Antibody complex, preparation method thereof, detection reagent and application

Abstract

The invention discloses a compound which comprises a bridging protein, and an antibody of a to-be-detected object or an antigen binding fragment of the antibody, which is connected to the bridging protein. The compound can be well suitable for biochemical reagents, especially immunoturbidimetric detection reagents such as glycosylated hemoglobin, complement C1q protein, a B factor, apolipoprotein E, a kappa light chain and a lambda light chain, can avoid use of a second antibody when being applied to the glycosylated hemoglobin detection reagents, reduces unstable factors, has good detection sensitivity, and can be used for preparing immunoturbidimetric detection reagents for detecting the glycosylated hemoglobin. Compared with a conventional biochemical detection method, the method has the advantages that the detection performance is remarkably improved, and the clinical application value is extremely high.

Description

Technical Field

[0001] The present invention relates to the field of medical testing, and in particular to an antibody complex and a preparation method, a detection reagent and applications thereof. Background Art

[0002] Currently, biochemical test reagents on the market are mainly divided into enzymatic test reagents and immunoturbidimetric test reagents. Immunoturbidimetric analysis is an analytical technique that combines precipitation reactions in the liquid phase with optical instruments and automated analysis technology. It uses the reaction of antigens and antibodies in a special dilution system to form turbidity. The content of the analyte in the sample can be calculated by correlating the turbidity with the content of the sample to be tested. Based on immunoturbidimetric analysis, latex-enhanced immunoturbidimetric analysis was derived to further improve the accuracy of test reagents. Its principle is to coat the antibody corresponding to the analyte on latex particles with a diameter of 10nm-1μm, increasing the volume of the antigen-antibody complex. After light passes through, the intensity changes of transmitted light and scattered light are more significant, thereby increasing the sensitivity of the test.

[0003] Glycated hemoglobin (HbA1c) is the product of the covalent binding of hemoglobin and glucose in human red blood cells. It is proportional to the glucose concentration in the blood and can reflect the blood glucose level of the human body 120 days ago. Measuring the concentration of glycated hemoglobin in whole blood is of great significance for the screening and monitoring of diabetes. Currently, the mainstream clinical reagent for measuring glycated hemoglobin is the latex-enhanced immunoturbidimetric method, which includes reagent one (containing latex microspheres) and reagent two (containing a monoclonal antibody for glycated hemoglobin and a secondary antibody that recognizes the monoclonal antibody for glycated hemoglobin, which is often a polyclonal antibody, and the two exist in the form of a complex). This method determines the percentage of glycated hemoglobin in total hemoglobin, has few interfering factors, and can be used with a biochemical analyzer for high-throughput testing. The specific protocol is as follows: The latex microspheres in Reagent 1 of the latex immunoturbidimetric kit are mixed with the whole blood to be tested. The latex microspheres fully adsorb the hemoglobin and glycated hemoglobin in the blood. Reagent 2 is then added to the kit. The glycated hemoglobin monoclonal antibody and secondary antibody complex in Reagent 2 specifically bind to the glycated hemoglobin on the microsphere surface. The bridging effect of the secondary antibody in the complex triggers microsphere aggregation, which in turn detects the glycated hemoglobin content through changes in particle turbidity. If the secondary antibody is a monoclonal antibody rather than a polyclonal antibody, it will only bind to the glycated hemoglobin monoclonal antibody at two sites, and its ability to exert a bridging effect is very limited. This means that the aggregated particle size of the "latex microsphere-glycated hemoglobin-glycated hemoglobin monoclonal antibody-secondary antibody" formed during the test is limited, resulting in insufficient detection sensitivity. Moreover, since the glycated hemoglobin antibody and the secondary antibody coexist in the same system for a long time, the interaction between the two may cause unstable tests. Many manufacturers package the glycated hemoglobin monoclonal antibody and the secondary antibody separately, namely reagent two and reagent three, and have the inspectors mix and test them on-site. The operation is cumbersome, increases operational errors, and causes differences in test results. Summary of the Invention

[0004] To address the above issues, the present invention provides a complex that is well-suited for the detection of glycated hemoglobin, eliminating the need for a secondary antibody, reducing instability, and exhibiting excellent sensitivity. Compared to conventional biochemical assays, it significantly improves the high-dose detection threshold. Furthermore, the inventors have discovered that the complex is also suitable for use in other biochemical assays, such as complement C1q protein, factor B, apolipoprotein E, kappa light chain, and lambda light chain immunoturbidimetric assays, significantly improving detection accuracy.

[0005] In order to achieve the above purpose, the present invention adopts the following technical means:

[0006] In one aspect, the present invention provides a bridging protein, wherein the bridging protein is selected from at least one of Protein A, Protein G, a protein obtained by performing any mutation optimization based on the amino acid sequence of Protein A, and a protein obtained by performing any mutation optimization based on the amino acid sequence of Protein G.

[0007] Preferably, the amino acid sequence of the bridging protein is selected from at least one of SEQ ID NO: 2 to SEQ ID NO: 5.

[0008] In another aspect, the present invention provides a complex comprising the bridging protein as described above, and an antibody to be detected or an antigen-binding fragment of the antibody to be detected linked to the bridging protein.

