Anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit and preparation method thereof

Abstract

The invention discloses an antistreptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit and a preparation method thereof. The preparation method comprises the following steps: S1, preparing a sample diluent; s2, respectively preparing fluorescent microspheres marked with ASO-1 recombinant antigen, fluorescent microspheres marked with denatured IgG-1, fluorescent microspheres marked with goat anti-human IgG and fluorescent microspheres marked with chicken IgY for later use; s3, preparing a conjugate pad; s4, preparing a nitrocellulose membrane, diluting the ASO-2 recombinant antigen, the denatured IgG-2, the cyclic citrullinated peptide antigen and the goat anti-chicken IgY with the coating liquid respectively, and then marking the diluted materials onto the nitrocellulose membrane; s5, preparing a reagent strip; and S6, preparing the kit. The kit provided by the invention can realize simultaneous quantitative detection of three items of antistreptolysin O, rheumatoid factors and anti-cyclic citrullinated peptide antibodies by one reagent strip, and has the advantages of high detection specificity, high repeatability and stability, convenient operation and low cost.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro reagent instant detection, and in particular relates to an anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit and a preparation method thereof. Background Art

[0002] Anti-streptolysin O (ASO) is an antibody produced by the body against the bacterial metabolite streptolysin O after the body is infected by group A β-hemolytic streptococci. Its presence and content can reflect the severity of the infection and can be seen to increase in the acute stage of rheumatoid disease. It is mainly used clinically for the auxiliary diagnosis of acute rheumatic fever.

[0003] Rheumatoid factors (RF) are a group of heterogeneous autoantibodies against antigenic determinants on the Fc region of IgG molecules. They are important for the diagnosis of rheumatoid arthritis, but can also be seen in other inflammatory rheumatic diseases and various non-rheumatic diseases. They can also be seen in clinically healthy individuals over the age of 60. Despite these limitations, the detection of rheumatoid factors is still a diagnostic criterion for the classification of rheumatoid arthritis. It is mainly used clinically for the auxiliary diagnosis of rheumatoid arthritis (RA).

[0004] Anti-cyclic citrullinated peptide antibodies are autoantibodies with synthetic cyclic peptide containing citrulline (CCP) as antigens. They have high sensitivity and specificity for rheumatoid arthritis (RA) and are a highly specific indicator for the early diagnosis of RA. The detection of anti-cyclic citrullinated peptide antibodies is highly specific for the diagnosis of rheumatoid arthritis and can be used for the early diagnosis of RA. In addition, anti-CCP antibodies are not only an indicator for the early diagnosis of RA, but also a sensitive indicator for distinguishing between invasive and non-invasive RA.

[0005] At present, the methods for testing anti-streptolysin O, rheumatoid factor, and anti-cyclic citrullinated peptide antibodies include immunoturbidimetry, enzyme-linked immunosorbent assay, chemiluminescent immunoassay, etc., but these methods have their own defects. For example, the immunoturbidimetry method requires a large amount of antibodies and has low sensitivity; the enzyme-linked immunosorbent assay has a long detection time, low sensitivity, and a narrow detection range; the reagents of chemiluminescent immunoassay are usually expensive, which increases the detection cost. Some chemiluminescent reactions may produce harmful substances, and attention should be paid to environmental protection. At the same time, chemiluminescent reactions require specific instruments and equipment, which increases the complexity of the equipment. In addition, there are currently no reagent products on the market that can simultaneously detect the three detection items of anti-streptolysin O, rheumatoid factor, and anti-cyclic citrullinated peptide antibodies. The existing products are all reagent products for separate detection items.

[0006] Compared with immunoturbidimetry and enzyme-linked immunosorbent assay, immunochromatography is more sensitive, simpler and faster. With the corresponding reader, reliable quantitative data can be obtained, and the linear range can also meet the clinical needs of many products. In addition, compared with chemiluminescence immunoassay, immunochromatography is more convenient, less expensive, and has no environmental pollution. More importantly, it can get results in a short time, which brings great benefits to clinical emergency and is one of the preferred methods for point-of-care testing (POCT) at this stage. How to use immunochromatography to simultaneously detect three test items: anti-streptolysin O, rheumatoid factor, and anti-cyclic citrullinated peptide antibodies has become a topic that needs to be studied. Summary of the invention

