of the analyte to be bound to the detection antibody. The amount of antibody. Therefore, the detection efficiency is greatly improved and the detection sensitivity is also increased. In antibody-based immunoassays such as sandwich ELISA or competition ELISA, the tandem repetitive proteins or cells of the invention can be immobilized on a solid support to increase the loading of the capture antibody on a solid support. Therefore, the amount of the analyte to be captured is increased and the detection sensitivity is also improved. In particular, the present invention relates to a recombinant protein called a tandem repetitive protein which is constructed from a fragment (Fc) binding domain of a protein by genetic engineering techniques. The tandem repetitive protein exhibits significantly greater binding efficiency and affinity for antibodies than monomeric proteins. By coating a tandem repetitive protein on a solid phase plate (eg, plate, membrane, and beads), the amount of antibody loaded on the plate can be significantly increased and the antigen-binding domain (Fab) of the antibody can be displayed on the plate. One-way (outward). Another application of the tandem repetitive protein is to generate a polyprotein/antibody complex by mixing a tandem repetitive protein with an antibody, which increases the cumulative amount of antibody on the antigenic region. Furthermore, the present invention relates to a cell which exhibits a tandem repetitive protein on its membrane which exhibits a tandem repetitive protein on the cell surface. By coating the cells on a solid phase plate, the amount of antibody loaded on the plate can be significantly increased and the display of the antigen binding domain (Fab) of the plated antibody can be unidirectional (outward). Another application of cells is to generate a cell/antibody complex by simply mixing cells with antibodies, which increases the cumulative amount of antibody on the antigenic region. Pairing with tandem repetitive proteins or cells allows the antibodies to be directly applied to the immunoassay without additional purification. A variety of antibodies can be used in the present invention to significantly improve the sensitivity and detection limits of immunoassays. Illustrative examples of the invention are shown below.The invention will be further understood by reference to the following non-limiting experimental examples.Instance Instance 1 Longitudinal repetitive protein of the present invention ( Polyprotein G) Cell line and its antibody capture capacity Polyprotein G Construction The C2 domain of Streptococcal protein G was sequenced and cloned to generate 8 repeats of 1 protein G-C2 domain and protein G-C2 domain (1 repeating amino acid sequence SEQ ID NO: 1). The B7 transmembrane protein was ligated to the C-terminus of the resulting domain to form protein G-mB7 and polyprotein G-mB7. The gene sequence is from the N-terminus to the C-terminus, respectively, HA-protein G-linker-mB7 and HA-polyprotein G-linker-mB7 (see Figure 1).Stable multiprotein G Cell line The gene sequence was inserted into a lentiviral vector and the resulting vector was transfected into a 3T3 cell line to form protein G cells and polyprotein G cells, respectively. Cell membrane proteins were collected and confirmed by Western blotting by adding mouse anti-HA antibody and HRP goat anti-mouse IgG Fcγ antibody to confirm whether protein G and polyprotein G were correctly expressed. As shown in Figure 2, the protein G-mB7 (molecular weight: 38 KDa) and the polyprotein G-mB7 (95 KDa) were correctly expressed on the cell membrane.Polyprotein G Cell-to-antibody capture FITC-conjugated goat anti-mouse immunoglobulin G Fcγ antibodies were separately added to protein G cells or polyprotein G cells for binding assay. Binding strength was measured by flow cytometry. The results showed that the fluorescence intensity of protein G cells and polyprotein G cells was 159.63 and 8058.42 (see Figure 3). This demonstrates that protein G cells or polyprotein G cells stably exhibit on the cell membrane and bind well to antibodies. Surprisingly, multiprotein G cells bind antibodies more than 50 times more efficiently than protein G cells.Antibody binding to multiple proteins G Specificity Protein G cells and polyprotein G cells were added to a 3.3 antibody (an anti-PEG antibody) and then FITC-conjugated 4-arm PEG was added. Flow cytometry was used to determine the intensity of fluorescence on the cell surface. The fluorescence intensity of protein G cells and polyprotein G cells were 21.11 and 138.99, respectively (see Figure 4). The results showed that the 3.3 antibody bound to protein