[0009] Preferably, the complex comprises 2-60 analyte antibodies or antigen-binding fragments thereof. Preferably, the analyte antibodies are monoclonal antibodies.

[0010] Preferably, the complex is a spherical complex, and preferably, the particle size of the complex is 15-60 nm.

[0011] Preferably, the titer of the complex is 4-60, preferably, the titer of the complex is 4, 12, 24, 60.

[0012] In another aspect, the present invention provides a method for preparing the above-mentioned composite, comprising the following steps:

[0013] (1) mixing the antibody to be tested or its antigen-binding fragment with the bridging protein;

[0014] (2) Purify the above mixture.

[0015] Preferably, in step (1), after mixing the antibody or antigen-binding fragment thereof to be tested with the bridging protein, the mixture is allowed to stand for 1-2 hours and then dialyzed.

[0016] Preferably, the purification in step (2) is to first perform ultrafiltration concentration on the mixture in step (1), and then perform molecular sieve purification.

[0017] In another aspect, the present invention provides a detection reagent, which includes the bridging protein as described above, the complex as described above, or the complex prepared by the preparation method as described above.

[0018] Preferably, the detection reagent is an immunoturbidimetric detection reagent, and more preferably, the reagent is a detection reagent for glycated hemoglobin, complement C1q protein, factor B, apolipoprotein E, kappa light chain, lambda light chain, etc.

[0019] In an optional embodiment, the detection reagent is a glycated hemoglobin detection reagent, including reagent R1 and reagent R2, and the reagent R2 includes the complex as described above, or the complex prepared by the preparation method as described above.

[0020] Preferably, the glycated hemoglobin detection reagent R1 includes latex microspheres and a buffer solution, and the reagent R2 also includes a buffer solution.

[0021] Preferably, the particle size of the latex microspheres is 100-300 nm.

[0022] Preferably, the reagent R1 further comprises a surfactant and a preservative, and the reagent R2 further comprises an inorganic salt, a surfactant, a stabilizer and a preservative.

[0023] Furthermore, in reagent R1, the buffer is selected from at least one of 4-hydroxyethylpiperazineethanesulfonic acid, borate, carbonate, citrate, Tris, and Good's buffer, the surfactant is selected from at least one of Tween 20, Tween 80, cocamidopropyl betaine, imidazole, phosphate betaine, and carboxylate betaine, and the preservative is selected from at least one of sodium azide, ProClin300, ProClin500, benzoates, and gentamicin.

[0024] Furthermore, in reagent R2, the buffer is selected from at least one of 4-hydroxyethylpiperazineethanesulfonic acid, borate, carbonate, citrate, Tris, and Good's buffer, the inorganic salt is selected from at least one of sodium sulfate, sodium chloride, potassium sulfate, and ammonium chloride, the surfactant is selected from at least one of Tween 20, Tween 80, cocamidopropyl betaine, imidazole, phosphate betaine, and carboxylate betaine, the stabilizer is selected from at least one of bovine serum albumin, sucrose, trehalose, mannitol, glycerol, and EDTA, and the preservative is selected from at least one of sodium azide, ProClin300, ProClin500, benzoates, and gentamicin.

[0025] Preferably, the pH of the reagent R1 is 6.0-9.0, and the pH of the reagent R2 is 5.0-9.0.

[0026] Furthermore, the pH of the reagent R1 is 6.5-8.5, and the pH of the reagent R2 is 5.5-8.5.

[0027] In an optional embodiment, the detection reagent is a complement C1q protein detection reagent, including reagent R1 and reagent R2, and the reagent R2 includes the complex described above, or the complex prepared by the preparation method described above.

[0028] In an optional embodiment, the detection reagent is a factor B detection reagent, including reagent R1 and reagent R2, and the reagent R2 includes the complex described above, or the complex prepared by the preparation method described above.

[0029] In an optional embodiment, the detection reagent is an apolipoprotein E detection reagent, including reagent R1 and reagent R2, and the reagent R2 includes the complex as described above, or the complex prepared by the preparation method as described above.

[0030] In an optional embodiment, the detection reagent is a κ light chain detection reagent, including reagent R1 and reagent R2, and the reagent R2 includes the complex as described above, or the complex prepared by the preparation method as described above.

[0031] In an optional embodiment, the detection reagent is a lambda light chain detection reagent, comprising reagent R1 and reagent R2, wherein the reagent R2 comprises the complex as described above, or the complex prepared by the preparation method as described above.

[0032] The beneficial effects of the present invention are as follows: by introducing a special antibody complex into the biochemical turbidimetric reagent project, the present invention can significantly improve the accuracy of reagent detection, and is suitable for immunoturbidimetric detection reagents such as glycated hemoglobin, complement C1q protein, B factor, apolipoprotein E, kappa light chain, and lambda light chain. In particular, when used in glycated hemoglobin detection reagents, the use of secondary antibodies can be avoided, reducing unstable factors, and also avoiding the cumbersome operation caused by some existing glycated hemoglobin detection kits that separate the glycated hemoglobin antibody and the secondary antibody into reagent two and reagent three, resulting in large operational errors and result differences. In addition, the present invention has good detection sensitivity, significantly improves detection performance compared with conventional biochemical detection methods, and has extremely high clinical application value. Specific embodiments

[0033] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0034] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which this disclosure belongs. The methods and materials described herein, as well as any similar or equivalent methods and materials, can be used in the practice or testing of the formulations or unit doses herein. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not restrictive.

