CN112798790A

CN112798790A - Kit for determining concentration of C-reactive protein and preparation method thereof

Info

Publication number: CN112798790A
Application number: CN202011553367.8A
Authority: CN
Inventors: 王兴红; 邹海涛
Original assignee: Shenzhen Comen Medical Instruments Co Ltd
Current assignee: Shenzhen Comen Medical Instruments Co Ltd
Priority date: 2020-12-24
Filing date: 2020-12-24
Publication date: 2021-05-14

Abstract

A kit for measuring the concentration of C-reactive protein and a preparation method thereof are provided, wherein a latex antibody compound in the kit is a latex microsphere coupled with an antibody, biotin and streptavidin, and the antibody is selected from antibodies capable of being combined with the C-reactive protein. The invention realizes the amplification of latex signals by utilizing streptavidin and biotin, and improves the sensitivity and specificity of the detection reagent.

Description

Kit for determining concentration of C-reactive protein and preparation method thereof

Technical Field

The invention relates to the technical field of medical detection, in particular to a kit for determining the concentration of C-reactive protein and a preparation method thereof.

Background

The existing method for determining the concentration of C-reactive protein mainly comprises the following two methods:

1. the latex microspheres are directly coupled with antibodies. The main defects are as follows: the sensitivity of the latex reagent test obtained in the mode is low, and the sensitivity of the low-value part of the test cannot meet the requirement of hypersensitivity.

2. The latex microspheres directly coat the polyclonal antibody. The main defects are as follows: the specificity tested in this way is not as good as that of the coated monoclonal antibody, and non-specific binding is easily generated.

Therefore, the prior art cannot accurately determine the concentration of C-reactive protein.

Disclosure of Invention

The invention provides a kit for determining the concentration of C-reactive protein and a preparation method thereof.

According to a first aspect, in one embodiment there is provided a latex antibody complex being a latex microsphere coupled with an antibody selected from the group consisting of antibodies that can bind to C-reactive protein, biotin, streptavidin.

According to a second aspect, there is provided in one embodiment a method of preparing the latex antibody complex of the first aspect, comprising:

an antibody-biotin compound preparation step, which comprises mixing an antibody and biotin to prepare an antibody-biotin compound;

a step of preparing a streptavidin-latex microsphere compound, which comprises mixing streptavidin and latex microspheres to prepare the streptavidin-latex microsphere compound;

and a coupling step, wherein the antibody-biotin compound and the streptavidin-latex microsphere compound are mixed to prepare the latex microsphere coupled with the antibody-biotin-streptavidin, namely the latex antibody compound.

According to a third aspect, in some embodiments, there is provided a reagent comprising the latex antibody complex of the first aspect.

According to a fourth aspect, in some embodiments, there is provided a combination of reagents comprising the latex antibody complex of the first aspect or the reagents of the third aspect, i.e. the R2 reagent.

According to a fifth aspect, in some embodiments, there is provided a kit comprising the combination of reagents of the fourth aspect.

According to a sixth aspect, in some embodiments there is provided the use of a latex antibody complex according to the first aspect, or a reagent according to the third aspect, or a combination of reagents according to the fourth aspect, or a kit according to the fifth aspect, for the detection of C-reactive protein.

According to the kit for measuring the concentration of the C-reactive protein and the preparation method thereof, the amplification of latex signals is realized by using the streptavidin and the biotin, and the sensitivity and the specificity of the detection reagent are improved.

Drawings

FIG. 1 is a graph showing the test results of example 1;

FIG. 2 is a graph showing the test results of example 2;

FIG. 3 is a graph showing the test results of example 3;

FIG. 4 is a graph showing the test results of example 4.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. Wherein like elements in different embodiments are numbered with like associated elements. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.

Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.

The numbering of the components as such, e.g., "first", "second", etc., is used herein only to distinguish the objects as described, and does not have any sequential or technical meaning. The term "connected" and "coupled" when used in this application, unless otherwise indicated, includes both direct and indirect connections (couplings).

Herein, C-reactive protein (CRP) is a protein (acute protein) which rises sharply in plasma when a body is infected or tissues are damaged, and plays an opsonizing role by activating complement and strengthening phagocytosis of phagocyte, and can remove pathogenic microorganisms invading the body and damaged, necrotic and apoptotic tissue cells.

Herein, streptavidin (referred to as "SA") is a protein with biological properties similar to those of avidin (referred to as "AV" hereinafter), and is a secretion of Streptomyces avidinii bacteria, and has a molecular weight and a biotin-binding ability similar to those of avidin in egg white, an isoelectric point of 6.0, and a nonspecific binding much lower than that of avidin. SA is a secreted product in the culture process of Streptomyces aviclrdi, and is mainly purified by 2-imino biotin affinity chromatography, and 10-60 mg of protein is contained in 1L of culture solution. Streptavidin is a tetrameric protein, 66KDa in size. One streptavidin molecule can bind highly specifically to four biotin molecules with very strong affinity, and the dissociation constant of the streptavidin-biotin complex is in the order of 10-14 mol/L. One of the complete SA peptide chains has 159 amino acid residues and a molecular weight of 16450.

Herein, Biotin (Biotin) is one of the B vitamins, which is also called vitamin H, vitamin B7, coenzyme r (coenzyme r), and the like. It is an essential substance for the synthesis of vitamin C, an indispensable substance for the normal metabolism of fats and proteins. Is a nutrient necessary for maintaining the natural growth and development of human bodies and the normal function and health of human bodies. Biotin can be detected by binding to avidin. During the detection, biotin is used only as a fixed link and not as a signal detector.

Herein, latex microspheres refer to polystyrene microspheres.

In some embodiments, using latex microspheres of CRP monoclonal antibody-biotin-streptavidin, sample types of serum, plasma, whole blood, etc. can be tested, resulting in a greatly increased sensitivity of the test while enhancing the specificity of the reagents.