G cells or polyprotein G cells could still bind to FITC-conjugated 4-arm PEG and thus demonstrated that the antibody maintained its specificity after binding to protein G cells or polyprotein G cells.Instance 2 Longitudinal repetitive protein of the present invention ( Polyprotein G) Bacterial cells and their antibody capture capacity Polyprotein G Construction The C2 domain of Streptococcal protein G was sequenced and colonized to generate 8 repeats of 1 protein G-C2 domain and protein G-C2 domain. An autotransporter adhesion (AIDA) membrane protein is ligated to the C-terminus of the resulting domain to form the protein G-AIDA and the polyprotein G-AIDA. The gene sequence is from the N-terminus to the C-terminus, respectively, HA-protein G-linker-AIDA and HA-polyprotein G-linker-AIDA (see Figure 5).Stable multiprotein G Cell line The gene sequence was inserted into the vector pET22b to form pET22b-protein G-AIDA and pET22b-polyprotein G-AIDA, respectively, and the resulting vector was transformed into E. coli BL21 (E. coli BL21) to form protein G bacteria and polyprotein G bacteria, respectively. Cell membrane proteins were collected and confirmed by Western blotting by adding anti-HA antibody and HRP goat anti-mouse IgG Fcγ antibody to confirm whether protein G and polyprotein G were correctly expressed. As shown in Figure 6, the protein G-AIDA (molecular weight: 70 KDa) and the polyprotein G-AIDA (125 KDa) were correctly expressed on the cell membrane.Polyprotein G Bacterial capture of antibodies Protein G bacteria and polyprotein G bacteria were each fixed in a 96-well plate. Will be 1μ g/mL horseradish peroxidase (HRP) conjugated goat anti-mouse IgG Fc antibody was added to the wells and then ABTS was added for catalytic coloration. The resulting mixture was measured at O.D. 405 nm. at 1μ The antibody concentration of g/mL showed that the absorbance of the polyprotein G bacteria was 18.9 times higher than that of the protein G bacteria (see Fig. 7).Instance 3 Repetitive protein G ( Polyprotein G) Construction and antibody capture ability Polyprotein G Sequence construction The C2 domain of Streptococcal protein G was sequenced and colonized to generate 8 repeats of the protein G-C2 domain. The histidine used as an identifier for protein purification was ligated to the C-terminus of the resulting domain. The gene sequence is HA-polyprotein G from the N-terminus to the C-terminus (see Figure 8).Polyprotein G Mass production To develop various types of polyprotein G used in immunoassays, construct a repetitive polyprotein G (8 repeats of the C2 domain of protein G) and then insert it into the retroviral vector pLNCX to form pLNCX-polyprotein G. . The resulting vector was transfected into Expi293 cells for mass production of the protein. The resulting polyprotein G was purified by Ni affinity column and then confirmed by Western blot (Fig. 9A) and 10% SDS-PAGE (non-reducing conditions) (Fig. 9B). The results showed that polyprotein G was correctly constructed and purified (65 KDa).Polyprotein G Capture of antibodies Will be different concentrations (1μ g/mL, 5μ g/mL and 10μ g/mL) polyprotein G is immobilized on a 96-well plate and will be 1μ g/mL horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG Fc antibody was added thereto. ABTS is then added for catalytic coloration. The resulting mixture was measured at O.D. 405 nm. at 1μ g/mL, 5μ g/mL or 10μ The antibody concentration of g/mL and the results showed that the polyprotein G was effective for capturing the antibody and the amount of the captured antibody was increased in a concentration-dependent manner (see Fig. 10).Instance 4 Polyprotein G Polyprotein G The cells greatly increase the amount of binding of the detection antibody to the antigenic site and thereby increase the sensitivity of the immunoassay Polyprotein G Bacterial increase sandwich ELISA Detection sensitivity Will 2μ A g/mL of MTI (an anti-IFNα capture antibody) was added to the ELISA plate and then 50 pg/mL, 30 pg/mL, 10 pg/mL, and 6 pg/mL of IFNα were added thereto. Biotin conjugate MT2 (an anti-IFN-α detection antibody) which is not bound or bound to the polyprotein G bacteria is added to the plate, and then the catalytically-catalyzed streptavidin-HRP and ABTS are added. The resulting mixture was measured at O.D. 405 nm. The results showed that the absorbance of the detection antibody bound to the polyprotein G bacteria was significantly higher than that of the detection antibody not bound to the polyprotein G, and thus the sensitivity of the sandwich ELISA for detecting IFNα was increased (see Fig. 11a). Will be 1μ g/mL of AGP4 (an anti-PEG capture antibody, IgM