[0036] As used herein, the terms "includes," "including," "having," "may," and variations thereof are intended to be open-ended, transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0037] definition

[0038] Unless otherwise specified, the terms used herein have the following meanings.

[0039] Antibodies are a class of immunoglobulins that can specifically bind to antigens, including monoclonal antibodies, engineered antibodies, chemically synthesized antibodies or recombinant antibodies, polyclonal antibodies, multivalent antibodies, and multispecific antibodies. In one embodiment, the present application relates to a monoclonal or polyclonal antibody. The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population; in other words, except for natural mutations that may exist in minimal amounts, the individual antibodies in the antibody population are considered to be identical. Monoclonal antibodies are highly specific for a single epitope. Such monoclonal antibodies can be produced by monoclonal B cells or hybridomas. Monoclonal antibodies can also be recombinant, that is, produced by protein engineering. Monoclonal antibodies can also be isolated from a phage antibody library.

[0040] Antigen-binding fragment is intended to mean any peptide, polypeptide, or protein that retains the ability of an antibody to bind to its target (also commonly referred to as an antigen). In one embodiment, such an "antigen-binding fragment" is selected from the group consisting of: Fv, scFv (sc stands for single chain), Fab, F(ab')2, Fab', scFv-Fc fragment, or bispecific antibody. The antigen-binding fragment has at least one CDR derived from its intact antibody.

[0041] The complex of the present invention

[0042] On one hand, the present invention provides a complex that can be used for detecting analytes in biological samples, and is particularly suitable for use as a biochemical latex turbidimetric detection reagent. The complex has better detection performance than traditional biochemical latex turbidimetric detection reagents.

[0043] In some embodiments, a complex is provided, comprising a bridging protein and at least two analyte antibodies or antigen-binding fragments thereof coupled to the bridging protein.

[0044] In some specific embodiments, the bridging protein can be selected from at least one of Protein A, Protein G, a protein obtained by any mutation based on the amino acid sequence of Protein A, and a protein obtained by any mutation based on the amino acid sequence of Protein G. The mutated protein can regularly non-covalently bind to human, mouse, or rabbit IgG antibodies to form a globular protein complex, and the region in the formed globular protein complex where the antibody binds to the corresponding antigen is exposed on the outside of the globule.

[0045] More specifically, bridging proteins are multi-polypeptide polymers that contain three domains: ① an Fc-binding domain; ② an oligomerization domain, which binds through non-covalent interactions to form polymers, such as dimers with C2-cycle symmetry, trimers with C3-cycle symmetry, tetramers with C4-cycle symmetry, or pentamers with C5-cycle symmetry; and ③ a monomeric polypeptide, which helps position the Fc-binding domain and oligomerization domain in the correct orientation to promote the formation of a globular complex.

[0046] In some specific embodiments, oligomer domains can self-associate through non-covalent interactions to form homopolymers of the same polypeptide.

[0047] In some specific embodiments, each monomeric polypeptide has two interfaces: ① an Fc binding interface and ② an oligomerization domain interface. Upon expression, the monomeric polypeptides of the present invention form symmetrical C2, C3, C4, and C5 circular oligomers through the cognate hook domains, i.e., the bridging proteins of the present invention.

[0048] In some specific embodiments, the bridging protein may comprise polypeptide monomers with certain amino acid differences, or all polypeptide monomers in the bridging protein may be identical and be homopolymers. The bridging protein may be a dimer, trimer, tetramer, or pentamer.

[0049] In some specific embodiments, when the polymer is a dimer, its monomeric polypeptides are selected from amino acids having at least 90% homology with the amino acid sequence of SEQ ID NO:2.

[0050] In some specific embodiments, when the polymer is a trimer, its monomeric polypeptides are selected from amino acids having at least 90% homology with the amino acid sequence of SEQ ID NO:3.

[0051] In some specific embodiments, when the polymer is a tetramer, its monomeric polypeptides are selected from amino acids having at least 90% homology with the amino acid sequence of SEQ ID NO:4.

[0052] In some specific embodiments, when the polymer is a pentamer, its monomeric polypeptides are selected from amino acids having at least 90% homology with the amino acid sequence of SEQ ID NO:5.

[0053] The complex of the present invention is formed by connecting multiple identical bridging proteins and multiple antibodies through non-covalent binding. The polyhedral structure is formed through the interaction between the Fc domain of the antibody and the Fc binding interface of the bridging protein, and the spherical complex is spontaneously assembled.

[0054] In some specific embodiments, the antibody or antigen-binding fragment thereof non-covalently binds to a polypeptide monomer of any bridging protein through its first Fc domain, and the antibody non-covalently binds to a polypeptide monomer of another bridging protein through its second Fc domain. More specifically, all polypeptide monomers of all bridging proteins in the complex are non-covalently bound to the Fc domain of the antibody or antigen-binding fragment thereof.

[0055] More specifically, the antibody to be detected can be selected from at least one of monoclonal antibodies, engineered antibodies, chemically synthesized antibodies or recombinant antibodies, polyclonal antibodies, multivalent antibodies, and multispecific antibodies, and is preferably a monoclonal antibody to be detected.