According to a first aspect, in some embodiments, there is provided a latex antibody complex, said latex antibody complex being a latex microsphere coupled with an antibody selected from the group consisting of antibodies that can bind to C-reactive protein, biotin, streptavidin. Latex antibody complexes may also be referred to as latex microsphere conjugates.

In some embodiments, the antibody includes, but is not limited to, at least one of a monoclonal antibody, a polyclonal antibody.

In some embodiments, the antibody includes, but is not limited to, at least one of a murine anti-human CRP monoclonal antibody, a rabbit anti-human CRP polyclonal antibody, a sheep anti-human CRP polyclonal antibody. This is merely an exemplary list, and other CRP monoclonal antibodies, CRP polyclonal antibodies with similar functions are also suitable for use in the present invention.

In some embodiments, the latex microspheres include, but are not limited to, polystyrene.

In some embodiments, the latex microspheres may have a particle size of 50-400 nm. Specifically, it may include, but not limited to, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, etc.

In some embodiments, the surface modifying group of the latex microsphere includes, but is not limited to, at least one of an amino group, a carboxyl group, an aldehyde group, and a hydroxyl group.

In some embodiments, the ratio of the mass of the antibody to the mass of biotin is (0.2-5): 1. specific examples may include, but are not limited to, 0.2: 1. 0.3: 1. 0.4: 1. 0.5: 1. 0.6: 1. 0.7: 1. 0.8: 1. 0.9: 1. 1: 1. 1.1: 1. 1.2: 1. 1.3: 1. 1.4: 1. 1.5: 1. 1.6: 1. 1.7: 1. 1.8: 1. 1.9: 1. 2: 1. 3: 1. 4: 1. 5: 1, etc.

In some embodiments, the ratio of the mass of the latex microspheres to the mass of streptavidin is (0.5-2): 1, specifically, may include but is not limited to 0.5: 1. 0.6: 1. 0.7: 1. 0.8: 1. 0.9: 1. 1: 1. 1.1: 1. 1.2: 1. 1.3: 1. 1.4: 1. 1.5: 1. 1.6: 1. 1.7: 1. 1.8: 1. 1.9: 1. 2: 1, etc.

In some embodiments, the latex-antibody complex is obtained by coupling a streptavidin-latex microsphere complex with an antibody-biotin complex, wherein the ratio of the mass of the streptavidin-latex microsphere complex to the mass of the antibody-biotin complex is (0.2-5): 1. specific examples may include, but are not limited to, 0.2: 1. 0.3: 1. 0.4: 1. 0.5: 1. 0.6: 1. 0.7: 1. 0.8: 1. 0.9: 1. 1: 1. 1.1: 1. 1.2: 1. 1.3: 1. 1.4: 1. 1.5: 1. 1.6: 1. 1.7: 1. 1.8: 1. 1.9: 1. 2: 1. 3: 1. 4: 1. 5: 1, etc.

According to a second aspect, in some embodiments, there is provided a method of preparing the latex antibody complex of the first aspect, comprising:

The preparation steps of the antibody-biotin compound and the streptavidin-latex microsphere compound are not in sequence, and any one of the two steps can be carried out first, or the two steps can be carried out simultaneously.

In some embodiments, the antibody-biotin compound preparation step includes diluting the antibody with a reaction solution to obtain an antibody diluent, dissolving biotin with an organic reagent to obtain a biotin solution, and mixing the antibody diluent with the biotin solution to react to obtain the antibody-biotin compound.

In some embodiments, the reaction solution includes, but is not limited to, at least one of morpholine ethanesulfonic acid (also known as MES, 2-morpholine ethanesulfonic acid, CAS number: 4432-31-9) buffer, borate buffer, phosphate buffer, carbonate buffer, sodium bicarbonate buffer.

In some embodiments, the organic reagent is selected from at least one of N, N-dimethylformamide, dimethylsulfoxide.

In some embodiments, the concentration of biotin in the biotin solution is 10 to 50 g/L.

In some embodiments, the concentration of antibody in the antibody dilution is 0.2-20 mg/mL. Specific examples include, but are not limited to, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL, 11mg/mL, 12mg/mL, 13mg/mL, 14mg/mL, 15mg/mL, 16mg/mL, 17mg/mL, 18mg/mL, 19mg/mL, 20mg/mL, and the like.

In some embodiments, the antibody diluent is mixed with a biotin dissolving solution, and after the reaction is finished, the mixture is filtered to remove redundant small molecules such as biotin, and the large molecules are collected to obtain the antibody-biotin compound.

In some embodiments, the apparatus used in filtration may include, but is not limited to, ultrafiltration tubes, dialysis bags.

In some embodiments, the step of preparing the streptavidin-latex microsphere composite comprises diluting the latex microspheres with a diluent to obtain a latex microsphere diluent, mixing the latex microsphere diluent with streptavidin, and reacting to obtain the streptavidin-latex microsphere composite.

In some embodiments, the diluent includes, but is not limited to, at least one of morpholine ethanesulfonic acid (MES) buffer, borate buffer, phosphate buffer, carbonate buffer, sodium bicarbonate buffer.

In some embodiments, the latex microsphere dilution is 0.02 wt% to 2 wt% solids content of the latex microspheres. Specifically, it may include, but is not limited to, 0.02 wt%, 0.03 wt%, 0.04 wt%, 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2 wt%, and the like. The initial solid content of the purchased latex microspheres is usually 5 wt% to 10 wt%, and when the method is used, the purchased latex microspheres are diluted to a solid content of 0.05 wt% to 0.2 wt% by using a buffer solution, and then the subsequent steps are carried out.

In some embodiments, in the step of preparing the streptavidin-latex microsphere composite, the latex microsphere diluent is mixed with streptavidin, and after the reaction is finished, the solid-liquid separation treatment is performed to collect the solid, so as to obtain the streptavidin-latex microsphere composite.

In some embodiments, after the solid-liquid separation treatment, the supernatant is discarded, and the solid is collected, i.e., the streptavidin-latex microsphere complex.