type) was added to the ELISA plate, and then 1000 pg/mL, 300 pg/mL, 100 pg/mL, and 30 pg/mL of PEGASYS (a type of PEG) The modified protein drug) is added thereto. Biotin conjugate 3.3 (an anti-PEG detection antibody, IgG type) which is not bound or bound to the polyprotein G bacteria is added to the plate, and then the catalytically catalyzed streptavidin-HRP and ABTS are added. The resulting mixture was measured at O.D. 405 nm. The results showed that the absorbance of the detection antibody bound to the polyprotein G bacteria was significantly higher than that of the detection antibody which was not bound to the polyprotein G bacteria, and thus the sensitivity of the sandwich ELISA to detect pyromycin was increased (see Fig. 11b).Polyprotein G Add sandwich ELISA Detection sensitivity Will 2μ g/mL MTI (an anti-IFNα capture antibody) was added to the ELISA plate and then 500 pg/mL, 100 pg/mL, 20 pg/mL, 4 pg/mL, and 0.8 pg/mL IFNα were added thereto. Biotin conjugate MT2 (an anti-IFN-α detection antibody) that is not bound or bound to polyprotein G is added to the plate, and then the catalytically-catalyzed streptavidin-HRP and ABTS are added. The resulting mixture was measured at O.D. 405 nm. The results showed that the absorbance of the detection antibody bound to the polyprotein G was significantly higher than that of the detection antibody not bound to the polyprotein G, and thus the sensitivity of the sandwich ELISA for detecting IFNα was increased (see Fig. 12a). Will be 1μ g/mL of AGP4 (an anti-PEG capture antibody, IgM type) was added to the ELISA plate and then 1000 pg/mL, 100 pg/mL, 10 pg/mL and 1 pg/mL of PEGASYS (a PEG-coated) The modified protein drug) is added thereto. Biotin conjugate 3.3 Ab (an anti-PEG detection antibody, IgG type) not bound or bound to polyprotein G was added to the plate, and then the catalytically catalyzed streptavidin-HRP and ABTS were added. The resulting mixture was measured at O.D. 405 nm. The results showed that the absorbance of the detection antibody bound to the polyprotein G was significantly higher than that of the detection antibody which was not bound to the polyprotein G, and thus the sensitivity of the sandwich ELISA to detect pyromycin was increased (see Fig. 12b).Polyprotein G Bacteria increase the detection sensitivity of western ink spots 50 ng/well, 10 ng/well, 2 ng/well, and 0.4 ng/well of PEGASYS (a PEG-modified protein drug) was subjected to 10% (w/v) SDS-PAGE electrolysis and then printed on On the nitrocellulose membrane. 2 that will not bind or bind to polyprotein G bacteriaμ A g/mL 3.3 Ab (an anti-PEG detection antibody, IgG type) was added to the membrane, and then HRP-conjugated goat anti-mouse antibody was added for coloration. The results showed that for PEGASYS, the detection limit of 3.3 Ab in combination with polyprotein G bacteria was enhanced to 0.4 ng/well, while the sensitivity of 3.3 Ab not bound to polyprotein G bacteria was 10 ng/well (see Figure 13). . The results show that polyprotein G bacteria do increase the sensitivity of Western blots and improve their detection limits.Polyprotein G Increase the detection sensitivity of western ink dots 20 ng/well, 10 ng/well, 5 ng/well, 2.5 ng/well, 1.3 ng/well, 0.6 ng/well, 0.3 ng/well, and 0.1 ng/well of PEGASYS (a PEG-modified The protein drug) was subjected to 10% (w/v) SDS-PAGE electrolysis and then printed on a nitrocellulose membrane. 2 that will not bind or bind to polyprotein Gμ A g/mL biotin-conjugated 3.3Ab (an anti-PEG detection antibody, IgG type) was added to the membrane, and then streptavidin-HRP was added for coloration. The results showed that for PEGASYS, the detection limit of biotin-3.3 Ab bound to polyprotein G was enhanced to 0.1 ng/well, while the sensitivity of biotin-3.3 Ab not bound to polyprotein G was 2.5 ng/well ( See Figure 14). The results show that polyprotein G does increase the sensitivity of Western blots and improve their detection limits.Instance 5 Polyprotein based G Plate and polyprotein based G Cell plate increases the load of capture antibody Polyprotein-based G Cell plate increases the load of capture antibody Polyprotein G cells were immobilized on the plate to produce a multi-protein G cell-based plate. Will be 0.0123μ g/mL, 0.0371μ g/mL, 0.11μ g/mL, 0.33μ g/mL and 1μ g/mL biotin-3.3 Ab (an anti-PEG capture antibody, IgG type) was added to a multi-protein G cell-based plate, a conventional polystyrene-based plate, and a commercial protein G-based plate, respectively. Streptavidin-HRP and ABTS for coloration catalysis were then added to the plates. The resulting mixture was measured at O.D. 405 nm. The result shows that at 