Preparation of rheumatoid factor fragment antigen, detection kit and preparation method

Abstract

The invention relates to the technical field of analytical chemistry, and provides a rheumatoid factor (RF) fragment antigen preparation method, a rheumatoid factor (RF) fragment antigen detection kit and a rheumatoid factor (RF) fragment antigen preparation method. Papain is used for enzyme digestion of an IgG solution to obtain a mixture of Fab and Fc fragments; and purifying the enzyme-digested product to obtain an Fc fragment, and diluting and storing the Fc fragment with a storage buffer solution. The prepared RF fragment antigen is applied to a detection kit of an RF-IgM subtype. The RF and subtype determination kit comprises an R1 magnetic particle working solution, an R2 chemiluminescent marker working solution and an R3 sample dilution buffer solution, and the R1 magnetic particle working solution contains an RF fragment antigen magnetic bead compound. Compared with the prior art, the reagent performance of the product can be greatly improved, the preparation method is simple, the preparation batch-to-batch difference is controllable, and large-scale industrial production can be carried out.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry and relates to a preparation of a rheumatoid factor fragment antigen, a detection kit and a preparation method. Background Art

[0002] Rheumatoid factor (RF) is an autoantibody with denatured IgG as its target antigen. RF mistakenly attacks its own cells and tissues through autoimmune reactions, producing immune complexes. These immune complexes are deposited in joints, blood vessel walls, and other body tissues, and trigger an inflammatory response, which can lead to joint swelling, pain, stiffness, and functional impairment. Common RF-induced diseases include autoimmune diseases such as rheumatoid arthritis (RA), Raynaud's phenomenon, systemic lupus erythematosus, or Sjögren's syndrome. Studies have shown that the positive rate of RF is only 2% in normal people, 5% in the elderly, and 80% in patients with rheumatoid arthritis (RA). Therefore, RF is an important indicator for the diagnosis of RA. RF can be divided into different subtypes, such as IgM, IgA, IgG, etc.

[0003] At present, the detection methods of RF and its subtypes are mainly based on the principles of enzyme-linked immunosorbent assay and latex immunoturbidimetry. Among them, the enzyme-linked immunosorbent assay has good repeatability, but poor sensitivity and specificity, and has many operation steps and takes a long time to detect; the latex immunoturbidimetry is used on a general biochemical platform and has a fast detection speed, but the equipment cost is high, and the total RF is detected, but different subtypes of RF are not typed. On the other hand, the prior art also has a technical solution for detecting RF through chemiluminescence immunoassay, which solves the problems of low sensitivity and low specificity, and can perform RF typing detection, but it also faces the new problems of narrow detection range and inability to quantify.

[0004] At present, the research on the antigenic epitope of RF is still not thorough, and it is impossible to make a recombinant antigen. The general practice is to denature human IgG. There are two denaturation methods: chemical denaturation and physical denaturation. Chemical denaturation will introduce chemical denaturants, and it is necessary to increase the purification step, which is not conducive to large-scale production; the physical denaturation method is simple, but the stability of the denatured protein is challenged, which brings a lot of troubles to the application end. For example, when applied to latex turbidimetric products, nonspecific agglutination reactions are prone to occur. Therefore, there is an urgent need for a preparation method of RF antigen with better effect, easy to promote and apply. Summary of the invention

[0005] In order to make up for the shortcomings of the prior art, the purpose of the present invention is to provide a method for preparing RF fragment antigens based on enzyme-cleaved antibodies, by removing the Fab segment of human IgG antibodies to fully expose the Fc segment, and the protease used for enzyme cleavage can be easily separated by purification. At the same time, a detection kit based on this RF fragment antigen was developed.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a method for preparing an Fc fragment antigen of a rheumatoid factor, and the specific steps are as follows: using papain to enzymatically digest an IgG solution to obtain a mixture of Fab and Fc fragments; purifying the enzymatic digestion product to obtain an Fc fragment, which is diluted with a storage buffer and stored.

[0008] The enzymatic cleavage step includes using an activation buffer to dissolve and activate papain, adding the activated papain to the IgG solution for sufficient reaction, and replacing the activation buffer to terminate the enzymatic cleavage reaction.

[0009] Furthermore, the components of the activation buffer include phosphate buffer, ethylenediaminetetraacetic acid and cysteine, and the pH value is 8.0.

[0010] Furthermore, the activation buffer comprises 0.01-0.2 mol / L phosphate buffer, 0.01-1 mol / L ethylenediaminetetraacetic acid and 1-100 mmol / L cysteine, and the pH value is 8.0.

[0011] Furthermore, the temperature for activating papain is 37°C.

[0012] Furthermore, the concentration of the activated papain solution is 0.1-10 mg / mL.

[0013] Furthermore, the ratio of activated papain to IgG solution in the sufficient reaction is 1:1-20.

[0014] Furthermore, the condition for sufficient reaction is a constant temperature water bath.

[0015] Furthermore, the conditions for the sufficient reaction are a constant temperature water bath at 37° C. and a reaction time of 0.5 to 12 hours.

[0016] Furthermore, the method of replacing the activation buffer to terminate the enzyme cleavage reaction includes dialysis and ultrafiltration.

[0017] The purification column used in the purification comprises at least one of a protein A affinity column, a protein G affinity column and a protein L affinity column.

[0018] The components of the storage buffer include phosphate buffer, L-arginine, trehalose, 2-methyl-4-isothiazoline-3-one, 5-chloro-2-methyl-4-isothiazoline-3-one, and purified water, and the pH value is 7.4.

[0019] Furthermore, the concentration of the storage buffer is 5 to 100 mg / mL.

[0020] Furthermore, the components of the storage buffer are 20-100 mmol / L phosphate buffer, 50-200 mM L-arginine, 10-60 g / L trehalose, 0.5-2 g / L 2-methyl-4-isothiazoline-3-one, 0.5-2 g / L 5-chloro-2-methyl-4-isothiazoline-3-one, and purified water, and the pH value is 7.4.

[0021] The second aspect of the present invention provides an Fc fragment antigen of rheumatoid factor, which is obtained according to the preparation method described in the first aspect of the present invention.