In some embodiments, the solid-liquid separation process includes, but is not limited to, a centrifugation process.

In some embodiments, the coupling step includes diluting the streptavidin-latex microsphere complex prepared in the streptavidin-latex microsphere complex preparation step to obtain a streptavidin-latex microsphere complex diluent, and mixing the streptavidin-latex microsphere complex diluent with the antibody-biotin complex to react to obtain the latex microsphere conjugate.

In some embodiments, the streptavidin-latex microsphere complex is diluted with a buffer.

In some embodiments, the buffer includes, but is not limited to, at least one of a morpholine ethanesulfonic acid buffer, a borate buffer, a phosphate buffer, a carbonate buffer, a sodium bicarbonate buffer.

According to a third aspect, in some embodiments, there is provided a reagent comprising a preservation solution and the latex antibody complex of the first aspect. The reagent can be used as an R2 reagent of a C-reactive protein detection kit. The role of the preservative solution mainly includes a liquid for storing the resulting latex antibody complex for long-term stability, and the preservative solution usually contains a buffer.

In some embodiments, the preservation solution contains at least one of a second buffer, a second surfactant, a second preservative, and a stabilizer.

In some embodiments, the preservation solution contains the following components in concentrations: 5-50mmol/L of second buffer, 0.5-10g/L of second surfactant, 0.5-5g/L of second preservative and 0.5-10g/L of stabilizer.

In some embodiments, the concentration of the second buffer in the preservation solution may specifically include, but is not limited to, 5mmol/L, 10mmol/L, 15mmol/L, 20mmol/L, 25mmol/L, 30mmol/L, 35mmol/L, 40mmol/L, 45mmol/L, 50mmol/L, and the like.

In some embodiments, the concentration of the second surfactant in the preservation solution may specifically include, but is not limited to, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, and the like.

In some embodiments, the concentration of the second preservative in the preservation solution may specifically include, but is not limited to, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, and the like.

In some embodiments, the concentration of the stabilizing agent in the preservation solution may specifically include, but is not limited to, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, and the like.

In some embodiments, the second buffer includes, but is not limited to, at least one of Phosphate Buffered Saline (PBS), tromethamine (Tris), glycine, borate, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid (MOPSO), morpholine ethanesulfonic acid (MES).

In some embodiments, the second surfactant includes, but is not limited to, at least one of polyvinylpyrrolidone, glycerol, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 10000, polyethylene glycol 20000, tween 20, tween 40, tween 80.

In some embodiments, the second preservative includes, but is not limited to, at least one of sodium azide, ProClin-950, ProClin-300, KroVin100, KroVin300, KroVin500, KroVin750, thimerosal.

In some embodiments, the stabilizing agent includes, but is not limited to, at least one of sucrose, trehalose, bovine serum albumin, casein, mannitol, gelatin.

In some embodiments, the solid content of the latex microspheres in the agent is 0.05 wt% to 0.2 wt%, and specifically may include, but is not limited to, 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.11 wt%, 0.12 wt%, 0.13 wt%, 0.14 wt%, 0.15 wt%, 0.16 wt%, 0.17 wt%, 0.18 wt%, 0.19 wt%, 0.2 wt%, and the like.

In some embodiments, further comprising an R1 reagent, the R1 reagent comprising at least one of a first buffer, a first surfactant, a first hemolysing agent, a first preservative.

In some embodiments, the R1 reagent contains the following components in the following concentrations: 5-50mmol/L of first buffer, 0.5-10g/L of first surfactant, 0.2-5g/L of first hemolytic agent and 0.5-5g/L of first preservative.

In some embodiments, the concentration of the first buffer in the R1 reagent includes, but is not limited to, 5mmol/L, 10mmol/L, 15mmol/L, 20mmol/L, 25mmol/L, 30mmol/L, 35mmol/L, 40mmol/L, 45mmol/L, 50mmol/L, and the like.

In some embodiments, the concentration of the first surfactant in the R1 reagent includes, but is not limited to, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, and the like.

In some embodiments, the concentration of the first hemolytic agent in the R1 reagent includes, but is not limited to, 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, and the like.

In some embodiments, the concentration of the first preservative in the R1 reagent includes, but is not limited to, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, and the like.

In some embodiments, the first buffer in the R1 reagent includes, but is not limited to, at least one of Phosphate Buffered Saline (PBS), tromethamine (Tris), glycine, borate, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid (MOPSO), morpholine ethanesulfonic acid (MES).

In some embodiments, the first surfactant in the R1 reagent includes, but is not limited to, at least one of polyvinylpyrrolidone (CAS accession No. 9003-39-8), glycerol (CAS accession No. 56-81-5), saponin, Triton100, Triton405, polyethylene glycol 6000(PEG6000), polyethylene glycol 8000(PEG8000), polyethylene glycol 10000(PEG10000), polyethylene glycol 20000(PEG20000), Tween 20(Tween-20), Tween 40(Tween-40), Tween 80 (Tween-80). Wherein the saponin has hemolytic effect, and can be combined with cholesterol to generate insoluble molecular complex, destroy permeability of red blood cell, and disintegrate.

In some embodiments, the first hemolytic agent in the R1 reagent is selected from at least one of a saponin, Triton X-100, Triton X-405.

In some embodiments, the first preservative in the R1 reagent includes, but is not limited to, at least one of sodium azide (CAS registry No.: 26628-22-8), ProClin-950, ProClin-300, KroVin100, KroVin300, KroVin500, KroVin750, Thimerosal (CAS registry No.: 54-64-8).

In some embodiments, the kit further comprises containers for separately holding the R1 reagent, R2 reagent.

Typically, the R1 reagent and the R2 reagent are separately dispensed into separate containers, or separate chambers of the same container, to form a kit product.

In some embodiments, the kit further comprises a package insert.