0.0123μ g/mL, 0.0371μ g/mL, 0.11μ g/mL, 0.33μ g/mL and 1μ At g/mL biotin-3.3Ab, the amount of antibody loaded on a multi-protein G cell-based plate was 18, 9.5, 6, 3.5, and 1.8 times higher than that of a conventional polystyrene-based plate, respectively. Commercially based on Protein G's boards, they were 3.5, 5.1, 2.4, 1.3 and 1.1 times higher, respectively. The results showed that the amount of antibody loaded on the polyprotein G cell-based plate was significantly higher than that of the conventional polystyrene-based plates and commercial protein G-based plates (see Figure 15).Polyprotein-based G Plate increases the load of capture antibody The polyprotein G was immobilized on a plate to produce a polyprotein G-based plate. Will be 0.0123μ g/mL, 0.0371μ g/mL, 0.11μ g/mL, 0.33μ g/mL and 1μ g/mL biotin-3.3Ab was added to a polyprotein G-based plate, a conventional polystyrene-based plate, and a commercial protein G-based plate, respectively. Streptavidin-HRP and ABTS for coloration catalysis were then added to the plates. The resulting mixture was measured at O.D. 405 nm. The result shows that at 0.0123μ g/mL, 0.0371μ g/mL, 0.11μ g/mL, 0.33μ g/mL and 1μ Under g/mL biotin-3.3Ab, the amount of antibody loaded on the polyprotein G-based plate was 14, 20, 10, 2.2, and 1.3 times higher than that of the conventional polystyrene-based plate, respectively, and was more commercial. The plates based on Protein G were 7.8, 5.8, 4, 1.3 and 1.15 times higher, respectively. The results showed that the amount of antibody loaded on the polyprotein G-based plate was significantly higher than that of the conventional polystyrene-based plates and commercial protein G-based plates (see Figure 16).Instance 6 Polyprotein based G Cell plate increases the binding amount of multiple antigen analytes Polyprotein-based G Increase in cell plate CTL4 Antigen binding amount Will be 0.0371μ g/mL, 0.11μ g/mL, 0.33μ g/mL, 1μ g/mL and 3μ g/mL of anti-CTLA4 antibodies were added to multi-protein G cell-based plates, traditional polystyrene-based plates, and commercial protein G-based plates, respectively. Will be 1μ g/mL of CTLA4-biotin, streptavidin-HRP and ABTS for coloration catalysis were sequentially added to the plate. The resulting mixture was measured at O.D. 405 nm. The result is displayed at 0.0371μ g/mL, 0.11μ g/mL, 0.33μ g/mL, 1μ The amount of CTLA4-biotin bound to the anti-CTLA4 antibody in the polyprotein G cell-based plate was significantly higher than that of the conventional polystyrene-based plate under g/mL and 3 μg/mL anti-CTLA4 antibody. At 0.0371μ g/mL and 0.11μ The amount of CTLA4-biotin bound to the anti-CTLA4 antibody in the polyprotein G cell-based plate was significantly higher than that of the commercial protein G-based plate under g/mL of anti-CTLA4 antibody (see Figure 17). Will be 1μ g/mL or 0.1 μg/mL anti-CTLA4 antibodies were added to multi-protein G cell-based plates, traditional polystyrene-based plates, and commercial protein G-based plates, respectively. Then, respectively, 0.0371μ g/mL, 0.11μ g/mL, 0.33μ g/mL, 1μ g/mL and 3 μg/mL of CTLA4-biotin were added to the plates. Streptavidin-HRP and ABTS for coloration catalysis were sequentially added to the plate. The resulting mixture was measured at O.D. 405 nm. The results show that at high concentrations (1μ g/mL) anti-CTLA4 antibody, CTLA4-biotin (0.0371) bound to anti-CTLA4 antibody in multi-protein G cell-based platesμ g/mL, 0.11μ g/mL, 0.33μ g/mL, 1μ The amount of g/mL and 3 μg/mL) is similar to that of commercial protein G-based plates, but significantly higher than conventional polystyrene-based plates (see Figure 18). However, at low concentrations (0.1μ g/mL) anti-CTLA4 antibody, CTLA4-biotin (0.0371) bound to anti-CTLA4 antibody in multi-protein G cell-based platesμ g/mL, 0.11μ g/mL, 0.33μ g/mL, 1μ The amount of g/mL and 3 μg/mL) was 6.5, 2.4, 2.2, 1.3, and 1.1 times higher than that of the commercial protein G-based plate, while the analyte was not detected by conventional polystyrene-based plates (see figure). 19).Polyprotein-based G Increase in cell plate PEG- Biotin binding amount Will be 0.0371μ g/mL, 0.11μ g/mL, 0.33μ g/mL, 1μ g/mL and 3 μg/mL of 3.3 Ab (an anti-PEG capture antibody, IgG type) were added to multi-protein G cell-based plates, conventional polystyrene-based plates, and commercial protein G-based plates, respectively. 