[0056] More specifically, the antigen-binding fragment may be selected from Fv, scFv (sc stands for single chain), Fab, F(ab')2, Fab', scFv-Fc fragment or bispecific antibody.

[0057] In some specific embodiments, when the bridging proteins in the complex are identical dimers, the complex is a dihedral structure assembled by two dimers and two antibodies or antigen-binding fragments thereof.

[0058] In some specific embodiments, when the bridging proteins in the complex are identical trimers, the complex is a tetrahedral structure assembled by four trimers and six antibodies or antigen-binding fragments thereof.

[0059] In some specific embodiments, when the bridging proteins in the complex are identical tetramers, the complex is an octahedral structure assembled by four tetramers and six antibodies or antigen-binding fragments thereof.

[0060] In some specific embodiments, when the bridging proteins in the complex are identical pentamers, the complex is an icosahedral structure assembled by 12 pentamers and 30 antibodies or antigen-binding fragments thereof.

[0061] In some specific embodiments, the complex is a spherical complex.

[0062] In some specific embodiments, the particle size of the complex is 15-60 nm.

[0063] In some specific embodiments, the complex has a titer of 4-60.

[0064] More specifically, the titer of the complex can be 4, 12, 24, or 60.

[0065] Method for producing the composite of the present invention

[0066] One embodiment of the present invention provides a method for preparing the above-mentioned complex.

[0067] Reagents of the present invention

[0068] One embodiment of the present invention provides a glycated hemoglobin detection reagent comprising the above-mentioned complex, which is used for detecting the glycated hemoglobin content in a biological sample.

[0069] More specifically, the reagent of the present invention comprises reagent R1 and reagent R2. In addition to the above-mentioned complex, the reagent of the present invention may also contain other components required for the detection of glycated hemoglobin in biological samples, such as a buffer, a preservative, an ionic strength regulator, etc. The reagent of the present invention may be in the form of an aqueous solution or in other suitable forms that are prepared into a solution with water before use.

[0070] Specifically, the reagent R1 includes latex microspheres and a buffer solution, and the reagent R2 also includes a buffer solution. Preferably, the particle size of the latex microspheres is 100-300 nm.

[0071] Furthermore, the reagent R1 further comprises a surfactant and a preservative, and the reagent R2 further comprises an inorganic salt, a surfactant, a stabilizer and a preservative; preferably, in the reagent R1, the buffer is selected from at least one of 4-hydroxyethylpiperazineethanesulfonic acid, borate, carbonate, citrate, Tris, and Good's buffer, the surfactant is selected from at least one of Tween 20, Tween 80, cocamidopropyl betaine, imidazole, phosphate betaine, and carboxylate betaine, and the preservative is selected from at least one of sodium azide, ProClin300, ProClin500, benzoates, and gentamicin; preferably, the reagent R 2, the buffer is selected from at least one of 4-hydroxyethylpiperazineethanesulfonic acid, borate, carbonate, citrate, Tris, and Good's buffer; the inorganic salt is selected from at least one of sodium sulfate, sodium chloride, potassium sulfate, and ammonium chloride; the surfactant is selected from at least one of Tween 20, Tween 80, cocamidopropyl betaine, imidazole, phosphate betaine, and carboxylate betaine; the stabilizer is selected from at least one of bovine serum albumin, sucrose, trehalose, mannitol, glycerol, and EDTA; and the preservative is selected from at least one of sodium azide, ProClin 300, ProClin 500, benzoates, and gentamicin.

[0072] Furthermore, the pH of the reagent R1 is 6.0-9.0, and the pH of the reagent R2 is 5.0-9.0. Preferably, the pH of the reagent R1 is 6.5-8.5, and the pH of the reagent R2 is 5.5-8.5.

[0073] Furthermore, the concentration of the latex microspheres is 0.01-1%, preferably 0.05-0.5%; the concentration of the buffer is 10-500mM, preferably 50-200mM; the concentration of the surfactant is 0.01-5%, preferably 0.1-2%; the concentration of the preservative is 0.01-5%, preferably 0.1-2%.

[0074] Application of the complex or reagent of the present invention

[0075] One embodiment of the present invention also provides use of the above-mentioned complex or reagent in detecting glycated hemoglobin, complement C1q protein, factor B, apolipoprotein E, kappa light chain, and lambda light chain in biological samples.

[0076] The features and performance of the present invention are further described in detail below with reference to specific embodiments.

[0077] Example 1 Preparation of bridging protein

[0078] a. Optimize the amino acid sequence of Protein A (Sequence ID NO. 1) to obtain four sequences: NPD2 (Seq ID NO. 2), NPT32 (Seq ID NO. 3), NPI52 (Seq ID NO. 4), and NPP52 (Seq ID NO. 5). For each of these sequences, select codons preferred by E. coli for full gene synthesis and embed them into the pET-41b vector. Transform each expression vector into BL21(DE3) cells, screen for positive strains using Ampicillin, and obtain expression strains. Store the constructed expression strains in a -80°C freezer until ready for use.

[0079] b. The strain obtained above was cultured and amplified at 37°C. When the OD 600 value reached 0.6-0.9, IPTG was added for induction. The expression was carried out at 25°C for 16 hours. The cells were collected and broken.