[0007] The problem to be solved by the present invention is to provide a preparation method of a detection kit for anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing a detection kit for anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibodies, comprising the following steps:

[0009] S1: Prepare sample diluent for diluting the sample to be tested during detection;

[0010] S2: preparing fluorescent microspheres labeled with ASO-1 recombinant antigen, fluorescent microspheres labeled with denatured IgG-1, fluorescent microspheres labeled with goat anti-human IgG, and fluorescent microspheres labeled with chicken IgY respectively, and setting them aside for use;

[0011] S3: preparing a conjugate pad, diluting the fluorescent microspheres labeled with ASO-1 recombinant antigen, the fluorescent microspheres labeled with denatured IgG-1, the fluorescent microspheres labeled with goat anti-human IgG, and the fluorescent microspheres labeled with chicken IgY in step S2 respectively, spraying them onto the conjugate pad, and drying them for later use;

[0012] S4: prepare nitrocellulose membrane, dilute ASO-2 recombinant antigen, denatured IgG-2, cyclic citrullinated peptide antigen and goat anti-chicken IgY with coating solution, and then streak onto the nitrocellulose membrane, and dry for later use;

[0013] S5: preparing a reagent strip, connecting and fixing the sample pad, conjugate pad, nitrocellulose membrane and absorbent paper to the bottom plate in sequence, and cutting and assembling them into a reagent strip;

[0014] S6: preparing a kit, packaging the sample diluent prepared in step S1 and the reagent strip prepared in step S5 and placing them in the kit to obtain the anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit.

[0015] The conjugate pad of the invention is provided with fluorescent microspheres for marking ASO-1 recombinant antigen, fluorescent microspheres for marking denatured IgG-1, fluorescent microspheres for marking goat anti-human IgG and fluorescent microspheres for marking chicken IgY, and the nitrocellulose membrane is provided with ASO-2 recombinant antigen, denatured IgG-2, cyclic citrullinated peptide antigen and goat anti-chicken IgY. The kit obtained by the preparation method enables the invention to realize quantitative detection of three items of anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody by one reagent strip at the same time, and the detection has high specificity, high repeatability and stability, convenient detection and low cost.

[0016] Furthermore, in step S2:

[0017] The preparation method of the fluorescent microspheres labeled with the ASO-1 recombinant antigen is as follows: activating the fluorescent microspheres with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, adding the labeled ASO-1 recombinant antigen, reacting, adding a blocking agent to block, centrifuging, and storing, thereby obtaining the fluorescent microspheres labeled with the ASO-1 recombinant antigen;

[0018] The preparation method of fluorescent microspheres labeled with denatured IgG-1 is as follows: activating the fluorescent microspheres with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, adding labeled denatured IgG-1, reacting, adding a blocking agent to block, centrifuging, and storing, thereby obtaining fluorescent microspheres labeled with denatured IgG-1;

[0019] The preparation method of the fluorescent microspheres labeled with goat anti-human IgG is as follows: activating the fluorescent microspheres with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, adding the labeled goat anti-human IgG, adding a blocking agent to block after the reaction, centrifuging, and storing, thereby obtaining the fluorescent microspheres labeled with goat anti-human IgG;

[0020] The preparation method of fluorescent microspheres labeled with chicken IgY is as follows: the fluorescent microspheres are activated with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, the labeled chicken IgY is added, after the reaction, a blocking agent is added for blocking, centrifugation is performed, and storage is performed to obtain fluorescent microspheres labeled with chicken IgY.

[0021] Furthermore, in step S2:

[0022] When the fluorescent microspheres of the ASO-1 recombinant antigen are labeled, the mass ratio of the fluorescent microspheres to the ASO-1 recombinant antigen is 1:2-1:10, and the reaction conditions are: 37° C. for 3 hours;

[0023] When the fluorescent microspheres are used to label denatured IgG-1, the mass ratio of the fluorescent microspheres to the denatured IgG-1 is 1:2-1:10, and the reaction conditions are: 37° C. for 3 hours;

[0024] When the fluorescent microspheres of goat anti-human IgG are labeled, the mass ratio of the fluorescent microspheres to goat anti-human IgG is 1:2-1:10, and the reaction conditions are: 37° C. for 3 hours;

[0025] When the fluorescent microspheres are labeled with chicken IgY, the mass ratio of the fluorescent microspheres to chicken IgY is 1:2-1:10, and the reaction conditions are: 37° C. for 3 hours.