1 μg/mL of PEG5K-biotin, streptavidin-HRP and ABTS for coloration catalysis were sequentially added to the plate. The resulting mixture was measured at O.D. 405 nm. The result is displayed at 0.0371μ g/mL, 0.11μ g/mL, 0.33μ g/mL, 1μ g/mL and 3μ The amount of PEG5K-biotin (1 μg/mL) bound to the anti-PEG antibody in the multi-protein G cell-based plate was significantly higher than that of the commercial protein-based plate and the traditional based on g/mL anti-PEG antibody. The amount of polystyrene board (see Figure 20).Polyprotein-based G Increase in cell plate PEG2K-Lipo-Dox Combination amount Will be 1μ g/mL of 3.3 Ab (an anti-PEG capture antibody, IgG type) was added to a multi-protein G cell-based plate and a conventional polystyrene-based plate, respectively. Will be 0.0256μ g/mL, 0.128μ g/mL, 3.2μ g/mL, 80μ g/mL and 400μ g/mL of PEG2K-Lipo-Dox was added to the plates, respectively. The binding amount of PEG2K-Lipo-Dox to multi-protein G cell-based plates and conventional polystyrene-based plates was measured by a fluorescence analyzer. The results showed that the amount of PEG2K-Lipo-Dox bound to the anti-PEG antibody in the polyprotein G cell-based plate was significantly higher than that of the conventional polystyrene-based plate (see Figure 21).Instance 7 Polyprotein based G Cell board greatly increases competition ELISA Sensitivity Will be 0.1μ g/mL of anti-CTLA4 antibodies were added to multi-protein G cell-based plates, traditional polystyrene-based plates, and commercial protein G-based plates, respectively. CTL4 analyte and CTLA4-biotin were added to the plate to competitively bind to the binding site of the anti-CTLA4 antibody. Streptavidin-HRP and ABTS were then added to the plates. The resulting mixture was measured at O.D. 405 nm. The results showed that CTL4 analytes were indeed detectable on multi-protein G cell-based plates, and the absorbance intensity of plates based on multi-protein G cells was significantly higher than that of conventional polystyrene-based plates, whereas traditional polystyrene-based plates could not be detected. Go to CTLA4 (Figure 22). Will be 1μ g/mL of anti-PEG antibody was added to a multi-protein G cell-based plate and a conventional polystyrene-based plate, respectively. PEG analytes and PEG-biotin were added to the plates to competitively bind to the binding sites of the anti-PEG antibodies. Streptavidin-HRP and ABTS were then added to the plates. The resulting mixture was measured at O.D. 405 nm. The results showed that PEG5K and PEG750 were detectable at 0.01 nM based on polyprotein G cell plates (Fig. 23, right panel), whereas PEG was not detectable on conventional polystyrene based plates (Fig. 23, left panel).Instance 8 Polyprotein G Cell plate greatly increases the sandwich ELISA Sensitivity Will be 1μ g/mL of 15.2 Ab (an anti-PEG antibody, IgG type) was added to a multi-protein G cell-based plate, a conventional polystyrene-based plate, and a commercial protein G-based plate, respectively. 0.1 nM, 1 nM, 10 nM and 100 nM PEG10K were added to the plates. AGP4-biotin (a biotin-conjugated anti-PEG detection antibody, IgM type), streptavidin-HRP, and ABTS were sequentially added to the plate. The resulting mixture was measured at O.D. 405 nm. The results showed that PEG10K was detectable on multi-protein G cell-based plates, whereas traditional polystyrene-based plates and commercial protein-based plates were not able to efficiently detect PEG10K (Figure 24). Will be 1μ g/mL of 15.2 Ab (an anti-PEG antibody, IgG type) was added to a multi-protein G cell-based plate, a conventional polystyrene-based plate, and a commercial protein G-based plate, respectively. PEG2K-Lipo-Dox of 0.1 nM, 1 nM, 10 nM, 100 nM and 1000 nM was added to the plate. AGP4-biotin (a biotin-conjugated anti-PEG detection antibody, IgM type), streptavidin-HRP, and ABTS were sequentially added to the plate. The resulting mixture was measured at O.D. 405 nm. The results show that the absorbance values of plates based on polyprotein G cells are significantly higher than those of conventional polystyrene based plates and commercial protein G based plates (Figure 25). Will be 1μ g/mL of 15.2 Ab (an anti-PEG antibody, IgG type) was added to a multi-protein G cell-based plate, a conventional polystyrene-based plate, and a commercial protein G-based plate, respectively. Picoxin (a PEG-modified protein drug) of 0.00021 nM, 0.002 nM, 0.02 nM, 0.2 nM, and 2 nM was added to the plate. AGP4-biotin (a biotin-conjugated anti-PEG detection antibody, IgM type), streptavidin-HRP, and ABTS were sequentially added to the plate. The resulting mixture was measured at O.D. 405 nm. The results showed that the absorbance values of the plates based on polyprotein G cells were significantly higher than those of commercial protein G-based plates, whereas the traditional polystyrene-based plates could not detect PEGASYS (Fig. 26). Will be 1μ g/mL of 15.2 Ab (an anti-PEG antibody, IgG type) was added to a multi-protein G cell-based plate, a conventional polystyrene-based plate, and a commercial protein G-based plate, respectively. 