[0022] The third aspect of the present invention provides a method for quantitatively detecting rheumatoid factor, which comprises detecting the Fc fragment antigen described in the second aspect of the present invention by chemiluminescence.

[0023] The method comprises the following steps: contacting the sample to be tested with the Fc fragment antigen coupled to the magnetic microparticles, then adding the anti-rheumatoid factor antibody coupled to the chemiluminescent marker, magnetically separating after the reaction is completed, adding the chemiluminescent substrate, detecting the chemiluminescent intensity, drawing a standard curve, and calculating the concentration of the rheumatoid factor in the sample to be tested.

[0024] Furthermore, the sample to be tested is plasma or serum.

[0025] Furthermore, the magnetic particles are carboxyl magnetic particles.

[0026] Furthermore, the concentration of the magnetic particles is 0.05-1 mg / mL.

[0027] Furthermore, the preparation method of the magnetic particle-coupled Fc fragment antigen comprises the following steps: washing and activating the carboxyl magnetic particles, coupling and binding with the Fc fragment antigen in a magnetic particle binding buffer, blocking in a magnetic particle blocking buffer, and washing to obtain the magnetic particle-coupled Fc fragment antigen.

[0028] Furthermore, the mass ratio of the magnetic particles to the Fc fragment antigen is 1000:5-40.

[0029] Furthermore, the rheumatoid factor includes at least one of rheumatoid factor-IgA, rheumatoid factor-IgG and rheumatoid factor-IgM.

[0030] Furthermore, the anti-rheumatoid factor antibody includes at least one of anti-rheumatoid factor-IgA, anti-rheumatoid factor-IgG and anti-rheumatoid factor-IgM.

[0031] Furthermore, the chemiluminescent markers include acridinium esters, acridinium ester derivatives, and alkaline phosphatase.

[0032] Furthermore, the method for preparing the anti-rheumatoid factor antibody coupled with the chemiluminescent marker comprises: incubating the chemiluminescent marker and the anti-rheumatoid factor antibody in a buffer solution and then dialysis purification to obtain the anti-rheumatoid factor antibody coupled with the chemiluminescent marker.

[0033] Furthermore, the concentration of the chemiluminescent marker is 0.1 mg / mL-2 mg / mL.

[0034] Furthermore, the chemiluminescent substrate includes alkaline hydrogen peroxide and AMPPD.

[0035] Furthermore, the method steps include: diluting the sample to be tested with a sample dilution buffer, adding it to a magnetic particle working solution and a chemiluminescent marker working solution in sequence, magnetically separating after the reaction is completed, adding a chemiluminescent substrate after washing, detecting the chemiluminescent intensity, drawing a standard curve, and calculating the concentration of rheumatoid factor in the sample to be tested.

[0036] The fourth aspect of the present invention provides a magnetic particle chemiluminescence kit for quantitatively detecting rheumatoid factor, which comprises the Fc fragment antigen described in the second aspect of the present invention.

[0037] Furthermore, the Fc fragment antigen is a magnetic particle. Further, the components of the R1 magnetic particle working solution are 0.05-1g / L magnetic particles coupled with Fc fragment antigen, 20-100mmol / L buffer, 2-10g / L stabilizer, 1-5g / L surfactant, 0.5-2g / L preservative and purified water, and the pH value is 5.5-8.0.

[0038] Furthermore, the preparation method of the R1 magnetic particle working solution is as follows: the carboxyl magnetic particles are washed and activated, coupled with the Fc fragment antigen in a magnetic particle binding buffer, blocked in a magnetic particle blocking buffer, and the Fc fragment antigen-carboxyl magnetic particle complex is obtained after washing, and a magnetic particle coupling diluent is added to dilute to obtain the R1 magnetic particle working solution.

[0039] Furthermore, the magnetic particle binding buffer is 0.1M 2-(N-morpholine)ethanesulfonic acid buffer.

[0040] Furthermore, the magnetic particle blocking buffer is 1% bovine serum albumin.

[0041] The components of the R2 chemiluminescent marker working solution include rheumatoid factor antibody coupled with a chemiluminescent marker, a buffer, a protein protective agent, a surfactant, glycerol, a preservative and purified water, and the pH value is 7.5-9.0.

[0042] Furthermore, the components of the R2 chemiluminescent marker working solution are 0.1-5 mg / L rheumatoid factor antibody coupled with a chemiluminescent marker, 20-100 mmol / L buffer, 2-10 g / L protein protectant, 0.5-2 g / L preservative and purified water, and the pH value is 6.0-7.5.

[0043] Furthermore, the preparation method of the R2 chemiluminescent marker working solution is as follows: the chemiluminescent marker and the anti-rheumatoid factor IgM antibody are incubated and combined in a buffer solution, and then dialyzed and purified to obtain the anti-rheumatoid factor IgM antibody coupled with the chemiluminescent marker, and then a diluent is added to dilute and obtain the R2 chemiluminescent marker working solution.

[0044] The components of the R3 sample dilution buffer include a buffer, a protein protectant, a preservative and purified water, and the pH value is 7.4-9.0.

[0045] Furthermore, the components of the R3 sample dilution buffer are 20-100 mmol / L buffer, 2-10 g / L protein protectant, 0.5-2 g / L preservative and purified water, and the pH value is 7.4-9.0.

[0046] Furthermore, the chemiluminescent substrate includes alkaline hydrogen peroxide and AMPPD.

[0047] The buffer comprises one of phosphate buffer, glycine buffer, 2-(N-morpholine)ethanesulfonic acid buffer, hydroxyethylpiperazineethanesulfonic acid buffer, 3-(N-morpholine)propanesulfonic acid buffer or tris(hydroxymethyl)aminomethane buffer and borate buffer.

[0048] The stabilizer includes one or more of bovine serum albumin, disodium ethylenediaminetetraacetate, and histidine.

[0049] The technical solution of the present invention adds a stabilizer to ensure that the Fc fragment and RF have an optimal reaction pH value, and on the other hand, ensure that the reagent has good stability during storage.

[0050] The protein protective agent includes one or more of bovine serum albumin, trehalose, sucrose, lactose, choline chloride, sorbitol, glycerol, glycine, and arginine.