[0080] b. Bacterial cell collection and disruption: Collect cells by centrifugation at 8000 rpm for 15 min, resuspend the cells in 50 mM phosphate buffer, pH 8.0, and disrupt the bacteria in the resuspension using a homogenizer. Maintain low temperature during the disruption process to avoid protein denaturation. Use a 4°C ice bath at a flow rate of 5000 ml / min. Disrupt the cells once at 300 bar and then at 800 bar. Repeat this cycle 3-4 times to collect the cell disruption solution. Centrifuge the collected cell disruption solution at 12500 g for 20 min, and collect the supernatant.

[0081] c. Nickel affinity chromatography: Load the sample between pH 6.5.5-8.5 and elute the target protein using an imidazole gradient. Equilibrate the column with 20mM phosphate buffer, pH 6.5.5-8.5; load the treated solution onto the nickel affinity column. After loading, rinse the column with 20mM phosphate buffer, pH 6.5.5-8.5. After the A280 absorbance reaches a plateau, perform gradient elution using Solution A (20mM phosphate buffer, pH 6.5.5-8.5) and Solution B (20mM phosphate buffer, pH 6.5.5-8.5 containing 0.5M imidazole).

[0082] d. Dialysis: Use 20 mM phosphate buffer, pH 6.5.5-8.5, 1:10 dialyze at 2-8 degrees, dialysis time 6-12 hours, 3-4 dialysis cycles, and harvest the target proteins NPD2, NPT32, NPI52, and NPP52 at the end of dialysis.

[0083] Example 2 Preparation of Glycated Hemoglobin Antibody Complex

[0084] In addition to the above-mentioned four bridging proteins NPD2, NPT32, NPI52 and NPP52, Protein A protein and Protein G (amino acid sequence as shown in Seq ID NO.6) were also selected as bridging proteins for complex coupling reaction, and complexes ag were synthesized respectively. The grouping is shown in the following table:

[0085] Table 1

[0086]

[0087]

[0088] *Except for the selection of the bridging protein, the preparation steps of complex ag are exactly the same.

[0089] The specific steps for preparing the complex are as follows:

[0090] a. The six bridging proteins and glycated hemoglobin monoclonal antibodies were dialyzed into a buffer solution of 50 mM Tris-HCl, 150 mM NaCl, 100 mM L-arginine, pH = 8.0. After dialysis for 18-24 hours, the concentrations of the bridging proteins and antibodies were detected.

[0091] b. Calculate the molar concentrations of the bridging protein and antibody based on their relative molecular weights, and mix the protein and antibody according to the molar concentration ratio. The specific ratios are shown in the table below:

[0092] Table 2

[0093] bridge proteins Molecular weight (kDa) Antibody and bridging protein mixing ratio Theoretical valence NPD2 39.3 1:1 4 NPT32 39.2 6:4 12 NPI52 38.6 30：12 60 NPP52 40.4 30：12 60 Protein A 4.2 1：1 2 Protein G 6.6 1：2 2

[0094] c. Mix the antibody and the bridging protein according to the proportions in the table, place the mixture at 4°C for 1-2 hours, remove it and dialyze it into PBS buffer solution;

[0095] d. The analyte-antibody complex, dialyzed into PBS, is concentrated by ultrafiltration to a concentration of at least 10 mg / ml and a volume of less than 2 ml. Purification is then performed using a Superdex 200 Prep Grade molecular sieve. Samples are collected in 5 ml tubes using an automated sample collection device. The collected samples are run on a gel for identification. Samples that meet the requirements are mixed, collected, and concentrated again to obtain the glycosylated hemoglobin-antibody complex that meets the requirements and can be used for subsequent testing.

[0096] Example 3 Glycated hemoglobin detection reagent

[0097] (1) Preparation of Glycated Hemoglobin Detection Reagent

[0098] The glycated hemoglobin detection reagent in this embodiment includes reagent R1 and reagent R2.

[0099] a. Preparation of Reagent R1

[0100] Prepare reagent R1 according to the following formula:

[0101] Latex microspheres 0.1%

[0102] HEPES buffer 100 mM Surfactant—Tween 20 0.2%

[0103] Preservative—ProClin-950 0.2%

[0104] pH 8.10

[0105] b. Preparation of Reagent R2

[0106] Prepare reagent R2 according to the following formula:

[0107] Glycated hemoglobin antibody complex 0.05g / L

[0108] Citric acid buffer monohydrate 50mM

[0109] Sodium chloride 1%

[0110] Tween 20 0.2%

[0111] Bovine serum albumin (BSA) 1g / L ProClin-300 0.2%

[0112] pH 6.20

[0113] (2) Glycated hemoglobin reagent detection plan a. Reagent detection parameter setting

[0114] Method: Endpoint method

[0115] Reaction direction: Ascending sample: reagent R1: reagent R2: 8:300:100

[0116] Main wavelength: 660nm

[0117] Sub-wavelength: 800nm

[0118] Reaction temperature: 37°C

[0119] Reaction time: 10 minutes

[0120] b. Sample addition method and measurement plan

[0121] Table 3 Assay protocol

[0122]

[0123]

[0124] c. Calculation method

[0125] A multi-point nonlinear / semi-logarithmic calibration mode was used with a spline function as the calculation mode. A dose / response curve was drawn based on the values ​​of the calibrators and the absorbance changes. The content of the target analyte in the sample could be calculated on the dose / response curve based on its absorbance changes.