0.0002 nM, 0.001 nM, 0.01 nM, 0.1 nM, and 1 nM PEG2K-quantum dots were added to the plates. AGP4-biotin (a biotin-conjugated anti-PEG detection antibody, IgM type), streptavidin-HRP, and ABTS were sequentially added to the plate. The resulting mixture was measured at O.D. 405 nm. The results show that the absorbance values of the plates based on polyprotein G cells are significantly higher than those of conventional polystyrene based plates and commercial protein G based plates (Figure 27). In view of the above, multi-protein G cell-based plates can be effectively used in immunoassays and provide the sensitivity of unexpected immunoassays.Instance 9 Unpurified capture antibodies can be used directly on polyproteins G Cell plate for immunoassay Will be 1μ g/mL of purified 15.2 Ab (an anti-PEG capture antibody, IgG type) and ascites -15.2 Ab (unpurified) were added to polyprotein G cell-based plates and conventional polystyrene-based plates, respectively. PEG2K-Lipo-Dox of 0.1 nM, 1 nM, 10 nM, 100 nM and 1000 nM was added to the plate. AGP4-biotin (a biotin-conjugated anti-PEG detection antibody, IgM type), streptavidin-HRP, and ABTS were sequentially added to the plate. The resulting mixture was measured at O.D. 405 nm. The results showed that the conventional polystyrene-based plate could detect PEG2K-Lipo-Dox when purified with 15.2 Ab, and it could not detect PEG2K-Lipo-Dox when using ascites -15.2 Ab (unpurified). However, polyprotein G cell-based plates were effective in detecting PEG2K-Lipo-Dox using purified 15.2 Ab or ascites-15.2 Ab (unpurified) (Fig. 28). This demonstrates that the use of unpurified capture antibodies in multi-protein G cell-based plates does not affect detection sensitivity, so protein G cell-based plates have significantly higher sensitivity and can reduce antibody purification steps to save cost.

Figure 1 shows the gene sequence of the polyprotein G of the present invention, which is expressed on the cell membrane. For protein G cells, the sequence includes human influenza hemagglutinin (HA), protein G (C2), linker (L), and mB7 from the N-terminus to the C-terminus. For polyprotein G cells, the sequence includes HA, C2, L, C2, L, C2, L, C2, L, C2, L, C2, L, C2, L, C2, L and mB7 from the N-terminus to the C-terminus. Figure 2 shows the expression of protein G in protein G cells and polyprotein G cells by western blots. Lane 1: 3T3 cells; Lane 2: protein G cells; and lane 3: polyprotein G cells. Figure 3 shows the ability of protein G cells and multiprotein G cells to capture antibodies. Left panel: 3T3 cells; middle panel: protein G cells; and right panel: polyprotein G cells. Figure 4 shows the antigen binding ability of antibodies captured by protein G cells and polyprotein G cells. Left panel: 3T3 cells; middle panel: protein G cells; and right panel: polyprotein G cells. Figure 5 shows the gene sequence of the polyprotein G of the present invention, which is expressed on the membrane of bacteria. For protein G cells, the sequence includes human influenza hemagglutinin (HA), protein G (C2), linker (L), and AIDA from the N-terminus to the C-terminus. For polyprotein G cells, the sequences include HA, C2, L, C2, L, C2, L, C2, L, C2, L, C2, L, C2, L, C2, L, and AIDA from the N-terminus to the C-terminus. Figure 6 shows the expression of protein G in protein G bacteria and polyprotein G bacteria by Western blots. Lane 1: BL21 bacteria; Lane 2: Protein G bacteria; and Lane 3: Polyprotein G bacteria. Figure 7 shows the ability of protein G bacteria and multi-protein G bacteria to capture antibodies. Figure 8 shows the gene sequence of the polyprotein G of the present invention. The sequence includes HA, C2, L, C2, L, C2, L, C2, L, C2, L, C2, L, C2, L, C2, L and 6 histidine acids from the N-terminus to the C-terminus. Figures 9A and 9B show Western blots (A) and SDS-PAGE (B) of polyprotein G. Lane 1: PBS as a negative control; and Lane 2: polyprotein G. Figure 10 shows the ability of polyprotein G to capture antibodies. Figures 11a and b show the sensitivity (error bars: mean = /- SD) of IFN-α (a) and PEGASYS (PEGASYS) (b) detected by using multi-protein G bacteria in a sandwich ELISA. Figures 12a and b show the sensitivity (error bars: mean = /- SD) of IFN-α (a) and PEGASYS (b) detected by using polyprotein G in a sandwich ELISA. Figure 13 shows that polyprotein G bacteria increase the sensitivity of Western blots and improve their detection limits. Figure 14 shows that polyprotein G increases the sensitivity of Western