[0051] Protein protectants play a key role in protecting protein stability and activity. By reducing oxidative damage, regulating temperature and pH, providing appropriate ion concentrations, promoting correct protein folding, and inhibiting nonspecific interactions, protein protectants can protect proteins from environmental and stress effects and maintain their integrity and functionality.

[0052] The surfactant includes one or more of Tween 20, Tween 80, Triton X-100, Triton X-405, polyethylene glycol octylphenyl ether, and ethylenediamine polyoxypropylene polyoxyethylene ether.

[0053] Surfactants have the functions of solubilization, sensitization, enhancement of luminescent signals, increase of system stability, reduction of nonspecific reactions and maintenance of pH stability of the solution system in chemiluminescent immunoassay. They can also fully expose the binding sites of rheumatoid factors in the sample to be tested, which is beneficial to the full recognition of antigen-antibody.

[0054] The preservative includes one or more of sodium azide, sodium nitrite, N-methyl-isothiazolone, N'-methylene-bis[N'-(3-hydroxymethyl-2,5-diketo-4-imidazolidinyl)]urea or a mixed solution of 2-methyl-4-isothiazoline-3-one and 5-chloro-2-methyl-4-isothiazoline-3-one.

[0055] During the long-term storage of protein, microbial growth may also lead to protein hydrolysis and inactivation, so preservatives need to be added, such as sodium azide, sodium thiothioate, ProClin series and antibiotics.

[0056] The pH value is adjusted by concentrated hydrochloric acid or sodium hydroxide solution.

[0057] Furthermore, the pH value is adjusted by 3 mol / L hydrochloric acid or 6 mol / L sodium hydroxide solution.

[0058] The fifth aspect of the present invention provides the use of the Fc fragment antigen described in the second aspect of the present invention, the method described in the third aspect of the present invention, and the magnetic particle chemiluminescence kit described in the fourth aspect of the present invention in the preparation of products for quantitative detection of rheumatoid factor and its subtypes, or in the preparation of products for diagnosis or auxiliary diagnosis of rheumatoid factor-related diseases.

[0059] Furthermore, the rheumatoid factor-related diseases include rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, dermatomyositis, scleroderma, mononucleosis, hepatitis, influenza, tuberculosis, bacterial endocarditis, malaria, schistosomiasis and certain tumors.

[0060] It should be noted that the detection method provided by the present invention is not for diagnosis and treatment purposes. The results obtained using the detection method of the present invention are only for quantifying the rheumatoid factor in the sample to be tested, and provide a certain reference value for the diagnosis of rheumatoid factor-related diseases, such as risk warnings, etc., and do not mean that the results can directly represent the diagnosis results of rheumatoid factor-related diseases. Therefore, the detection method provided by the present invention is not for diagnosis and treatment purposes.

[0061] Advantages and beneficial effects of the present invention:

[0062] The rheumatoid factor Fc fragment antigen prepared by the method of the present invention has strong specificity, good stability and small batch-to-batch difference, and the magnetic particle chemiluminescence kit for quantitative detection of rheumatoid factor prepared by the method has the advantages of wide linear range, high sensitivity, good stability, etc. The method of the present invention for preparing the rheumatoid factor fragment antigen and the rheumatoid factor quantitative detection kit is simple and easy, the batch-to-batch difference is controllable, and large-scale industrial production can be carried out, which has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 It is a reaction curve diagram of Example 2 of the present invention and Comparative Example 1 when performing RF detection;

[0064] Figure 2 This is a graph showing the linearity test results of the kit of the present invention. DETAILED DESCRIPTION

[0065] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0066] In the context of the present invention, the term "antibody" is used in the broadest sense in the present invention, and explicitly covers monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, and multispecific antibodies (e.g., bivalent antibodies) formed from at least two complete antibodies, as long as they exhibit the desired biological activity. Antibodies generally refer to immunoglobulins (Ig), which are composed of two identical light chains and two identical heavy chains. Each chain contains a variable region (V region) and a constant region (C region), wherein the variable region provides antigen binding sites and specificity, and the constant region constitutes the framework of the immunoglobulin and has a biological effector function. IgG is the main antibody component in serum, accounting for 75% of the total Ig in serum. IgG plays a major role in the body's immune protection. Most antibacterial, antiviral, and antitoxin antibodies belong to immunoglobulin G antibodies, and its indicators are meaningful for diagnosing certain diseases. IgG can be divided into three functional regions by digesting it with papain, namely two identical antigen binding fragments (fragment antigen binding, Fab) and a crystallizable fragment (fragment crystalline, Fc). The Fc fragment is located in the constant region and consists of the carboxyl terminal half of the two heavy chains. Since the Fc segment is far away from the antigen binding site, it provides an area that binds to the antibody without affecting the antigen-antibody reaction, and is the most effective epitope region for secondary antibody reagents to bind. The Fc fragment is highly conservative in sequence structure relative to the Fab part of the IgG antibody molecule, so the antibodies obtained by immunization with the Fc fragment have higher specificity than those obtained by immunization with the whole IgG molecule.

[0067] The term "antigen" (Ag) is used in the broadest sense in the present invention, and refers to a substance that can cause the production of antibodies, and is any substance that can induce an immune response. Foreign molecules can be recognized by immunoglobulins on B cells or processed by antigen-presenting cells and combined with major histocompatibility complexes to form complexes that reactivate T cells and trigger continuous immune responses. Antigenic substances have two important characteristics: immunogenicity and immunoreactivity. Immunogenicity refers to the ability of an antigen to induce a specific immune response in the body and produce antibodies and / or sensitized lymphocytes; immunoreactivity refers to the ability to specifically bind to corresponding immune effector substances (antibodies or sensitized lymphocytes) in vivo and in vitro.