[0026] Furthermore, in step S2, when the fluorescent microspheres are activated, the mass ratio of the fluorescent microspheres to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide is 1:2:2-1:10:10, and the reaction conditions are: 37° C. for 25 min.

[0027] Furthermore, in the step S3, the diluents used for diluting the fluorescent microspheres labeled with ASO-1 recombinant antigen, the fluorescent microspheres labeled with denatured IgG-1, the fluorescent microspheres labeled with goat anti-human IgG, and the fluorescent microspheres labeled with chicken IgY are prepared as follows: Tris buffer with a concentration of 50mM-100mM, sucrose with a mass ratio of 2%-20%, Tween20 with a volume ratio of 0.5%-2%, and ProClin 300 with a volume ratio of 0.1%-0.5% are added to a container, mixed evenly, fixed to volume, and after mixing, the pH is adjusted to 8.5±0.2 to obtain the diluent.

[0028] Furthermore, in step S3, the fluorescent microspheres labeled with ASO-1 recombinant antigen, the fluorescent microspheres labeled with denatured IgG-1, the fluorescent microspheres labeled with goat anti-human IgG, and the fluorescent microspheres labeled with chicken IgY are diluted and sprayed onto the conjugate pad at a spray volume of 1-2 μL / cm.

[0029] Furthermore, in step S4, the coating solution used for diluting the ASO-2 recombinant antigen, denatured IgG-2, cyclic citrullinated peptide antigen and goat anti-chicken IgY with the coating solution is prepared as follows: adding phosphate buffer with a concentration of 10mM-100mM, sucrose with a mass ratio of 2%-20% and ProClin 300 with a volume ratio of 0.1%-0.5% to a container, mixing evenly, fixing the volume, and adjusting the pH to 7.4±0.2 after mixing to obtain the coating solution.

[0030] Furthermore, in step S4, the ASO-2 recombinant antigen, denatured IgG-2, cyclic citrullinated peptide antigen and goat anti-chicken IgY are respectively diluted with coating solution and streaked onto the nitrocellulose membrane with a coating amount of 0.5-1 μL / cm; the ASO-2 recombinant antigen, denatured IgG-2 and cyclic citrullinated peptide antigen are respectively streaked as a detection line, and the goat anti-chicken IgY is streaked as a quality control line.

[0031] Furthermore, in the step S1, the sample diluent is prepared by adding citrate with a final concentration of 10mM-100mM and ProClin 300 with a volume ratio of 0.1%-0.5% into a container, mixing evenly, fixing the volume, and adjusting the pH to 7.4±1 after mixing to obtain the sample diluent.

[0032] Another problem to be solved by the present invention is to provide a detection kit for anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibodies.

[0033] In order to solve the above technical problems, the scheme adopted is: an anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit obtained by the preparation method of the above-mentioned anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit, comprising a sample diluent and a reagent strip; the nitrocellulose membrane of the reagent strip is coated with a detection line and a quality control line, the detection line includes three lines, which are respectively coated with ASO-2 recombinant antigen, denatured IgG-2 and cyclic citrullinated peptide antigen, and the quality control line is coated with goat anti-chicken IgY; the binding pad of the reagent strip is sprayed with fluorescent microspheres marked with ASO-1 recombinant antigen, fluorescent microspheres marked with denatured IgG-1, fluorescent microspheres marked with goat anti-human IgG and fluorescent microspheres marked with chicken IgY.

[0034] Compared with the prior art, the present invention can realize quantitative detection of three items, namely anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody, by one reagent strip at the same time, and has high detection specificity, high repeatability and stability, convenient detection and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The following is further described in detail with reference to the accompanying drawings and embodiments of the present invention:

[0036] Figure 1 It is a schematic diagram of the structure of the reagent strip in the kit of this application;

[0037] 1-sample pad; 2-binding pad; 3-nitrocellulose membrane; 4-absorbent paper; 31-test line 1; 32-test line 2; 33-test line 3; 34-quality control line. DETAILED DESCRIPTION

[0038] The present invention provides a method for preparing an anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit, comprising the following steps:

[0039] S1: Prepare sample diluent for diluting the sample to be tested during detection;