blots and improves their detection limits. Figure 15 shows capture antibody loading (error bars: mean = /- SD) based on polyprotein G cell plates, conventional polystyrene based plates, and commercial protein G based plates. Figure 16 shows capture antibody loading (error bars: mean = /- SD) based on polyprotein G plates, conventional polystyrene based plates, and commercial protein G based plates. Figure 17 shows CTLA4-biotin binding of multi-protein G cell-based plates, conventional polystyrene-based plates, and commercial protein G-based plates that capture serially diluted anti-CTLA4 antibodies at the same CTLA4-biotin concentration. (Error Bar: Average = / - SD). Figure 18 shows CTLA4-biotin binding of multi-protein G cell-based plates, traditional polystyrene-based plates, and commercial protein G-based plates that capture high concentrations of anti-CTLA4 antibodies at serially diluted CTLA4-biotin concentrations. Quantity (error bar: average = / - SD). Figure 19 shows CTLA4-biotin binding of multi-protein G cell-based plates, traditional polystyrene-based plates, and commercial protein G-based plates that capture low concentrations of anti-CTLA4 antibodies at serially diluted CTLA4-biotin concentrations. Quantity (error bar: average = / - SD). Figure 20 shows PEG5K-biotin binding of multi-protein G cell-based plates, conventional polystyrene-based plates, and commercial protein G-based plates that capture serially diluted anti-PEG antibodies at the same PEG5K-biotin concentration. (Error Bar: Average = / - SD). Figure 21 shows the amount of PEG2K-LipoDox binding of multi-protein G cell-based plates and conventional polystyrene-based plates that capture anti-PEG antibodies at serially diluted PEG2K-Lipo-Dox concentrations (error bars: mean = / - SD). Figure 22 shows the detection sensitivity of CTLA4 on multi-protein G cell-based plates, conventional polystyrene-based plates, and commercial protein G-based plates in a competition ELISA (error bars: mean = /- SD). Figure 23 shows the detection sensitivity of PEG 5K and PEG 750 based on polyprotein G cell plates and conventional polystyrene based plates in a competition ELISA (error bars: mean = / - SD). Figure 24 shows the detection sensitivity of PEG10K based on polyprotein G cell plates, conventional polystyrene based plates, and commercial protein G based plates in a sandwich ELISA (error bars: mean = / - SD). Figure 25 shows the detection sensitivity of PEG2K-Lipo-Dox based on polyprotein G cell plates, conventional polystyrene based plates, and commercial protein G based plates in a sandwich ELISA (error bars: mean = / - SD ). Figure 26 shows the detection sensitivity of the PEGASYS (a PEG-modified protein drug) based on a multi-protein G cell plate, a conventional polystyrene-based plate, and a commercial protein G-based plate in a sandwich ELISA. : Average = / - SD). Figure 27 shows the detection sensitivity of PEG2K-quantum dots based on polyprotein G cell plates, conventional polystyrene-based plates, and commercial protein G-based plates in a sandwich ELISA (error bars: mean = /- SD) . Figure 28 shows the detection sensitivity of PEG2K-Lipo-Dox based on multi-protein G cell plates and conventional polystyrene-based plates using unpurified capture antibodies and purified capture antibodies (error bars: mean = / - SD ).

Claims (28)

A tandem repetitive protein comprising at least two repeats of an amino acid sequence of an antibody IgG binding protein or fragment thereof and one or more linkers that link the repeats to each other. The tandem repetitive protein of claim 1, wherein the antibody IgG binding protein is a protein A, protein G, protein A/G, protein L or Fc receptor or a fragment, combination or fragment thereof. A tandem repetitive protein according to claim 1 which comprises from 2 to 20 repeats of the amino acid sequence of protein A, protein G, protein A/G, protein L or Fc receptor or a fragment, combination or combination thereof. The tandem repetitive protein of claim 1, wherein the amino acid sequence comprises the sequence of a C1, C2 or C3 fragment of protein G. The tandem repetitive protein of claim 1, wherein the amino acid sequence comprises the sequence of the C2 fragment of protein G. The tandem repetitive protein of claim 1, wherein the amino acid sequence comprises the amino acid sequence of SEQ ID NO: 1. The tandem repetitive protein of claim 1, wherein the tandem repetitive protein comprises 8 repeats of the amino acid sequence of SEQ ID NO: 1. The tandem repetitive protein of claim 1, wherein the linkers used in the tandem repetitive protein are the same or different. The tandem repetitive protein