In some embodiments, the latex antibody complex of the first aspect, or the reagent of the third aspect, or the combination of reagents of the fourth aspect, or the kit of the fifth aspect, is used to detect the concentration of C-reactive protein in a sample.

In some embodiments, the sample includes, but is not limited to, at least one of whole blood, serum, plasma.

In some embodiments, the invention utilizes streptavidin and biotin to realize latex signal amplification, improves sensitivity, can make the linear range of whole blood sample detection be 0.1-400mg/L, can make the linear range of serum and plasma sample detection be 0.1-280mg/L, and improves the specificity of the reagent.

The following examples were all measured for absorbance at room temperature (23 ℃. + -. 2 ℃).

In the following examples, the reagents used for adjusting the pH were aqueous sodium hydroxide solution and hydrochloric acid.

Example 1

The detection reagent of the present embodiment includes R1 reagent and R2 reagent.

The latex microsphere has a selected particle size of 80nm and a surface modification group of carboxyl.

The pH of the R1 and R2 reagents was 8.0.

This example provides a method for preparing a kit for determining the concentration of C-reactive protein, comprising the steps of:

1. preparation of R1 reagent

Adding a proper amount of water into a preparation tank, adjusting a stirrer to a proper rotating speed, sequentially adding a first buffering agent, a first surfactant, a first hemolytic agent and a first preservative, stirring for 10-20 minutes until the first buffering agent, the first surfactant, the first hemolytic agent and the first preservative are completely dissolved, adjusting the pH value to 8.0 after the solution is clear and transparent and has no precipitate, and fixing the volume to the final volume to prepare the R1 reagent containing the following final concentration components: dissolving 5mmol/L of first buffer, 0.5g/L of first surfactant, 0.2g/L of first hemolytic agent and 0.5g/L of first preservative, filtering through a microporous filter membrane, collecting filtrate, placing the filtered R1 reagent finished product in a storage container, and carrying out subpackage marking. The first buffer is Tris (tromethamine, also known as Tris), the first surfactant is PEG6000, the first hemolytic agent is saponin, and the first preservative is ProClin-300. The saponin has hemolytic effect, can be combined with cholesterol to generate insoluble molecular compound, destroys the permeability of red blood cells to disintegrate, and the addition of the saponin enables the kit to be used for detecting the concentration of C-reactive protein in a whole blood sample.

2. Preparation of R2 reagent

2.1 preparation of CRP murine anti-human monoclonal antibody-Biotin Complex

Diluting the CRP mouse anti-human monoclonal antibody by using a sodium bicarbonate buffer solution to obtain a CRP mouse anti-human monoclonal antibody dilution solution with the CRP mouse anti-human monoclonal antibody concentration of 2mg/mL, dissolving biotin by using DMF (dimethylformamide) to obtain a biotin solution with the biotin concentration of 10g/L, and according to the CRP mouse anti-human monoclonal antibody: biotin 0.2: 1, mixing the dilution solution of the CRP mouse anti-human monoclonal antibody with a biotin dissolving solution, filtering by using an ultrafiltration tube after mixing for a certain time, successfully collecting the CRP mouse anti-human monoclonal antibody combined with biotin as a macromolecule, and discarding other micromolecules such as biotin and the like to obtain the CRP mouse anti-human monoclonal antibody-biotin compound.

2.2 preparation of CRP mouse anti-human monoclonal antibody-biotin-streptavidin-latex microsphere Complex

Latex microspheres were diluted with MES (i.e., morpholinoethanesulfonic acid, CAS number: 4432-31-9) buffer to make a 20mM MES solution, and the latex microspheres in the latex dilution were 0.05 wt% solids, as latex microspheres: streptavidin ═ 1: 1, adding streptavidin into a latex diluent, mixing, centrifuging at the rotating speed of 15000rpm for 1 hour, removing supernatant to obtain a streptavidin-latex microsphere compound, adding MES buffer solution, carrying out ultrasonic resuspension, and repeating for 3 times to obtain a solution containing the streptavidin-latex microsphere compound, wherein the solid content of latex microspheres in the solution is 0.05 wt%. The MES buffer used in the subsequent examples was the same as in this example.

And (2) taking the streptavidin-latex microsphere compound solution prepared in the last step, dropwise adding the CRP monoclonal antibody-biotin compound while stirring, and mixing according to the weight ratio of the streptavidin-latex microsphere compound: CRP murine anti-human monoclonal antibody-biotin complex ═ 5: 1, reacting for a certain time, adding a 10% BSA solution (bovine serum albumin solution) as a sealant, reacting for a certain time, centrifuging the mixed solution at 15000rpm for 1 hour, removing the supernatant to obtain a CRP mouse anti-human monoclonal antibody-biotin-streptavidin-latex microsphere complex, adding a preservation solution, ultrasonically resuspending, repeating for 3 times, wherein the solid content of the latex microspheres in the obtained mixed solution is 0.05 wt%, filtering, subpackaging, and marking. The preservation solution used in this example contained the following components at final concentrations: the preservative comprises a second buffer of 5mmol/L, a second surfactant of 0.5g/L, a second preservative of 0.5g/L and a stabilizer of 1g/L, wherein the second buffer is Tris (Tris hydroxymethyl aminomethane), the second surfactant is Tween-20, the second preservative is ProClin-300, the stabilizer is a mixture of trehalose and bovine serum albumin, and the concentrations of the trehalose and the bovine serum albumin in the preservative solution are both 0.5 g/L.

The reagents R1 and R2 of this example were used to measure the concentration of C-reactive protein in whole blood samples.

The turbidity is proportional to the content of C-reactive protein in the whole blood sample when a sufficient amount of antibody is present, and the content of C-reactive protein in the whole blood sample can be quantitatively detected by comparing with the whole blood sample with a known concentration at a wavelength of 540 nm. Whole blood samples of known concentrations (obtained by using Aristo full-automatic specific protein analyzer test assignment of shenzhen city mystery limited) were tested as experimental group 1 using R1, R2 reagents prepared by the method of this example; the same whole blood samples were tested using the same R1 reagent as in this example and the R2 reagent without biotin or streptavidin added (the other components and concentrations of the R2 reagent were the same as those of the R2 reagent in this example) as control 1, and the results of the two measurements were compared.