[0068] In the present invention, the term "rheumatoid factor" (RF) is an autoantibody with the Fc fragment of denatured IgG as the target antigen, and is one of the autoantibodies associated with rheumatoid arthritis. RF can be any type of five immunoglobulins, which can be divided into IgA-RF, IgG-RF, IgM-RF and IgE-RF. Among them, IgM type is the most common, and has the characteristics of high agglutination and easy precipitation, so IgM-RF is mainly measured clinically. IgG-RF and IgA-RF are also relatively common, but they are often not detected by agglutination method, and enzyme-linked immunosorbent assay and radioimmunoassay are required. IgG-RF is difficult to detect, and about 50% of IgG-RF is missed, which is one of the reasons for "latent rheumatoid factor". RF not only reacts with denatured IgG molecules, but also reacts with self-IgG or allogeneic IgG molecules, and can also cross-react with other antigens such as nucleoprotein. RF is produced in peripheral lymph nodes, joint synovium, tonsillar lymph follicles and bone marrow. The terminal galactose residues of the Fc fragment polysaccharide terminus of IgG are missing in rheumatoid arthritis, which can be seen in both serum and synovial fluid. RF can enhance the affinity of IgG to its antigens present in immune complexes, or mediate the formation of immune complexes by directly binding IgG together. RF can be seen in a variety of systemic autoimmune diseases and infectious diseases, and can also be detected in healthy people. It is common in rheumatoid arthritis, Sjögren's syndrome and mixed cryoglobulinemia. RF can be detected in 60% to 80% of patients with rheumatoid arthritis, and the positive rate is also about 70% in Sjögren's syndrome and mixed cryoglobulinemia. 5% of normal people, especially the elderly, may be positive for RF, and the positive rate may increase with age. The false positive rate of RF in the elderly over 75 years old can reach 2% to 25%. The RF produced by normal people and the RF produced by rheumatoid arthritis have different cellular basis and pathogenicity. CD5+B cells are the cellular basis of naturally occurring RF. Peripheral blood, lymph nodes and tonsil lymph follicles are the main sites for the synthesis of naturally occurring RF, which can regulate immune response, activate complement and clear immune complexes.

[0069] In the present invention, the term "enzymatic cleavage" refers to the cutting of the sticky-end DNA molecules and vector molecules by restriction endonucleases to obtain the corresponding sticky-end connections; or the enzymatic hydrolysis of protein molecules by specific proteases to obtain specific fragments with clear molecular weights, and the presence of the target protein is determined by its molecular weight and labeling method. In a specific embodiment of the present invention, papain is used to enzymatically hydrolyze IgG in an IgG solution to decompose it into two identical Fab fragments and an Fc fragment, thereby separating the Fc fragment from the enzymatic cleavage product.

[0070] In the present invention, the term "purification" refers to protein purification, and its principle is that different proteins have different amino acid sequences and spatial structures, resulting in differences in their physical, chemical and biological properties. The selection and combination of purification techniques can develop a fastest way to obtain products of the desired purity. Protein purification can be achieved by methods such as column chromatography or electrophoresis. In a specific embodiment of the present invention, protein A, G or L columns can be used to purify the enzyme cleavage product to obtain an Fc fragment.

[0071] In the present invention, the term "dialysis" refers to protein dialysis, which is a commonly used method for separating and purifying proteins. The principle is to use a semipermeable membrane to screen and separate proteins and other small molecules in a solution. During the dialysis process, small molecule solutes pass through the semipermeable membrane, while proteins are retained inside the semipermeable membrane, thereby achieving protein purification and concentration. In a specific embodiment of the present invention, a dialysis bag is used, and a dialysis buffer matched with a dialysis device is used to filter and remove impurities. The dialysis buffer is preferably 20mmol / L phosphate buffer (PBS) with a pH value of 7.4.

[0072] In the context of the present invention, the term "magnetic particle chemiluminescence" refers to magnetic bead chemiluminescence technology, which is an immunological technology with chemiluminescent agents as substrates. At the same time, nano-scale magnetic particles are used as solid phase carriers to increase the adsorption area, so that antigens and antibodies can be combined to the maximum extent, and the binding reaction is carried out under conditions close to the liquid phase. It has the advantages of both chemiluminescence and immunological technology and is a non-radioactive detection method that has developed rapidly in recent years. Chemiluminescent immunoassay (CLIA) is famous for its high sensitivity, can detect analytes at low concentrations, and provides a good detection limit in a wide dynamic range. Immunomagnetic beads (IMB) have the advantages of simple operation, high separation efficiency, large specific surface area and good physical stability. It is a magnetic separation carrier with excellent performance and a universal tool for purification, detection and quantitative analysis of complex analytes. Common types of magnetic beads include carboxyl magnetic beads, tosyl magnetic beads and streptavidin magnetic beads. In a specific embodiment of the present invention, the magnetic particle chemiluminescence kit uses carboxyl magnetic particles, which are high-performance magnetic beads for chemiluminescent reagents. The carboxyl functional group is used for protein or nucleic acid coupling, and can exert outstanding performance in enzyme immunoassay, immunoprecipitation, and protein imprint analysis.

[0073] In the present invention, the term "chemiluminescent marker" refers to a marker that can emit light and be detected in a chemiluminescent immunoassay (CLIA). Commonly used markers include isoluminol, acridinium ester, acridinium ester derivatives, horseradish peroxidase (HRP), alkaline phosphatase (ALP), terpyridine ruthenium, etc. The chemiluminescent marker used in the present invention is preferably acridinium ester, acridinium ester derivative or alkaline phosphatase, more preferably an acridinium ester derivative.

[0074] In the present invention, the term "sample to be tested" refers to a composition from a subject, which contains cells and / or other molecular entities to be characterized and / or identified based on, for example, physical, biochemical, chemical and / or physiological characteristics. For example, a sample refers to any sample derived from a subject that is expected or known to contain the subject's metabolome. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell cultures, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymphatic fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, sputum, tears, sweat, mucus, saliva, tissue culture fluid, tissue extracts, homogenized tissues, cell extracts and combinations thereof. In a specific embodiment of the present invention, the sample is plasma or serum.

[0075] The present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Simple improvements to the present invention made according to the essence of the present invention all belong to the scope of protection claimed in the present invention. It should be noted that the experimental methods described in the following embodiments, unless otherwise specified, are conventional methods, and the reagents and materials, unless otherwise specified, can be obtained from commercial sources.

[0076] Example 1 Preparation of RF fragment antigen

[0077] 1. Prepare activation buffer: prepare 10mmol / L phosphate buffer (PBS), 0.05mol / L ethylenediaminetetraacetic acid (EDTA) and 50mmol / L cysteine, pH 8.0, filter with 0.22μm filter membrane to make 1L activation buffer.