of claim 1, wherein the linker comprises an amino acid sequence of GGGSG (SEQ ID NO: 2) or GGGGSGGGGSV (SEQ ID NO: 3). The tandem repetitive protein of claim 1, wherein the tandem repetitive protein comprises 8 repeats of the amino acid sequence of protein G or a fragment thereof and the amino acid comprising SEQ ID NO: 2 or SEQ ID NO: The linker of the sequence. The tandem repetitive protein of claim 1, wherein the tandem repetitive protein comprises 8 repeats of the amino acid sequence of SEQ ID NO: 1, and the repeats are ligated to each other comprising SEQ ID NO: 2 or SEQ ID NO: a linker of the amino acid sequence of 3. The tandem repetitive protein of claim 1, wherein the tandem repetitive protein comprises the amino acid sequence of SEQ ID NO: 4. A cell comprising a tandem repetitive protein of claim 1 which is expressed on the cell membrane, wherein the tandem repetitive protein is fused to a transmembrane protein of the cell. An antibody-repetitive protein complex comprising a plurality of antibodies or fragments thereof that bind to a tandem repetitive protein of claim 1 or to a cell of claim 13. The antibody-repetitive protein complex of claim 14, wherein the anti-system detects the antibody or capture antibody. A method for detecting an analyte in a sample, comprising using a tandem repetitive protein of claim 1, a cell of claim 13 or an antibody of claim 15 in an immunoassay or antibody-coated immunoassay - The repetitive protein complex is used to capture the analyte in the sample and to qualitatively or quantitatively detect the analyte. The method of claim 16, comprising the steps of: providing a solid support coated with an analyte as appropriate; binding the plurality of detection antibodies to the tandem repetitive protein of claim 1 or the cell of claim 13 Forming a detection antibody complex; binding the detection antibody complex to the analyte coated in the solid support; and qualitatively or quantitatively detecting the analyte. The method of claim 16, comprising the steps of: providing a solid support; immobilizing the capture antibody on the solid support; capturing the analyte in the sample by the capture antibody; and binding the plurality of detection antibodies to the request a tandem repetitive protein or a cell of claim 14 to form a detection antibody complex; the detection antibody complex is added to bind to the analyte; and the analyte is qualitatively or quantitatively detected. The method of claim 16, comprising the steps of: providing a solid support; immobilizing the tandem repetitive protein of claim 1 or the cell of claim 13 on the solid support; and binding the plurality of capture antibodies to the longitudinal Sorting the repetitive protein or the cell; capturing the analyte in the sample by capturing the antibody complex; adding a detection antibody to bind to the analyte; and qualitatively or quantitatively detecting the analyte. The method of claim 16, comprising the steps of: providing a solid support; immobilizing the tandem repetitive protein G of claim 1 or a fragment thereof or the cell of claim 13 on the solid support; Binding to the tandem repetitive protein or the cell to form a capture antibody complex; mixing the signal-labeled analyte having a predetermined concentration with the analyte in the sample to form a mixture; by the capture antibody complex The analyte of the mixture (signal labeled analyte/analyte in the sample) is captured; and the analyte is qualitatively or quantitatively detected. A kit for detecting an analyte in a sample, comprising a solid carrier coated with an antigen, the analyte or a capture antibody, as appropriate; and a tandem repetitive protein of claim 1 or as claimed in claim 13 cell. A kit of claim 21, further comprising a detection antibody or a capture antibody. A kit of claim 22, wherein the detection antibody is further labeled. A kit of claim 21, wherein the solid support is a pellet, bead, assay plate or test tube. A kit according to claim 21, wherein the solid support is a 96-well microtiter plate. A kit according to claim 21, which is for use in a western blot or ELISA, comprising a solid support coated with an analyte as appropriate and a tandem repetitive protein of claim 1 or as claimed in claim 13 cell. A kit according to claim 21, which is for use in a sandwich ELISA comprising a solid support; and a tandem repetitive protein of claim 1 or a cell of claim 13. A kit of claim 21 for use in a competition ELISA comprising a solid carrier coated with a tandem repetitive protein of claim 1 or a cell of claim 13; and tandem repeatability as claimed in claim 1. Protein or cell as claimed in item 13.