As shown in FIG. 1, the abscissa of the graph is the content of C-reactive protein in the sample in mg/L, and the ordinate of the graph is the signal value of the solution at a wavelength of 540nm (the signal value is automatically converted by the analyzer according to absorbance), it can be seen that the linear range of the detection of the whole blood sample in the present embodiment is 0.1-400mg/L, which is significantly wider than the linear range of the control group using the R2 reagent without adding biotin and streptavidin, the hook effect occurs when the concentration of the C-reactive protein is greater than 200mg/L in the control group, and the linear range of the control group is only 0.1-200 mg/L.

The control group mentioned in the subsequent examples was the same as that of the present example.

Example 2

Reference is made to example 1 with the difference that: the mass ratio of antibody to biotin, and the mass ratio of streptavidin-latex microsphere complex to antibody-biotin complex were different, and the component concentrations of the basic buffer were slightly adjusted.

The pH of the R1 and R2 reagents was 8.0.

1. preparation of R1 reagent

Adding a proper amount of water into a preparation tank, adjusting a stirrer to a proper rotating speed, sequentially adding a first buffering agent, a first surfactant, a first hemolytic agent and a first preservative, stirring for 10-20 minutes until the first buffering agent, the first surfactant, the first hemolytic agent and the first preservative are completely dissolved, adjusting the pH value to 8.0 after the solution is clear and transparent and has no precipitate, and fixing the volume to the final volume to prepare the R1 reagent containing the following final concentration components: 20mmol/L of first buffer, 2g/L of first surfactant, 1g/L of first hemolytic agent and 1g/L of first preservative, filtering through a microporous filter membrane after dissolving, collecting filtrate, placing the filtered R1 reagent finished product in a storage container, and performing subpackage marking. The first buffer is Tris (Tris hydroxymethyl aminomethane), the first surfactant is PEG6000, the first hemolytic agent is saponin, and the first preservative is ProClin-300. The saponin has hemolytic effect, can be combined with cholesterol to generate insoluble molecular compound, destroys the permeability of red blood cells to disintegrate, and the addition of the saponin enables the kit to be used for detecting the concentration of C-reactive protein in a whole blood sample.

2. Preparation of R2 reagent

2.1 preparation of CRP murine anti-human monoclonal antibody-Biotin Complex

Diluting the CRP monoclonal antibody by using a sodium bicarbonate buffer solution to prepare a CRP mouse anti-human monoclonal antibody diluent with the CRP monoclonal antibody concentration of 2mg/mL, dissolving biotin by using DMF (dimethylformamide) to obtain a biotin solution with the biotin concentration of 10g/L, and according to the CRP monoclonal antibody: biotin 5: 1, mixing the dilution solution of the CRP mouse anti-human monoclonal antibody with a biotin dissolving solution, filtering by using an ultrafiltration tube after mixing for a certain time, successfully collecting the CRP monoclonal antibody combined with biotin as a macromolecule, and discarding other micromolecules such as biotin to obtain the CRP mouse anti-human monoclonal antibody-biotin compound.

Diluting the latex microspheres with MES buffer solution to obtain latex diluent with the solid content of the latex microspheres of 0.05 wt%, according to the latex microspheres: streptavidin ═ 1: 1, adding streptavidin into the latex diluent, mixing, centrifuging at the speed of 15000rpm for 1 hour after mixing, removing supernatant to obtain a streptavidin-latex microsphere complex, adding MES buffer solution, carrying out ultrasonic resuspension, and repeating for 3 times to obtain a solution containing the streptavidin-latex microsphere complex, wherein the solid content of latex microspheres in the solution is 0.05 wt%.

Taking the streptavidin-latex microsphere compound solution prepared in the previous step, dropwise adding the CRP mouse anti-human monoclonal antibody-biotin compound while stirring, according to the conditions of the streptavidin-latex microsphere compound: CRP murine anti-human monoclonal antibody-biotin complex ═ 0.5: 1, reacting for a certain time, adding a 10% BSA solution (bovine serum albumin solution) as a sealant, reacting for a certain time, centrifuging the mixed solution at 15000rpm for 1 hour, removing the supernatant to obtain a CRP mouse anti-human monoclonal antibody-biotin-streptavidin-latex microsphere complex, adding a preservation solution, ultrasonically resuspending, repeating for 3 times, wherein the solid content of the latex microspheres in the obtained mixed solution is 0.05 wt%, filtering, subpackaging, and marking. The preservation solution used in this example contained the following components at final concentrations: the preservative comprises a second buffer agent of 20mmol/L, a second surfactant of 2g/L, a second preservative of 1g/L and a stabilizer of 1g/L, wherein the second surfactant is Tween-20, the second preservative is ProClin-300, the stabilizer is a mixture of trehalose and bovine serum albumin, the concentrations of the trehalose and the bovine serum albumin in the preservation solution are both 0.5g/L, and the second buffer agent is Tris (Tris).

The turbidity is proportional to the content of C-reactive protein in the whole blood sample when a sufficient amount of antibody is present, and the content of C-reactive protein in the whole blood sample can be quantitatively detected by comparing with the whole blood sample with a known concentration at a wavelength of 540 nm. Whole blood samples of known concentrations (obtained by using Aristo full-automatic specific protein analyzer test assignment of shenzhen city mystery limited) were tested as experimental group 2 using R1 and R2 reagents prepared by the method of this example; the results were compared with those of control 1.