[0078] 2. Prepare dialysis buffer: prepare 20 mmol / L phosphate buffer (PBS), adjust the pH to 7.4, and filter with a 0.22 μm filter membrane to prepare dialysis buffer.

[0079] 3. Prepare storage buffer: 10 mmol / L phosphate buffer (PBS), 100 mM L-arginine, 40 g / L trehalose, 0.5 g / L 2-methyl-4-isothiazoline-3-one and 0.5 g / L 5-chloro-2-methyl-4-isothiazoline-3-one, purified water, pH 7.4; filter with a 0.22 μm filter membrane to prepare 1 L storage buffer.

[0080] 4. Activate papain: Dissolve 10 mg / mL papain in activation buffer and activate at 37°C;

[0081] 5. Enzyme cleavage: Add activated papain to the IgG solution and allow to react fully in a 37°C constant temperature water bath for 5 hours.

[0082] 6. Dialysis and fluid replacement to terminate the reaction: Use dialysis buffer to dialyze the enzyme digestion product at 2-8℃ for 16h, replace the fluid 2-3 times in the middle, replace the buffer, and terminate the enzyme digestion reaction;

[0083] 7. Purification: Use protein G gravity column to purify the enzyme cleavage product to obtain Fc fragment;

[0084] 8. Storage: Collect the concentrated reaction solution and dilute it to 1-10 mg / mL with storage buffer to obtain RF fragment antigen, which can be stored at -20℃ for a long time.

[0085] Example 2 Preparation of RF fragment antigen Magnetic microparticle chemiluminescence kit

[0086] 1. Experimental Materials

[0087] Main materials: RF fragment antigen (prepared in Example 1), magnetic particles (2.8 μm, with carboxyl groups).

[0088] 2. Reagent Preparation

[0089] 1. Preparation of R1

[0090] 1) Washing: Take a certain amount of magnetic particles (1.5 mg) into a 2 mL centrifuge tube and wash once with SDS (20 mM, dissolved in 0.1 M MES (pH = 5.0)) (100 μl);

[0091] 2) Activation of magnetic particles: discard the supernatant, wash with 0.1M MES (pH=5.0) solution for 3 times, add 0.1mg EDC and 0.1mg NHS (10mg / mL, dissolved in 0.1M MES (pH=5.0), freshly prepared before use), make up to 100μl with 0.1M MES, and react at room temperature for 0.5h on a reagent mixer;

[0092] 3) Coupling: discard the supernatant, wash twice with magnetic particle binding buffer 0.1M MES, add 150μl magnetic particle binding buffer 0.1M MES and a certain amount of antigen (15μg), and react at room temperature for 3h on a reagent mixer;

[0093] 4) Blocking: discard the supernatant, wash twice with magnetic particle blocking buffer (1% BSA, dissolved in 0.01M PBS, freshly prepared before use), add 200 μl magnetic particle blocking buffer and block at room temperature for 2 h;

[0094] 5) Storage: discard the supernatant, wash twice with magnetic particle coupling diluent, dilute to R1 magnetic particle working solution (magnetic particle concentration: 0.2 mg / mL; antigen concentration: 2 μg / mL) according to a certain concentration, and store at 2-8°C.

[0095] 2. Preparation of R2

[0096] 1) Pretreatment: Take a certain amount (100 μg) of anti-rheumatoid factor-IgM antibody, add 0.02M pH 7.4PB to 100 μL, and dialyze with 0.02M pH 7.4PB at 2-8°C (time 2-3 hours);

[0097] 2) Labeling: Acridinium ester derivative (AE) was diluted to 1 mM with 0.02 M pH 7.4 PB (antibody: AE molar ratio was 1:30) and reacted at room temperature for 1 hour on a reagent mixer;

[0098] 3) Dialysis separation: The reaction solution was transferred to a dialysis bag (molecular weight cut-off 8000-14000), with a maximum dialysis volume of 2 mg per bag. The dialyzate was dialyzed for at least 12 hours in a 0.02M pH 7.4PB dialysis solution at 2-8°C, with the dialysis solution replaced at least 3-4 times, with each replacement interval greater than 2 hours. The chemiluminescence signal value of the dialysate in the dialyzed cup was tested to be within 100,000.

[0099] 4) Mix the collected samples and detect the concentration using an ultra-micro spectrophotometer. Add an equal volume of glycerol and store at -20°C. Use 0.5 times the detection concentration as the identification concentration, and then use R2 buffer to dilute to the working concentration.

[0100] 3. Preparation of rheumatoid factor calibrator

[0101] Take 995 mL of water, add 50 mM PBS buffer, 0.9% sodium chloride, 0.5% disodium ethylenediaminetetraacetic acid, 1.0% bovine serum albumin, and 0.1% sodium azide in sequence, stir each time the material is added until it is completely dissolved, and adjust the pH value to 7.0-7.8, preferably 7.4; then add rheumatoid factor-IgM to a series of concentrations, filter through a 0.22 μm filter membrane, and obtain a rheumatoid factor calibrator solution prepared in a series of concentrations.

[0102] Comparative Example 1 Preparation of RF Complete Antigen

[0103] 1) Prepare 40 w / v% sucrose solution using 10 mmol / L PBS (pH 7.4) buffer;

[0104] 2) Dilute IgG (50 mg / ml) to 10 mg / ml using the buffer in step 1);

[0105] 3) placing the solution in step 2) in a 63°C constant temperature water bath for 60 minutes;

[0106] 4) placing the heated solution in step 3) in an ice water bath and protecting from light for 2 h;

[0107] 5) The product in step 4) is dialyzed with 10 mmol / L PBS (pH 7.4) buffer at 2-8°C for at least 12 hours, with the dialysate replaced 2-3 times to remove high concentration sucrose. The product collected in the dialysis bag is the complete RF antigen.