[0040] S2: Prepare fluorescent microspheres (manufacturer: Bangs Laboratories) labeled with ASO-1 recombinant antigen (manufacturer: Jiangsu Huakong Biotechnology Co., Ltd.), fluorescent microspheres labeled with denatured IgG-1 (manufacturer: Jiangsu Huakong Biotechnology Co., Ltd.), fluorescent microspheres labeled with goat anti-human IgG (manufacturer: Jiangsu Huakong Biotechnology Co., Ltd.), and fluorescent microspheres labeled with chicken IgY (manufacturer: Jiangsu Huakong Biotechnology Co., Ltd.) respectively, and set aside for use;

[0041] S3: preparing a conjugate pad 2, diluting the fluorescent microspheres labeled with ASO-1 recombinant antigen, the fluorescent microspheres labeled with denatured IgG-1, the fluorescent microspheres labeled with goat anti-human IgG, and the fluorescent microspheres labeled with chicken IgY in step S2 respectively, and spraying them onto the conjugate pad 2, and drying them for later use;

[0042] S4: prepare nitrocellulose membrane 3, dilute ASO-2 recombinant antigen (manufacturer: Jiangsu Huakong Biotechnology Co., Ltd.), denatured IgG-2 (manufacturer: Jiangsu Huakong Biotechnology Co., Ltd.), cyclic citrullinated peptide antigen (manufacturer: Jiangsu Huakong Biotechnology Co., Ltd.) and goat anti-chicken IgY (manufacturer: Jiangsu Huakong Biotechnology Co., Ltd.) with coating solution, and then streak onto nitrocellulose membrane 3, and dry for later use;

[0043] S5: Prepare a reagent strip, connect and fix the sample pad 1, the conjugate pad 2, the nitrocellulose membrane 3 and the absorbent paper 4 to the bottom plate in sequence, cut and assemble into a reagent strip, such as Figure 1 As shown;

[0044] S6: preparing a kit, packaging the sample diluent prepared in step S1 and the reagent strip prepared in step S5 and placing them in the kit to obtain the anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit.

[0045] The conjugate pad of the invention is provided with fluorescent microspheres for marking ASO-1 recombinant antigen, fluorescent microspheres for marking denatured IgG-1, fluorescent microspheres for marking goat anti-human IgG and fluorescent microspheres for marking chicken IgY, and the nitrocellulose membrane is provided with ASO-2 recombinant antigen, denatured IgG-2, cyclic citrullinated peptide antigen and goat anti-chicken IgY, so that the invention can realize quantitative detection of three items of anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody by one reagent strip at the same time, and the detection has high specificity, high repeatability and stability, convenient detection and low cost.

[0046] In the step S2: the preparation method of the fluorescent microspheres labeled with the ASO-1 recombinant antigen is as follows: the fluorescent microspheres are activated with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (manufacturer: Sigma-Aldrich) and N-hydroxysulfosuccinimide (manufacturer: Sigma-Aldrich), the labeled ASO-1 recombinant antigen is added, after the reaction, a blocking agent is added for blocking, centrifugation is performed, and storage is performed to obtain the fluorescent microspheres labeled with the ASO-1 recombinant antigen;

[0047] The preparation method of fluorescent microspheres labeled with denatured IgG-1 is as follows: activating the fluorescent microspheres with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, adding labeled denatured IgG-1, reacting, adding a blocking agent to block, centrifuging, and storing, thereby obtaining fluorescent microspheres labeled with denatured IgG-1;

[0048] The preparation method of the fluorescent microspheres labeled with goat anti-human IgG is as follows: activating the fluorescent microspheres with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, adding the labeled goat anti-human IgG, adding a blocking agent to block after the reaction, centrifuging, and storing, thereby obtaining the fluorescent microspheres labeled with goat anti-human IgG;

[0049] The preparation method of fluorescent microspheres labeled with chicken IgY is as follows: the fluorescent microspheres are activated with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, the labeled chicken IgY is added, after the reaction, a blocking agent is added for blocking, centrifugation is performed, and storage is performed to obtain fluorescent microspheres labeled with chicken IgY.