[0126] (4) Performance evaluation of glycosylated hemoglobin detection reagents

[0127] To verify the effects of the six complexes in Example 1 on the performance of the glycated hemoglobin detection reagent, the following six groups of reagents were set up for verification. Group AG reagents had identical component formulas except for the selection of complexes. The grouping is shown in the table below. A commercially available glycated hemoglobin latex-enhanced immunoturbidimetric detection reagent was set as Group G reagent.

[0128] Table 4

[0129] A B C D E F Complex a b c d e f

[0130] *Except for the selection of the complex, the other component formulas of complexes AF are exactly the same. For example, the glycated hemoglobin antibody complex in group A reagent is complex a in Example 1, and the same applies to group BF reagent.

[0131] a. Calibration and analytical sensitivity test results

[0132] Table 5

[0133]

[0134]

[0135] The above results show that compared with the commercially available reagents (Group G) and the reagents prepared by complex coupling with Protein A protein (Group E) and Protein G (Group F) as bridging proteins, the analytical sensitivity of the reagents prepared by complex coupling with four bridging proteins NPD2, NPT32, NPI52 and NPP52 (Group AD) was greatly improved from S2 point to S5 point.

[0136] b. Accuracy test

[0137] Table 6

[0138] Group A B C D E F G Quality control test 1 10.02 9.87 10.09 10.08 9.51 9.41 9.67 Quality Control Test 2 9.89 10.00 10.10 10.13 9.22 9.28 9.24 Quality Control Test 3 9.91 10.09 10.12 10.05 10.13 9.28 9.35 Test mean 9.94 9.99 10.10 10.09 9.62 9.32 9.42 Quality control target value 10.00 10.00 10.00 10.00 10.00 10.00 10.00 Relative deviation -0.60％ -0.13％ 1.03％ 0.87％ -3.80％ -6.77％ -5.80％

[0139] The above results show that compared with the commercially available reagent (Group G) and the reagent prepared by complex coupling with Protein A protein (Group E) and Protein G (Group F) as bridging proteins, the accuracy of the glycated hemoglobin detection reagent using this complex (Group AD) is better, mainly manifested in that the relative deviation of Group EG is >3% and that of Group AF is <1.5%.

[0140] c. Precision test

[0141] Table 7

[0142]

[0143]

[0144] The above results show that compared with the commercially available reagent (Group G) and the reagent prepared by complex coupling with Protein A protein (Group E) and Protein G (Group F) as bridging proteins, the precision of the glycated hemoglobin detection reagent using this complex (Group AD) is better, mainly manifested in that the CV of Group EG is >3% and that of Group AF is <1.5%.

[0145] The present invention's glycated hemoglobin detection kit comprises a first reagent and a second reagent, wherein the first reagent comprises latex microspheres, and the second reagent comprises the present invention's glycated hemoglobin-antibody complex. While the secondary antibody in conventional glycated hemoglobin detection kits can only bind to two glycated hemoglobin antibodies, resulting in limited bridging effects, the present invention's glycated hemoglobin-antibody complexes contain two to thirty glycated hemoglobin antibodies. This significant multivalent effect facilitates the aggregation of the "latex microsphere-glycated hemoglobin-glycated hemoglobin-antibody complex," resulting in larger microspheres and faster aggregation, resulting in higher sensitivity than conventional glycated hemoglobin detection kits.

[0146] Example 4 Complement C1q protein detection reagent

[0147] (1) Preparation of complement C1q protein detection reagent

[0148] The complement C1q protein detection reagent described in this embodiment includes reagent R1 and reagent R2.

[0149] a. Preparation of complement C1q protein-antibody complex

[0150] The NPD2 bridging protein prepared in Example 1 was mixed with the C1q protein antibody at a molar concentration ratio of 1:1, and the mixture was allowed to stand at 4°C for 1-2 hours, then removed and dialyzed into a PBS buffer solution. The analyte-antibody complex dialyzed into PBS was concentrated by ultrafiltration to a concentration of 10 mg / ml or more and a volume of less than 2 ml. It was then purified using a molecular sieve Superdex 200 Prep Grade. Samples were collected in 5 ml tubes using an automatic sample collection device. The collected samples were run on a gel for identification. Samples that met the requirements were mixed, collected, and concentrated again to obtain the C1q protein-antibody complex that met the requirements, which could be used for subsequent testing.

[0151] b. Preparation of Reagent R1

[0152] Prepare reagent R1 according to the following formula:

[0153] Tris buffer 80mM

[0154] Surfactant—Tween 80 0.2%

[0155] Sodium chloride 1.2%

[0156] Preservative—ProClin-950 0.2%

[0157] pH 6.50

[0158] c. Preparation of Reagent R2

[0159] C1q protein antibody complex 40g / L

[0160] Citric acid buffer monohydrate 60mM

[0161] Sodium chloride 1.2%

[0162] Cocamidopropyl Betaine 0.2%

[0163] ProClin-300 0.5%

[0164] pH 6.50

[0165] (2) Performance verification of complement C1q protein detection reagent

[0166] The commercially available C1q latex-enhanced immunoturbidimetric reagent was used as a control for accuracy verification. The test results showed that the relative deviation (CV value) of the detection mean value of the C1q detection reagent prepared in this example from the target value of the quality control product was less than 2.0%, while the CV value of the control reagent was less than 4.0%.