[0108] The method for preparing a rheumatoid factor chemiluminescent detection kit using RF complete antigen adopts the following steps:

[0109] 1. Prepare reagent R1:

[0110] 1) Washing: Take a certain amount of magnetic particles (1.5 mg) into a 2 mL centrifuge tube and wash once with SDS (20 mM, dissolved in 0.1 M MES (pH = 5.0)) (100 μl);

[0111] 2) Activation of magnetic particles: discard the supernatant, wash with 0.1M MES (pH=5.0) solution for 3 times, add 0.1mg EDC and 0.1mg NHS (10mg / mL, dissolved in 0.1M MES (pH=5.0), freshly prepared before use), make up to 100μl with 0.1M MES, and react at room temperature for 0.5h on a reagent mixer;

[0112] 3) Coupling: discard the supernatant, wash twice with magnetic particle binding buffer 0.1M MES, add 150μl magnetic particle binding buffer 0.1M MES and a certain amount of RF complete antigen (15μg), and react at room temperature for 3h on a reagent mixer;

[0113] 4) Blocking: discard the supernatant, wash twice with magnetic particle blocking buffer (1% BSA, dissolved in 0.01M PBS, freshly prepared before use), add 200 μl magnetic particle blocking buffer and block at room temperature for 2 h;

[0114] 5) Storage: discard the supernatant, wash twice with magnetic particle coupling diluent, dilute to R1 magnetic particle working solution (magnetic particle concentration: 0.2 mg / mL; antigen concentration: 2 μg / mL) according to a certain concentration, and store at 2-8°C.

[0115] 2. Prepare reagent R2:

[0116] 1) Pretreatment: Take a certain amount (100 μg) of anti-rheumatoid factor-IgM antibody, add 0.02M pH 7.4PB to 100 μL, and dialyze with 0.02M pH 7.4PB at 2-8°C (time 2-3 hours);

[0117] 2) Labeling: Acridinium ester derivatives were diluted to 1 mM with 0.02 M pH 7.4 PB (antibody: AE molar ratio was 1:30) and reacted at room temperature for 1 hour on a reagent mixer;

[0118] 3) Dialysis separation: The reaction solution was transferred to a dialysis bag (molecular weight cut-off 8000-14000), with a maximum dialysis volume of 2 mg per bag. The dialyzate was dialyzed for at least 12 hours in a 0.02M pH 7.4PB dialysis solution at 2-8°C, with the dialysis solution replaced at least 3-4 times, with each replacement interval greater than 2 hours. The chemiluminescence signal value of the dialysate in the dialyzed cup was tested to be within 100,000.

[0119] 4) Mix the collected samples and detect the concentration using an ultra-micro spectrophotometer. Add an equal volume of glycerol and store at -20°C. Use 0.5 times the detection concentration as the identification concentration, and then use R2 buffer to dilute to the working concentration.

[0120] 3. The chemiluminescent detection reagents prepared in Example 2 and Comparative Example 1 were used for RF detection, and the detection tool was Yingkai fully automatic chemiluminescent immunoassay analyzer:

[0121] Test results such as Figure 1 As shown (the horizontal axis is RF concentration, the unit is U / mL, and the vertical axis is the luminescence value), it can be seen from the figure that the RF fragment antigen preparation magnetic microparticle chemiluminescence kit prepared in Example 2 is superior to the comparative example in terms of luminescence value, linear range, etc.

[0122] Example 3 Performance test of the kit for quantitative determination of rheumatoid factor

[0123] 1. Detection tool: Yingkai fully automatic chemiluminescence immunoassay analyzer.

[0124] 2. Detection method:

[0125] Take 10 μl of the 50-fold diluted serum sample, add 25 μl of R1, incubate at 37°C for 5 min, then add 25 μl of reagent R2, and read the luminescence value after reacting for 10 min.

[0126] Reaction direction: rising.

[0127] Calculation method: Make a standard curve based on the luminescence value and the different concentrations of the calibrator. Test the serum sample to obtain the absorbance, and calculate the sample content based on the standard curve.

[0128] 3. Linearity test of the kit of the present invention

[0129] Linear analysis was performed on the calibrators with concentrations of 8.54U / mL, 16.65U / mL, 29.11U / mL, 59.88U / mL, 118.80U / mL, and 225.60U / mL, and the linear correlation coefficient was calculated. The test results are as follows: Figure 2 As shown, R 2 =0.9945. In addition, the linear range of the kit for detecting rheumatoid factor samples is 10-240U / mL.

[0130] 4. Test on the positive and negative coincidence rate of samples tested by the kit of the present invention and the imported control kit

[0131] The kit of Example 2 and the imported control kit were used to test 197 serum samples respectively, and the positive and negative coincidence rates and the total coincidence rates of the sample tests were compared.

[0132] The results are shown in Table 1. Taking the test results of the imported test kit as a control, the positive compliance rate of the test kit in Example 2 was 97.2%, the negative compliance rate was 96.4%, and the total compliance rate was 97.0%.

[0133] Table 1. Statistical table of samples tested by the kit of the present invention and the imported control kit

[0134]

[0135] 5. Batch difference test of the kit of the present invention

[0136] According to the experimental method described in Example 1, three batches of RF fragment antigens were repeatedly prepared and used to prepare three batches of rheumatoid factor chemiluminescent kits described in Example 2. Two samples with different concentrations were tested respectively, and the comparison of the intra-batch coefficient of variation (CV) and the inter-batch coefficient of dispersion is shown in Table 2. The results show that the rheumatoid factor chemiluminescent kit prepared using the fragment antigen of the present invention has small inter-batch difference and good repeatability.

[0137] Table 2. Dispersion coefficients of different batches of kits for two samples of different concentrations

[0138] Different batches of coated magnetic particles CV-Batch 1 CV-Batch 2 CV-Batch 3 Inter-batch coefficient of dispersion 4.11 (IU / ml) 4.5％ 3.5％ 3.2％ 8.6％ 81.95 (IU / ml) 3.7％ 2.7％ 8.2％ 6.3％

[0139] 6. Anti-interference test of the kit of the present invention

[0140] Serum samples of healthy people with normal physical examinations (without obvious hemolysis, jaundice, and chyle) were taken to make mixed serum, and bilirubin, hemoglobin, and chyle were accurately weighed and dissolved in the above-mentioned human serum matrix to make 3 groups of high-concentration interfering substance samples. The high-concentration interfering substance samples were accurately measured and mixed with two serum samples (the volume ratio of the high-concentration interfering substance samples to the serum samples was less than 1:10) to make mixed serum samples containing a certain concentration of interfering substances (the concentration of interfering substances is shown in Table 3). Each sample was measured 3 times and the average value was taken. The interference degree was calculated as follows:

[0141] Interference degree = mean value of samples with interference / mean value of samples without interference × 100%

[0142] The acceptable interference range is between 90% and 110%.