[0050] Furthermore, in the step S2: when labeling the fluorescent microspheres of the ASO-1 recombinant antigen, the mass ratio of the fluorescent microspheres to the ASO-1 recombinant antigen is 1:2-1:10, and the reaction conditions are: 37° C. for 3 hours;

[0051] When the fluorescent microspheres are used to label denatured IgG-1, the mass ratio of the fluorescent microspheres to the denatured IgG-1 is 1:2-1:10, and the reaction conditions are: 37° C. for 3 hours;

[0052] When the fluorescent microspheres of goat anti-human IgG are labeled, the mass ratio of the fluorescent microspheres to goat anti-human IgG is 1:2-1:10, and the reaction conditions are: 37° C. for 3 hours;

[0053] When the fluorescent microspheres are labeled with chicken IgY, the mass ratio of the fluorescent microspheres to chicken IgY is 1:2-1:10, and the reaction conditions are: 37° C. for 3 hours.

[0054] In the step S2, when the fluorescent microspheres are activated, the mass ratio of the fluorescent microspheres to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide is 1:2:2-1:10:10, and the reaction conditions are: 37° C. for 25 min.

[0055] In the step S3, the diluents used for diluting the fluorescent microspheres labeled with ASO-1 recombinant antigen, the fluorescent microspheres labeled with denatured IgG-1, the fluorescent microspheres labeled with goat anti-human IgG, and the fluorescent microspheres labeled with chicken IgY are prepared as follows: Tris buffer with a concentration of 50mM-100mM, sucrose with a mass ratio of 2%-20%, Tween20 with a volume ratio of 0.5%-2%, and ProClin 300 with a volume ratio of 0.1%-0.5% are added to a container, mixed evenly, fixed to volume, and after mixing, the pH is adjusted to 8.5±0.2 to obtain the diluent.

[0056] Furthermore, in step S3, the fluorescent microspheres labeled with ASO-1 recombinant antigen, the fluorescent microspheres labeled with denatured IgG-1, the fluorescent microspheres labeled with goat anti-human IgG, and the fluorescent microspheres labeled with chicken IgY are diluted and sprayed onto the conjugate pad 2 at a spray volume of 1-2 μL / cm.

[0057] In the step S4, the coating solution used for diluting the ASO-2 recombinant antigen, the denatured IgG-2, the cyclic citrullinated peptide antigen and the goat anti-chicken IgY with the coating solution is prepared as follows: phosphate buffer with a concentration of 10mM-100mM, sucrose with a mass ratio of 2%-20%, and ProClin 300 with a volume ratio of 0.1%-0.5% are added to a container, mixed evenly, fixed to volume, and adjusted to pH 7.4±0.2 after mixing to obtain the coating solution.

[0058] Furthermore, in step S4, when the ASO-2 recombinant antigen, denatured IgG-2, cyclic citrullinated peptide antigen and sheep anti-chicken IgY are diluted with coating solution and then streaked onto the nitrocellulose membrane 3, the streaking coating amount is 0.5-1 μL / cm; the ASO-2 recombinant antigen, denatured IgG-2, and cyclic citrullinated peptide antigen are streaked as detection line one 31, detection line two 32, and detection line three 33, respectively, and the sheep anti-chicken IgY is streaked as the quality control line 34.

[0059] Furthermore, in the step S1, the sample diluent is prepared by adding citrate with a final concentration of 10mM-100mM and ProClin 300 with a volume ratio of 0.1%-0.5% into a container, mixing evenly, fixing the volume, and adjusting the pH to 7.4±1 after mixing to obtain the sample diluent.

[0060] The present invention also provides an anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit obtained by adopting the preparation method of the anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit, comprising a sample diluent and a reagent strip; the reagent strip comprises a bottom plate and a sample pad 1, a conjugation pad 2, a nitrocellulose membrane 3 and a blotting paper 4 arranged on the bottom plate, wherein the sample pad 1 is arranged above the conjugation pad 2, the conjugation pad 2 and the blotting paper 4 are respectively arranged at two ends above the nitrocellulose membrane 3 and are on the same horizontal plane, the nitrocellulose membrane 3 is coated with a detection line and a quality control line 34, the detection line comprises three lines, which are respectively coated with ASO-2 recombinant antigen, denatured IgG-2 and cyclic citrullinated peptide antigen, and the quality control line 34 is coated with goat anti-chicken IgY; the conjugation pad 2 of the reagent strip is sprayed with fluorescent microspheres marked with ASO-1 recombinant antigen, fluorescent microspheres marked with denatured IgG-1, fluorescent microspheres marked with goat anti-human IgG and fluorescent microspheres marked with chicken IgY.