[0167] Example 5 Factor B detection reagent

[0168] (1) Preparation of Factor B Detection Reagent

[0169] The factor B detection reagents described in this embodiment include reagent R1 and reagent R2.

[0170] a. Preparation of complement factor B antibody complex

[0171] The NPT32 bridging protein prepared in Example 1 was mixed with the factor B antibody at a molar concentration ratio of 4:6. The mixture was allowed to stand at 4°C for 1-2 hours, then dialyzed into a PBS buffer solution. The analyte-antibody complex dialyzed into PBS was concentrated by ultrafiltration to a concentration of 10 mg / ml or more and a volume of less than 2 ml. The complex was then purified using a Superdex 200 Prep Grade molecular sieve. Samples were collected in 5 ml tubes using an automatic sample collection device. The collected samples were run on a gel for identification. Qualified samples were mixed, collected, and concentrated again to obtain qualified factor B antibody complexes suitable for subsequent testing.

[0172] b. Preparation of Reagent R1

[0173] Prepare reagent R1 according to the following formula:

[0174] Boric acid buffer 100mM

[0175] Surfactant—Betaine Phosphate 0.2%

[0176] Preservative—Sodium Azide 0.9%

[0177] pH 7.50

[0178] c. Preparation of Reagent R2

[0179] Factor B antibody complex 15g / L

[0180] Boric acid buffer 100mM

[0181] Trehalose 10g / L

[0182] Sodium chloride 1.5%

[0183] Tween 20 0.2%

[0184] Sodium azide 0.9%

[0185] pH 6.10

[0186] Example 6 Apolipoprotein E Detection Reagent

[0187] (1) Preparation of apolipoprotein E detection reagent

[0188] The apolipoprotein E detection reagent described in this embodiment includes reagent R1 and reagent R2.

[0189] a. Preparation of Apolipoprotein E Antibody Complex

[0190] The NPI52 bridging protein prepared in Example 1 was mixed with an apolipoprotein E antibody at a molar concentration ratio of 12:30, and the mixture was allowed to stand at 4°C for 1-2 hours, then taken out for dialysis into a PBS buffer solution; the analyte-antibody complex dialyzed into PBS was concentrated by ultrafiltration to a concentration of 10 mg / ml or more, and after the volume was less than 2 ml, it was purified using a molecular sieve Superdex 200 Prep Grade, and samples were collected in 5 ml tubes using an automatic sample collection device. The collected samples were run on a gel for identification, and the samples that met the requirements were mixed, collected, and concentrated again to obtain the apolipoprotein E antibody complex that met the requirements, which could be used for subsequent testing.

[0191] b. Preparation of Reagent R1

[0192] Prepare reagent R1 according to the following formula:

[0193] HEPES buffer 80mM

[0194] Surfactant—Tween 20 0.5%

[0195] Preservative—ProClin-300 0.2%

[0196] pH 6.90

[0197] c. Preparation of Reagent R2

[0198] Apolipoprotein E antibody complex 33g / L

[0199] HEPES buffer 80mM

[0200] Tween 20 0.2%

[0201] ProClin-300 0.5%

[0202] pH 6.90

[0203] (2) Performance verification of apolipoprotein E detection reagent

[0204] Accuracy verification was performed using a commercially available apolipoprotein E latex-enhanced immunoturbidimetric reagent as a control. The test results showed that the relative deviation (CV value) of the detection mean value of the apolipoprotein E detection reagent prepared in this example from the target value of the quality control product was less than 3.0%, while the CV value of the control reagent was less than 5.0%.

[0205] Example 7 κ light chain detection reagent

[0206] (1) Preparation of κ light chain detection reagent

[0207] The κ light chain detection reagent described in this embodiment includes reagent R1 and reagent R2.

[0208] a. Preparation of κ light chain antibody complex

[0209] The NPP52 bridging protein prepared in Example 1 was mixed with a κ light chain antibody at a molar concentration ratio of 12:30, and the mixture was placed at 4°C for standing. After standing for 1-2 hours, it was taken out and dialyzed into a PBS buffer solution; the test antibody complex dialyzed into PBS was ultrafiltrated and concentrated to more than 10 mg / ml. After the volume was less than 2 ml, it was purified using a molecular sieve Superdex 200 Prep Grade, and samples were collected in 5 ml tubes using an automatic sample collection device. The collected samples were run on a gel for identification, and the samples that met the requirements were mixed and collected. After concentration again, the κ light chain antibody complex that met the requirements was obtained and could be used for subsequent testing.