As shown in FIG. 2, the abscissa of the graph is the content of C-reactive protein in the sample, and the ordinate is the signal value of the solution at a wavelength of 540nm (the signal value is automatically converted by the analyzer according to absorbance), it can be seen that the linear range of the detection of the whole blood sample in this example is 0.1-400mg/L, which is significantly larger than the linear range of the control group using the R2 reagent without biotin and streptavidin, the hook effect occurs when the concentration of C-reactive protein is greater than 200mg/L in the control group, and the linear range of the control group is only 0.1-200 mg/L.

Example 3

Reference is made to example 1 with the difference that: different from the antibody used, polyclonal antibody was used in example 3.

The pH of the R1 and R2 reagents was 8.0.

1. preparation of R1 reagent

Adding a proper amount of water into a preparation tank, adjusting a stirrer to a proper rotating speed, sequentially adding a first buffering agent, a first surfactant, a first hemolytic agent and a first preservative, stirring for 10-20 minutes until the first buffering agent, the first surfactant, the first hemolytic agent and the first preservative are completely dissolved, adjusting the pH value to 8.0 after the solution is clear and transparent and has no precipitate, and fixing the volume to the final volume to prepare the R1 reagent containing the following final concentration components: 40mmol/L of first buffer, 5g/L of first surfactant, 5g/L of first hemolytic agent and 3g/L of first preservative, filtering through a microporous filter membrane after dissolving, collecting filtrate, placing the filtered R1 reagent finished product in a storage container, and carrying out subpackage marking. The first buffer is Tris (Tris hydroxymethyl aminomethane), the first surfactant is PEG6000, the first hemolytic agent is TritonX405, and the first preservative is ProClin-300. The saponin has hemolytic effect, can be combined with cholesterol to generate insoluble molecular compound, destroys the permeability of red blood cells to disintegrate, and the addition of the saponin makes the kit used for detecting the concentration of C-reactive protein in whole blood.

2. Preparation of R2 reagent

2.1 preparation of CRP goat anti-human polyclonal antibody-biotin Complex

Diluting CRP goat anti-human polyclonal antibody with sodium bicarbonate buffer solution to obtain CRP monoclonal antibody dilution solution with CRP goat anti-human polyclonal antibody concentration of 2mg/mL, dissolving biotin with DMF (dimethylformamide) to obtain biotin solution with biotin concentration of 10g/L, according to CRP goat anti-human polyclonal antibody: biotin 0.2: 1, mixing the CRP goat anti-human polyclonal antibody diluent and the biotin dissolving solution, filtering by using an ultrafiltration tube after mixing for a certain time, successfully collecting the CRP goat anti-human polyclonal antibody combined with biotin as a macromolecule, and discarding other micromolecules such as biotin to obtain the CRP monoclonal antibody-biotin compound.

2.2 preparation of CRP monoclonal antibody-biotin-streptavidin-latex microsphere Complex

Diluting the latex microspheres with MES buffer solution to obtain latex diluent in which the solid content of the latex microspheres is 0.05 wt%, according to the weight percentage of the latex microspheres: streptavidin ═ 1: 1, adding streptavidin into a latex diluent, mixing, centrifuging at the rotating speed of 15000rpm for 1 hour, removing supernatant to obtain a streptavidin-latex microsphere complex, adding MES buffer solution, carrying out ultrasonic resuspension, and repeating for 3 times to obtain a solution containing the streptavidin-latex microsphere complex, wherein the solid content of latex microspheres in the solution is 0.05 wt%.

Taking the streptavidin-latex microsphere compound solution prepared in the last step, dropwise adding the CRP goat anti-human polyclonal antibody-biotin compound while stirring, according to the conditions of the streptavidin-latex microsphere compound: CRP goat anti-human polyclonal antibody-biotin complex 5: 1, adding a 10% BSA solution (bovine serum albumin solution) as a sealant after reacting for a certain time, centrifuging the mixed solution at the speed of 15000rpm for 1 hour, removing supernatant to obtain a CRP goat anti-human polyclonal antibody-biotin-streptavidin-latex microsphere complex, adding a preserving solution, ultrasonically resuspending, repeating for 3 times, wherein the concentration of the latex microspheres in the obtained mixed solution is 0.05 wt%, filtering, subpackaging, and marking. The preservation solution used in this example contained the following components at final concentrations: the preservative comprises a 40mmol/L second buffer, a 5g/L second surfactant, a 3g/L second preservative and a 5g/L stabilizer, wherein the second surfactant is Tween-20, the second preservative is ProClin-300, the stabilizer is a mixture of trehalose and bovine serum albumin, the concentrations of the trehalose and the bovine serum albumin in the preservation solution are both 2.5g/L, and the second buffer is Tris (Tris).

The turbidity changes in the presence of a sufficient amount of antibody in proportion to the amount of C-reactive protein in the whole blood sample, and the amount of C-reactive protein in the whole blood sample can be quantitatively detected by comparing with the whole blood sample with a known concentration at a wavelength of 540 nm. Whole blood samples of known concentrations (obtained by the test assignment using Aristo full-automatic specific protein analyzer of shenzhen city mystery limited) were tested as experimental group 3 using the R1, R2 reagents prepared by the method of this example; the results were compared with those of control 1.

As shown in FIG. 3, the abscissa of the graph is the content of C-reactive protein in the sample in mg/L, and the ordinate of the graph is the signal value of the solution at a wavelength of 540nm (the signal value is automatically converted by the analyzer according to absorbance), it can be seen that the linear range of the detection of the whole blood sample in the present embodiment is 0.1-400mg/L, which is significantly larger than the linear range of the control group using the R2 reagent without biotin and streptavidin, the hook effect occurs when the concentration of C-reactive protein is greater than 200mg/L in the control group, and the linear range of the control group is only 0.1-200 mg/L.

Example 4

Reference is made to example 3 with the difference that: the mass ratio of antibody to biotin, and the mass ratio of streptavidin-latex microsphere complex to antibody-biotin complex were different, and the component concentrations of the basic buffer were slightly adjusted.

The pH of the R1 and R2 reagents was 8.0.