[0061] When testing samples, dilute the sample to be tested with sample diluent as required, and then add the diluted sample to the reagent strip through the sample addition port. Due to capillary action, the sample passes through the sample pad 1, the conjugate pad 2 and the nitrocellulose membrane 3 in sequence. After passing through the conjugate pad 2 and the nitrocellulose membrane 3, the waste liquid finally enters the absorbent paper 4. After the sample is added, the reagent strip is left to react for 10-15 minutes, and the fluorescent immunoassay analyzer reads the test results.

[0062] Compared with the prior art which can only detect one item separately, the present invention adopts immunochromatography to realize quantitative detection of three items of anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody with one reagent strip at the same time, and has high detection specificity, high repeatability and stability, convenient operation and low cost. The present application adopts immunochromatography to detect anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody, and its performance index comparison with other detection methods is shown in Table 1 below.

[0063] Table 1 Comparison of performance indicators of immunochromatography and other detection methods

[0064]

[0065] It can be seen from Table 1 above that the immunochromatography method has higher sensitivity, accuracy and repeatability than the immunoturbidimetry method and the enzyme-linked immunosorbent assay, and has a shorter detection time than the chemiluminescence method, and is easy to operate and low in cost. Comprehensively speaking, the immunochromatography method has a higher cost-effectiveness.

[0066] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing a kit for detecting anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibodies, characterized in that: The following steps are involved: S1: Prepare sample diluent for diluting the sample to be tested during detection; S2: preparing fluorescent microspheres labeled with ASO-1 recombinant antigen, fluorescent microspheres labeled with denatured IgG-1, fluorescent microspheres labeled with goat anti-human IgG, and fluorescent microspheres labeled with chicken IgY respectively, and setting them aside for use; S3: preparing a conjugate pad, diluting the fluorescent microspheres labeled with ASO-1 recombinant antigen, the fluorescent microspheres labeled with denatured IgG-1, the fluorescent microspheres labeled with goat anti-human IgG, and the fluorescent microspheres labeled with chicken IgY in step S2 respectively, spraying them onto the conjugate pad, and drying them for later use; S4: prepare nitrocellulose membrane, dilute ASO-2 recombinant antigen, denatured IgG-2, cyclic citrullinated peptide antigen and goat anti-chicken IgY with coating solution, then streak onto the nitrocellulose membrane, dry and set aside; S5: preparing a reagent strip, connecting and fixing the sample pad, conjugate pad, nitrocellulose membrane and absorbent paper to the bottom plate in sequence, and cutting and assembling them into a reagent strip; S6: preparing a kit, packaging the sample diluent prepared in step S1 and the reagent strip prepared in step S5 and placing them in the kit to obtain the anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit.

2. The method for preparing the anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit according to claim 1, characterized in that: In step S2: The preparation method of the fluorescent microspheres labeled with the ASO-1 recombinant antigen is as follows: activating the fluorescent microspheres with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, adding the labeled ASO-1 recombinant antigen, reacting, adding a blocking agent to block, centrifuging, and storing, thereby obtaining the fluorescent microspheres labeled with the ASO-1 recombinant antigen; The preparation method of fluorescent microspheres labeled with denatured IgG-1 is as follows: activating the fluorescent microspheres with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, adding labeled denatured IgG-1, reacting, adding a blocking agent to block, centrifuging, and storing, thereby obtaining fluorescent microspheres labeled with denatured IgG-1; The preparation method of the fluorescent microspheres labeled with goat anti-human IgG is as follows: activating the fluorescent microspheres with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, adding the labeled goat anti-human IgG, adding a blocking agent to block after the reaction, centrifuging, and storing, thereby obtaining the fluorescent microspheres labeled with goat anti-human IgG; The preparation method of fluorescent microspheres labeled with chicken IgY is as follows: the fluorescent microspheres are activated with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, the labeled chicken IgY is added, after the reaction, a blocking agent is added for blocking, centrifugation is performed, and storage is performed to obtain fluorescent microspheres labeled with chicken IgY.