[0210] b. Preparation of Reagent R1

[0211] Prepare reagent R1 according to the following formula:

[0212] Citric acid buffer monohydrate 100mM

[0213] Surfactant—Tween 80 0.2%

[0214] Preservative—ProClin-300 0.2%

[0215] pH 7.10

[0216] c. Preparation of Reagent R2

[0217] κ light chain antibody complex 16g / L

[0218] Citric acid buffer monohydrate 80mM

[0219] Sodium chloride 0.9%

[0220] Tween 80 0.2%

[0221] ProClin-300 0.5%

[0222] pH 7.10

[0223] (2) Performance verification of κ light chain detection reagents

[0224] A commercially available κ light chain latex-enhanced immunoturbidimetric reagent was used as a control for accuracy verification. The test results showed that the relative deviation (CV value) of the detection mean of the κ light chain detection reagent prepared by this embodiment from the target value of the quality control product was less than 3.0%, while the CV value of the control reagent was less than 6.0%.

[0225] Example 8 λ light chain detection reagent

[0226] (1) Preparation of λ light chain detection reagent

[0227] The lambda light chain detection reagents described in this embodiment include reagent R1 and reagent R2.

[0228] a. Preparation of λ light chain antibody complex

[0229] The NPP52 bridging protein prepared in Example 1 was mixed with the λ light chain antibody at a molar concentration ratio of 12:30, and the mixture was placed at 4°C for standing. After standing for 1-2 hours, it was taken out and dialyzed into a PBS buffer solution; the test antibody complex dialyzed into PBS was ultrafiltrated and concentrated to more than 10 mg / ml. After the volume was less than 2 ml, it was purified using a molecular sieve Superdex 200 Prep Grade, and samples were collected in 5 ml tubes using an automatic sample collection device. The collected samples were run on a gel for identification, and the samples that met the requirements were mixed, collected, and concentrated again to obtain the λ light chain antibody complex that met the requirements, which could be used for subsequent testing.

[0230] b. Preparation of Reagent R1

[0231] Prepare reagent R1 according to the following formula:

[0232] Tris buffer 80mM

[0233] Surfactant—Cocamidopropyl Betaine 0.2%

[0234] Preservative—ProClin-950 0.2%

[0235] pH 7.10

[0236] c. Preparation of Reagent R2

[0237] Lambda light chain antibody complex 18g / L

[0238] Tris buffer 80mM

[0239] Tween 80 0.2%

[0240] ProClin-950 0.5%

[0241] pH 7.10

[0242] (2) Performance verification of λ light chain detection reagent

[0243] A commercially available λ light chain latex-enhanced immunoturbidimetric reagent was used as a control for accuracy verification. The test results showed that the relative deviation (CV value) of the detection mean of the λ light chain detection reagent prepared by this embodiment from the target value of the quality control product was less than 3.0%, while the CV value of the control reagent was less than 6.0%.

[0244] All data, reagents and steps herein should be understood as illustrative and non-restrictive, although described the present invention in conjunction with above-mentioned specific embodiment, many modifications and other variations are apparent to those skilled in the art.All such modifications and other variations also fall within the scheme of the present invention and protection scope.

Claims

1. A bridging protein, characterized in that The bridging protein is selected from at least one of Protein A, Protein G, a protein obtained by performing any mutation optimization on the amino acid sequence of Protein A, and a protein obtained by performing any mutation optimization on the amino acid sequence of Protein G.

2. The bridging protein according to claim 1, characterized in that The amino acid sequence of the bridging protein is selected from at least one of SEQ ID NO: 2 to SEQ ID NO:

5.

3. A composite, characterized in that The method comprises the bridging protein according to any one of claims 1 to 2, and an antibody to be detected or an antigen-binding fragment to be detected connected to the bridging protein.

4. The composite according to claim 3, characterized in that The complex comprises 2-60 analyte antibodies or analyte antigen-binding fragments. Preferably, the analyte antibodies are monoclonal antibodies.

5. The composite according to claim 3, characterized in that The complex is a spherical complex. Preferably, the particle size of the complex is 15-60 nm.

6. The composite according to claim 3, characterized in that The titer of the complex is 4-60, preferably, the titer of the complex is 4, 12, 24, or 60.

7. The method for preparing the composite according to any one of claims 3 to 6, characterized in that: The steps include: (1) mixing the antibody to be tested or its antigen-binding fragment with the bridging protein; (2) Purify the above mixture.

8. The preparation method according to claim 7, characterized in that: In step (1), after mixing the antibody or antigen-binding fragment thereof to be tested with the bridging protein, the mixture is allowed to stand for 1-2 hours and then dialyzed; Preferably, the purification in step (2) is to first perform ultrafiltration concentration on the mixture in step (1), and then perform molecular sieve purification.

9. A detection reagent, characterized in that The reagent includes the bridging protein according to any one of claims 1 to 2, or the complex according to any one of claims 3 to 6, or the complex prepared by the preparation method according to any one of claims 7 to 8; Preferably, the reagent is an immunoturbidimetric detection reagent; More preferably, the reagent is a detection reagent for glycated hemoglobin, complement C1q protein, factor B, apolipoprotein E, kappa light chain, or lambda light chain.

10. Use of the bridging protein according to any one of claims 1-2, the complex according to any one of claims 3-6, or the reagent according to claim 9 in the preparation of an in vitro diagnostic test reagent; Preferably, the in vitro diagnostic test reagents are glycated hemoglobin, complement C1q protein, factor B, apolipoprotein E, kappa light chain, and lambda light chain detection reagents.

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