[0143] Table 3. Anti-interference test results

[0144] Sample 1 Sample 2 Bilirubin, added at 0.4 mg / mL 93％ 99％ Hemoglobin, added at 10 mg / mL 96％ 98％ Chyle, added at 20 mg / mL 100％ 97％

[0145] The results show that when the bilirubin concentration is less than or equal to 0.4 mg / mL, the hemoglobin concentration is less than or equal to 10 mg / mL, and the chyle concentration is less than or equal to 20 mg / mL, the interfering substances have no obvious effect on the test results of the kit of the present invention.

[0146] 7. Test on the stability of the kit of the present invention during shelf life

[0147] The reagents and calibrators of the kit were accelerated at 37°C for 7 days, and the luminescence values ​​after acceleration at 4°C and 37°C were compared and tested, and each sample was measured twice to obtain the average value (as shown in Table 4). The results show that the deviation values ​​of the detection reagent signals after the kit of the present invention was accelerated at 4°C and 37°C for 7 days were less than 15%.

[0148] Table 4. Test values ​​under different storage conditions

[0149]

[0150] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing an Fc fragment antigen of a rheumatoid factor, characterized in that: Here are the steps: The IgG solution is enzymatically digested with papain to obtain a mixture of Fab and Fc fragments; the enzymatic digestion product is purified to obtain the Fc fragment, which is diluted with a storage buffer and stored.

2. The preparation method according to claim 1, characterized in that: The enzymatic cleavage step includes using an activation buffer to dissolve and activate papain, adding the activated papain to the IgG solution for sufficient reaction, and replacing the activation buffer to terminate the enzymatic cleavage reaction; The purification column used in the purification comprises at least one of a protein A affinity column, a protein G affinity column and a protein L affinity column; The storage buffer comprises phosphate buffer, L-arginine, trehalose, 2-methyl-4-isothiazoline-3-one, 5-chloro-2-methyl-4-isothiazoline-3-one, and purified water, and has a pH value of 7.4; Preferably, the components of the activation buffer include phosphate buffer, ethylenediaminetetraacetic acid and cysteine, and the pH value is 8.0; Preferably, the activation buffer comprises 0.01-0.2 mol / L phosphate buffer, 0.01-1 mol / L ethylenediaminetetraacetic acid and 1-100 mmol / L cysteine, and the pH value is 8.0; Preferably, the temperature for activating papain is 37°C; Preferably, the concentration of the activated papain solution is 0.1 to 10 mg / mL; Preferably, the ratio of activated papain to IgG solution in the sufficient reaction is 1:1-20; Preferably, the sufficient reaction condition is a constant temperature water bath; Preferably, the conditions for the sufficient reaction are a 37°C constant temperature water bath and a reaction time of 0.5 to 12 hours; Preferably, the method of replacing the activation buffer to terminate the enzyme cleavage reaction comprises dialysis and ultrafiltration. Preferably, the concentration of the storage buffer is 5 to 100 mg / mL; Preferably, the components of the storage buffer are 20-100 mmol / L phosphate buffer, 50-200 mM L-arginine, 10-60 g / L trehalose, 0.5-2 g / L 2-methyl-4-isothiazoline-3-one, 0.5-2 g / L 5-chloro-2-methyl-4-isothiazoline-3-one, and purified water, and the pH value is 7.

4.

3. An Fc fragment antigen of a rheumatoid factor, characterized in that: Obtained according to the preparation method according to claim 1 or 2.

4. A method for quantitatively detecting rheumatoid factor, characterized in that: The method comprises detecting the Fc fragment antigen according to claim 3 by chemiluminescence.

5. The method according to claim 4, characterized in that The method comprises the following steps: contacting the sample to be tested with the Fc fragment antigen coupled to the magnetic microparticles, then adding the anti-rheumatoid factor antibody coupled to the chemiluminescent marker, magnetically separating after the reaction is completed, adding the chemiluminescent substrate, detecting the chemiluminescent intensity, drawing a standard curve, and calculating the concentration of the rheumatoid factor in the sample to be tested; Preferably, the sample to be tested is plasma or serum; Preferably, the magnetic particles are carboxyl magnetic particles; Preferably, the concentration of the magnetic particles is 0.05 to 1 mg / mL; Preferably, the method for preparing the magnetic particle-coupled Fc fragment antigen comprises the following steps: washing and activating the carboxyl magnetic particles, coupling and binding with the Fc fragment antigen in a magnetic particle binding buffer, blocking in a magnetic particle blocking buffer, and washing to obtain the magnetic particle-coupled Fc fragment antigen; Preferably, the mass ratio of the magnetic particles to the Fc fragment antigen is 1000:5-40; Preferably, the rheumatoid factor includes at least one of rheumatoid factor-IgA, rheumatoid factor-IgG and rheumatoid factor-IgM; Preferably, the anti-rheumatoid factor antibody comprises at least one of anti-rheumatoid factor-IgA, anti-rheumatoid factor-IgG and anti-rheumatoid factor-IgM; Preferably, the chemiluminescent marker comprises acridinium ester, acridinium ester derivative, alkaline phosphatase; Preferably, the method for preparing the anti-rheumatoid factor antibody coupled to the chemiluminescent marker comprises: incubating the chemiluminescent marker and the anti-rheumatoid factor antibody in a buffer solution, and then dialysis purification to obtain the anti-rheumatoid factor antibody coupled to the chemiluminescent marker; Preferably, the concentration of the chemiluminescent marker is 0.1 mg / mL-2 mg / mL; Preferably, the chemiluminescent substrate comprises alkaline hydrogen peroxide, AMPPD; Preferably, the method steps include: diluting the sample to be tested with a sample dilution buffer, adding it to a magnetic particle working solution and a chemiluminescent marker working solution in sequence, magnetically separating after the reaction is completed, adding a chemiluminescent substrate after washing, detecting the chemiluminescent intensity, drawing a standard curve, and calculating the concentration of rheumatoid factor in the sample to be tested.