3. The method for preparing the anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit according to claim 2, characterized in that: In step S2: When the fluorescent microspheres of the ASO-1 recombinant antigen are labeled, the mass ratio of the fluorescent microspheres to the ASO-1 recombinant antigen is 1:2-1:10, and the reaction conditions are: 37° C. for 3 hours; When the fluorescent microspheres are used to label denatured IgG-1, the mass ratio of the fluorescent microspheres to the denatured IgG-1 is 1:2-1:10, and the reaction conditions are: 37° C. for 3 hours; When the fluorescent microspheres of goat anti-human IgG are labeled, the mass ratio of the fluorescent microspheres to goat anti-human IgG is 1:2-1:10, and the reaction conditions are: 37° C. for 3 hours; When the fluorescent microspheres are labeled with chicken IgY, the mass ratio of the fluorescent microspheres to chicken IgY is 1:2-1:10, and the reaction conditions are: 37° C. for 3 hours.

4. The method for preparing the anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit according to claim 2, characterized in that: In the step S2, when the fluorescent microspheres are activated, the mass ratio of the fluorescent microspheres to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide is 1:2:2-1:10:10, and the reaction conditions are: 37° C. for 25 min.

5. The method for preparing the anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit according to claim 1, characterized in that: In the step S3, the diluents used for diluting the fluorescent microspheres labeled with ASO-1 recombinant antigen, the fluorescent microspheres labeled with denatured IgG-1, the fluorescent microspheres labeled with goat anti-human IgG, and the fluorescent microspheres labeled with chicken IgY are prepared as follows: Tris buffer with a concentration of 50mM-100mM, sucrose with a mass ratio of 2%-20%, Tween20 with a volume ratio of 0.5%-2%, and ProClin 300 with a volume ratio of 0.1%-0.5% are added to a container, mixed evenly, fixed to volume, and after mixing, the pH is adjusted to 8.5±0.2 to obtain the diluent.

6. The method for preparing the anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit according to claim 5, characterized in that: In step S3, the fluorescent microspheres labeled with ASO-1 recombinant antigen, the fluorescent microspheres labeled with denatured IgG-1, the fluorescent microspheres labeled with goat anti-human IgG, and the fluorescent microspheres labeled with chicken IgY are diluted and sprayed onto the conjugate pad at a spray volume of 1-2 μL / cm.

7. The method for preparing the anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit according to claim 1, characterized in that: In step S4, the coating solution used for diluting the ASO-2 recombinant antigen, denatured IgG-2, cyclic citrullinated peptide antigen and goat anti-chicken IgY with the coating solution is prepared as follows: phosphate buffer with a concentration of 10mM-100mM, sucrose with a mass ratio of 2%-20% and ProClin 300 with a volume ratio of 0.1%-0.5% are added to a container, mixed evenly, fixed to volume, and adjusted to pH 7.4±0.2 after mixing to obtain the coating solution.

8. The method for preparing the anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit according to claim 7, characterized in that: In the step S4, the ASO-2 recombinant antigen, denatured IgG-2, cyclic citrullinated peptide antigen and goat anti-chicken IgY are respectively diluted with coating solution and then streaked onto the nitrocellulose membrane with a coating amount of 0.5-1 μL / cm; the ASO-2 recombinant antigen, denatured IgG-2 and cyclic citrullinated peptide antigen are respectively streaked as a detection line, and the goat anti-chicken IgY is streaked as a quality control line.

9. The method for preparing the anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit according to claim 1, characterized in that: In step S1, the sample diluent is prepared by adding citrate with a final concentration of 10 mM-100 mM and ProClin 300 with a volume ratio of 0.1%-0.5% into a container, mixing evenly, fixing the volume, and adjusting the pH to 7.4±1 after mixing to obtain the sample diluent.

10. An anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit obtained by the preparation method of the anti-streptolysin O, rheumatoid factor and anti-cyclic citrullinated peptide antibody detection kit according to any one of claims 1 to 9, characterized in that: The kit comprises a sample diluent and a reagent strip; a detection line and a quality control line are coated on the nitrocellulose membrane of the reagent strip, wherein the detection line comprises three lines, which are respectively coated with ASO-2 recombinant antigen, denatured IgG-2 and cyclic citrullinated peptide antigen, and the quality control line is coated with goat anti-chicken IgY; and a conjugation pad of the reagent strip is sprayed with fluorescent microspheres marked with ASO-1 recombinant antigen, fluorescent microspheres marked with denatured IgG-1, fluorescent microspheres marked with goat anti-human IgG and fluorescent microspheres marked with chicken IgY.

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