1. preparation of R1 reagent

Adding a proper amount of water into a preparation tank, adjusting a stirrer to a proper rotating speed, sequentially adding a first buffering agent, a first surfactant, a first hemolytic agent and a first preservative, stirring for 10-20 minutes until the first buffering agent, the first surfactant, the first hemolytic agent and the first preservative are completely dissolved, adjusting the pH value to 8.0 after the solution is clear and transparent and has no precipitate, and fixing the volume to the final volume to prepare the R1 reagent containing the following final concentration components: 50mmol/L of first buffer, 10g/L of first surfactant, 5g/L of first hemolytic agent and 5g/L of first preservative, filtering through a microporous filter membrane after dissolving, collecting filtrate, placing the filtered R1 reagent finished product in a storage container, and carrying out subpackage marking. The first buffer is Tris (Tris hydroxymethyl aminomethane), the first surfactant is PEG6000, the first hemolytic agent is Triton100, and the first preservative is ProClin-300. The saponin has hemolytic effect, can be combined with cholesterol to generate insoluble molecular compound, destroys the permeability of red blood cells to disintegrate, and the addition of the saponin enables the kit to be used for detecting the concentration of C-reactive protein in a whole blood sample.

2. Preparation of R2 reagent

2.1 preparation of CRP goat anti-human polyclonal antibody-biotin Complex

Diluting CRP goat anti-human polyclonal antibody with sodium bicarbonate buffer solution to obtain CRP monoclonal antibody dilution solution with CRP goat anti-human polyclonal antibody concentration of 2mg/mL, dissolving biotin with DMF (dimethylformamide) to obtain biotin solution with biotin concentration of 10g/L, according to CRP goat anti-human polyclonal antibody: biotin 5: 1, mixing the CRP goat anti-human polyclonal antibody diluent and the biotin dissolving solution, filtering by using an ultrafiltration tube after mixing for a certain time, successfully collecting the CRP monoclonal antibody combined with biotin as a macromolecule, and discarding other micromolecules such as biotin to obtain the CRP monoclonal antibody-biotin compound.

2.2 preparation of CRP goat anti-human polyclonal antibody-biotin-streptavidin-latex microsphere Complex

Diluting the latex microspheres with MES buffer solution to obtain latex diluent in which the solid content of the latex microspheres is 0.05 wt%, according to the weight percentage of the latex microspheres: streptavidin ═ 1: 1, adding streptavidin into the latex diluent, mixing, centrifuging at the speed of 15000rpm for 1 hour after mixing, removing supernatant to obtain a streptavidin-latex microsphere complex, adding MES buffer solution, carrying out ultrasonic resuspension, and repeating for 3 times to obtain a solution containing the streptavidin-latex microsphere complex, wherein the solid content of latex microspheres in the solution is 0.05 wt%.

And (2) taking the streptavidin-latex microsphere compound solution prepared in the last step, dropwise adding the CRP monoclonal antibody-biotin compound while stirring, and mixing according to the weight ratio of the streptavidin-latex microsphere compound: CRP goat anti-human polyclonal antibody-biotin complex 0.5: 1, adding a 10% BSA solution (bovine serum albumin solution) as a sealant after reacting for a certain time, centrifuging the mixed solution at the speed of 15000rpm for 1 hour, removing supernatant to obtain a CRP goat anti-human polyclonal antibody-biotin-streptavidin-latex microsphere compound, adding a preserving solution, ultrasonically resuspending, repeating for 3 times, wherein the solid content of the latex microspheres in the obtained mixed solution is 0.05 wt%, filtering, subpackaging, and marking. The preservation solution used in this example contained the following components at final concentrations: the preservative comprises a 50mmol/L second buffer, a 10g/L second surfactant, a 5g/L second preservative and a 10g/L stabilizer, wherein the second surfactant is Tween-20, the second preservative is ProClin-300, the stabilizer is a mixture of trehalose and bovine serum albumin, the concentrations of the trehalose and the bovine serum albumin in the preservative solution are both 5g/L, and the second buffer is Tris (Tris).

The turbidity is proportional to the content of C-reactive protein in the whole blood sample when a sufficient amount of antibody is present, and the content of C-reactive protein in the whole blood sample can be quantitatively detected by comparing with the whole blood sample with a known concentration at a wavelength of 540 nm. Whole blood samples of known concentrations (obtained by the test assignment using Aristo full-automatic specific protein analyzer of shenzhen city mystery limited) were tested as experimental group 4 using the R1, R2 reagents prepared by the method of this example; the results were compared with those of control 1.

As shown in FIG. 4, the abscissa of the graph is the content of C-reactive protein in the sample in mg/L, and the ordinate of the graph is the signal value of the solution at a wavelength of 540nm (the signal value is automatically converted by the analyzer according to absorbance), it can be seen that the linear range of the detection of the whole blood sample in the present embodiment is 0.1-400mg/L, which is significantly wider than the linear range of the control group using the R2 reagent without adding biotin and streptavidin, the hook effect occurs when the concentration of the C-reactive protein is greater than 200mg/L in the control group, and the linear range of the control group is only 0.1-200 mg/L.

The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.

Claims

1. A latex antibody complex, wherein the latex antibody complex is a latex microsphere coupled with an antibody, biotin or streptavidin, and the antibody is selected from antibodies capable of binding to C-reactive protein.

2. The latex antibody complex of claim 1, wherein said antibody is selected from at least one of a monoclonal antibody, a polyclonal antibody;

and/or, the antibody is selected from at least one of mouse anti-human CRP monoclonal antibody, rabbit anti-human CRP polyclonal antibody and sheep anti-human CRP polyclonal antibody;

and/or, the latex microspheres are selected from polystyrene;

and/or the particle size of the latex microsphere is 50-400 nm;

and/or the surface modification group of the latex microsphere is selected from at least one of amino, carboxyl, aldehyde group and hydroxyl.