6. A magnetic chemiluminescence kit for quantitative detection of rheumatoid factor, characterized in that: The magnetic particle chemiluminescence kit comprises the Fc fragment antigen according to claim 3.

7. The magnetic particle chemiluminescence kit according to claim 6, characterized in that: The Fc fragment antigen is coupled to magnetic particles; the magnetic particle chemiluminescence kit also includes a chemiluminescent marker, an anti-rheumatoid factor antibody, and a rheumatoid factor calibrator; Preferably, the magnetic particles are carboxyl magnetic particles; Preferably, the particle size of the magnetic particles is 2 to 3 μm; Preferably, the anti-rheumatoid factor antibody is a monoclonal antibody labeled with a chemiluminescent marker; Preferably, the anti-rheumatoid factor antibody comprises at least one of anti-rheumatoid factor-IgA, anti-rheumatoid factor-IgG and anti-rheumatoid factor-IgM; Preferably, the chemiluminescent marker comprises acridinium ester, acridinium ester derivative, alkaline phosphatase.

8. The magnetic particle chemiluminescence kit according to claim 6, characterized in that: The magnetic particle chemiluminescence kit comprises R1 magnetic particle working solution, R2 chemiluminescence marker working solution, R3 sample dilution buffer solution, and rheumatoid factor calibrator; The components of the R1 magnetic particle working solution include magnetic particles coupled with Fc fragment antigens, buffer, stabilizer, surfactant, preservative and purified water, and the pH value is 5.5-8.0; Preferably, the components of the R1 magnetic particle working solution are 0.05-1 g / L magnetic particles coupled with Fc fragment antigen, 20-100 mmol / L buffer, 2-10 g / L stabilizer, 1-5 g / L surfactant, 0.5-2 g / L preservative and purified water, and the pH value is 5.5-8.0; Preferably, the preparation method of the R1 magnetic microparticle magnetic bead working solution is as follows: washing and activating the carboxyl magnetic microparticle magnetic beads, coupling and binding with the Fc fragment antigen in a magnetic microparticle binding buffer, blocking in a magnetic microparticle blocking buffer, washing to obtain an Fc fragment antigen-carboxyl magnetic microparticle magnetic bead complex, adding a magnetic microparticle coupling diluent, and diluting to obtain an R1 magnetic microparticle working solution; Preferably, the magnetic particle binding buffer is 0.1M 2-(N-morpholine)ethanesulfonic acid buffer; Preferably, the magnetic particle blocking buffer is 1% bovine serum albumin; The components of the R2 chemiluminescent marker working solution include anti-rheumatoid factor antibodies coupled with chemiluminescent markers, buffer, protein protective agent, surfactant, glycerol, preservative and purified water, and the pH value is 7.5-9.0; Preferably, the components of the R2 chemiluminescent marker working solution are 0.1-5 mg / L anti-rheumatoid factor antibody coupled with a chemiluminescent marker, 20-100 mmol / L buffer, 2-10 g / L protein protective agent, 1-5 g / L surfactant, 1-20 g / L glycerol, 0.5-2 g / L preservative and purified water, and the pH value is 6.0-7.5; Preferably, the preparation method of the R2 chemiluminescent marker working solution is as follows: the chemiluminescent marker and the anti-rheumatoid factor IgM antibody are incubated and combined in a buffer solution, and then dialyzed and purified to obtain the anti-rheumatoid factor IgM antibody coupled with the chemiluminescent marker, and then a diluent is added to dilute and obtain the R2 chemiluminescent marker working solution; The components of the R3 sample dilution buffer include a buffer, a protein protectant, a preservative and purified water, and the pH value is 7.4 to 9.0; Preferably, the components of the R3 sample dilution buffer are 20-100 mmol / L buffer, 2-10 g / L protein protectant, 0.5-2 g / L preservative and purified water, and the pH value is 7.4-9.0; Preferably, the chemiluminescent substrate comprises alkaline hydrogen peroxide or AMPPD.

9. The magnetic particle chemiluminescence kit according to claim 8, characterized in that: The buffer comprises one of phosphate buffer, glycine buffer, 2-(N-morpholine)ethanesulfonic acid buffer, hydroxyethylpiperazineethanesulfonic acid buffer, 3-(N-morpholine)propanesulfonic acid buffer or tris(hydroxymethyl)aminomethane buffer and borate buffer; The stabilizer includes one or more of bovine serum albumin, disodium ethylenediaminetetraacetate, and histidine; The protein protective agent includes one or more of bovine serum albumin, trehalose, sucrose, lactose, choline chloride, sorbitol, glycerol, glycine, and arginine; The surfactant includes one or more of Tween 20, Tween 80, Triton X-100, Triton X-405, polyethylene glycol octylphenyl ether, and ethylenediamine polyoxypropylene polyoxyethylene ether; The preservative includes one or more of sodium azide, sodium nitrite, N-methyl-isothiazolone, N'-methylene-bis[N'-(3-hydroxymethyl-2,5-diketo-4-imidazolidinyl)]urea or a mixed solution of 2-methyl-4-isothiazoline-3-one and 5-chloro-2-methyl-4-isothiazoline-3-one; The pH value is adjusted by concentrated hydrochloric acid or sodium hydroxide solution; Preferably, the pH value is adjusted by 3 mol / L concentrated hydrochloric acid or 6 mol / L sodium hydroxide solution.

10. Use of the Fc fragment antigen according to claim 3, the method according to claim 4 or 5, or the magnetic particle chemiluminescence kit according to any one of claims 6 to 9 in preparing a product for quantitative detection of rheumatoid factor, or in preparing a product for diagnosis or auxiliary diagnosis of rheumatoid factor-related diseases; Preferably, the rheumatoid factor-related diseases include rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, dermatomyositis, scleroderma, mononucleosis, hepatitis, influenza, tuberculosis, bacterial endocarditis, malaria, schistosomiasis and certain tumors.

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