And/or the ratio of the mass of the antibody to the mass of biotin is (0.2-5): 1;

and/or the mass ratio of the latex microspheres to the streptavidin is (0.5-2): 1;

and/or the latex-antibody complex is obtained by coupling a streptavidin-latex microsphere complex and an antibody-biotin complex, wherein the ratio of the mass of the streptavidin-latex microsphere complex to the mass of the antibody-biotin complex is (0.2-5): 1.

3. the method of preparing a latex antibody complex according to any one of claims 1 to 2, comprising:

4. The method according to claim 3, wherein the antibody-biotin compound preparation step comprises diluting an antibody with a reaction solution to obtain an antibody diluent, dissolving biotin with an organic reagent to obtain a biotin solution, and mixing the antibody diluent with the biotin solution to react and obtain an antibody-biotin compound;

and/or the reaction solution is selected from at least one of morpholine ethanesulfonic acid buffer solution, borate buffer solution, phosphate buffer solution, carbonate buffer solution and sodium bicarbonate buffer solution;

and/or the organic reagent is selected from at least one of N, N-dimethylformamide and dimethyl sulfoxide;

and/or the concentration of biotin in the biotin dissolving solution is 10-50 g/L;

and/or, in the antibody dilution, the concentration of the antibody is 0.2-20 mg/mL;

and/or mixing the antibody diluent with a biotin solution, filtering after the reaction is finished, and collecting to obtain an antibody-biotin compound;

and/or in the step of preparing the streptavidin-latex microsphere compound, diluting latex microspheres with diluent to obtain latex microsphere diluent, mixing the latex microsphere diluent with streptavidin, and reacting to obtain the streptavidin-latex microsphere compound;

and/or the diluent is selected from at least one of morpholine ethanesulfonic acid buffer solution, borate buffer solution, phosphate buffer solution, carbonate buffer solution and sodium bicarbonate buffer solution;

and/or, in the latex microsphere diluent, the solid content of the latex microspheres is 0.02 wt% -2 wt%;

and/or in the step of preparing the streptavidin-latex microsphere compound, mixing the latex microsphere diluent with streptavidin, carrying out solid-liquid separation treatment after the reaction is finished, and collecting solids to obtain the streptavidin-latex microsphere compound;

and/or in the coupling step, diluting the streptavidin-latex microsphere complex prepared in the streptavidin-latex microsphere complex preparation step to obtain a streptavidin-latex microsphere complex diluent, mixing the streptavidin-latex microsphere complex diluent with the antibody-biotin complex, and reacting to obtain the latex microsphere conjugate;

and/or, diluting the streptavidin-latex microsphere complex with a buffer;

and/or the buffer solution is selected from at least one of morpholine ethanesulfonic acid buffer solution, borate buffer solution, phosphate buffer solution, carbonate buffer solution and sodium bicarbonate buffer solution.

5. A reagent comprising a storage solution and the latex antibody complex according to any one of claims 1 to 2.

6. The reagent according to claim 5, wherein the preservation solution contains at least one of a second buffer, a second surfactant, a second preservative, and a stabilizer;

and/or the preservation solution contains the following components in concentration: 5-50mmol/L of second buffer, 0.5-10g/L of second surfactant, 0.5-5g/L of second preservative and 0.5-10g/L of stabilizer;

and/or the second buffer is selected from at least one of phosphate buffered saline solution, tromethamine, glycine, borate, 4-hydroxyethyl piperazine ethanesulfonic acid, 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid and morpholine ethanesulfonic acid;

and/or the second surfactant is at least one selected from polyvinylpyrrolidone, glycerol, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 10000, polyethylene glycol 20000, tween 20, tween 40 and tween 80;

and/or, the second preservative is at least one selected from sodium azide, ProClin-950, ProClin-300, KroVin100, KroVin300, KroVin500, KroVin750 and thimerosal;

and/or, the stabilizer is at least one selected from sucrose, trehalose, bovine serum albumin, casein, mannitol and gelatin;

and/or the solid content of the latex microspheres in the reagent is 0.05 wt% to 0.2 wt%.

7. A combination of reagents comprising the latex antibody complex of any one of claims 1 to 2, or the reagent of any one of claims 5 to 6, wherein the reagent of any one of claims 5 to 6 is the R2 reagent.

8. The reagent combination of claim 7, further comprising a R1 reagent, wherein the R1 reagent comprises at least one of a first buffer, a first surfactant, a first hemolytic agent, a first preservative;

and/or, the R1 reagent contains the following components in concentration: 5-50mmol/L of first buffer, 0.5-10g/L of first surfactant, 0.2-5g/L of first hemolytic agent and 0.5-5g/L of first preservative;

and/or, the first buffer in the R1 reagent is selected from at least one of phosphate buffered saline, tromethamine, glycine, borate, 4-hydroxyethyl piperazine ethanesulfonic acid, 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid and morpholine ethanesulfonic acid;

and/or the first surfactant in the R1 reagent is selected from at least one of polyvinylpyrrolidone, glycerol, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 10000, polyethylene glycol 20000, Tween 20, Tween 40 and Tween 80;

and/or, the first hemolytic agent in the R1 reagent is selected from at least one of saponin, Triton X-100 and Triton X-405;

and/or, the first preservative in the R1 reagent is at least one selected from sodium azide, ProClin-950, ProClin-300, KroVin100, KroVin300, KroVin500, KroVin750 and thimerosal.

9. A kit comprising the combination of reagents of claim 7 or 8;

and/or, the kit further comprises containers for separately containing the R1 reagent, R2 reagent;

and/or, the kit further comprises a package insert.

10. Use of a latex antibody complex according to any one of claims 1 to 2, or a reagent according to any one of claims 5 to 6, or a combination of reagents according to claims 7 to 8, or a kit according to claim 9 for the detection of C-reactive protein;

and/or, the application is detecting the concentration of C-reactive protein in the sample;

and/or, the sample is selected from at least one of whole blood, serum and plasma.