Buffer solution for reducing rheumatoid factor interference in IgM antibody capture method detection

Abstract

The invention provides a buffer solution for reducing interference of rheumatoid factors in IgM antibody capture method detection, the buffer solution contains 0.05-5% by mass of poly-human IgG, and the polymerization degree of the poly-human IgG is 8-14. The buffer solution has the best binding capacity with RF, and when the buffer solution is used as a buffer diluent for IgM antibody detection, the accuracy of IgM antibody detection can be ensured.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and particularly relates to a buffer for reducing the interference of rheumatoid factor in IgM antibody capture assay, a kit including the same, and a using method thereof. Background Art

[0002] Labeled immunoassay technology is a determination technology based on the reaction of high affinity and high specificity between antigen and antibody, combined with substances that can be detected microscopically (radionuclide, enzyme, fluorescein, luminescent substance, etc.) for labeling, and indirectly reflects the content of the analyte.

[0003] The common modes of labeled immunoassay technology include sandwich method, indirect method, competitive method and capture method. The sensitivity and specificity of the double antibody sandwich method for detecting macromolecular antigens (such as HBsAg detection) or the double antigen sandwich method for detecting antibodies (such as the detection of the third-generation HCV antibody and HIV antibody, which have high requirements for the purity of raw materials) are superior to general methods. The indirect method uses a labeled secondary antibody to detect the test antibody that has bound to the solid-phase antigen, and is commonly used for the detection of some pathogen IgG. The competitive method is commonly used for the detection of small molecules. Since small molecules have few binding sites and cannot be detected by the sandwich method, for some antibody detection items, due to the difficulty in removing impurities in the raw materials, such as HBeAb and HBcAb, the competitive method is also used for detection, but the competitive method has many problems. The capture method is mostly used for the determination of IgM antibodies. First, all serum IgM (including specific IgM and non-specific IgM) is immobilized on the solid phase through anti-human μ chain, and then a specific labeled antigen is added for detection.

[0004] IgM is the earliest antibody to appear in the primary humoral immune response and is the vanguard of the body's anti-infection defense. Detection of IgM in serum indicates a recent infection. Therefore, IgM antibodies are commonly used for early detection of pathogens. In the diagnosis of acute pathogen infections, it is usually necessary to detect IgM antibodies, such as the detection of serum anti-HBc IgM in hepatitis B virus infection and the series of IgM detections in the torch project. The detection of IgM usually has indirect methods and solid-phase capture methods. When using the indirect method to detect IgM antibodies, it is usually necessary to pretreat the sample with anti-human IgG to remove the interference of IgG in the sample, which is not only cumbersome but also affects sensitivity and specificity. The common steps for the capture method to detect IgM are as follows: Bind anti-human IgM antibodies to a solid-phase carrier to form solid-phase anti-human IgM antibodies, and wash away the unbound antibodies. Add the diluted test specimen and incubate at an appropriate temperature for a period of time to allow the IgM antibodies contained in the specimen to be captured by the solid-phase antibodies, forming a solid-phase antibody-IgM antibody complex. Wash away the unbound substances. Add enzyme-labeled specific antigen and continue to incubate for a period of time. It only binds to the specific IgM on the solid phase to form an antibody-IgM antibody-specific antigen-enzyme complex. Wash away the unbound enzyme-labeled specific antigen. Add the substrate, and the enzyme in the complex catalyzes the substrate to produce a colored product. Qualitative judgment or quantitative analysis of the test IgM antibodies is carried out according to the color depth.

[0005] Rheumatoid factor (RF) is an autoantibody produced in the body with denatured IgG as the antigen, usually IgM, which binds to the Fc fragment of IgG. Approximately 90% of patients with rheumatoid arthritis are positive for RF, and other autoimmune diseases also have a certain positive rate. About 5% of normal people are positive.

[0006] When using the capture method to detect IgM antibodies, it is particularly susceptible to interference from RF (IgM-class antibodies) and other non-specific IgM antibodies. Due to the low titer of non-specific IgM, the interference can be removed by moderately diluting the sample. However, rheumatoid factor has a relatively high titer in some autoimmune populations and can competitively bind to the IgM antibodies in the test sample against the anti-u chain, resulting in false positives, and this false positive cannot be well removed by conventional dilution, which becomes a major defect in the capture method for detecting IgM antibodies. Summary of the Invention

[0007] To solve the problems and deficiencies of the existing technology, the present invention provides a buffer solution for reducing the interference of rheumatoid factor in the detection of IgM antibodies. The buffer solution contains polyclonal human IgG with a mass percentage of 0.05-5%, and the degree of polymerization of the polyclonal human IgG is 8-14.

[0008] Compared with conventional IgG, the conformation of polyclonal human IgG has changed, the number and accessibility of its active sites have increased, and the binding ability of polyclonal human IgG with a specific degree of polymerization to RF has been enhanced. When the degree of polymerization of polyclonal human IgG is less than 8, its binding ability to RF is poor, and it cannot achieve the effect of reducing the interference of rheumatoid factor in IgM antibody detection; when polyclonal human IgG is over-polymerized, for example, when the degree of polymerization reaches more than 14, it is easy to cause reagent jump values and will also affect the actual detection. When the degree of polymerization of polyclonal human IgG is in the range of 8-14, its binding ability to RF is the best, and when used as a buffer diluent for IgM antibody detection, it can ensure the accuracy of IgM antibody detection.

[0009] As a further improvement of the above technical solution, the buffer also contains calf serum with a volume percentage of 8-12% and bovine serum albumin with a mass percentage of 2-4%. Calf serum can eliminate the interference of anti-bovine antibodies in some samples, and bovine serum albumin can play a role in blocking blank solid-phase sites and reducing non-specific adsorption.

[0010] As a further improvement of the above technical solution, the buffer also contains NaCl with a weight percentage of 0.08-0.1% and sodium caseinate with a weight percentage of 2-4%. Antigen-antibody reactions require a certain concentration of physiological saline environment; the role of sodium caseinate is to better block and protect buffer free radical oxidation.

[0011] As a further improvement of the above technical solution, the buffer also contains 10 mM disodium EDTA and Triton X-100 with a volume percentage of 0.05%. Disodium EDTA is used to chelate metal ions and protect specific proteins, and Triton eliminates the binding of non-specific proteins and reduces non-specific reactions.

[0012] As a further improvement of the above technical solution, the specific composition of the buffer is:

[0013] 50 mM Tris with a pH of 7.2;

[0014] 0.09 wt% NaCl;

[0015] 10 mM disodium EDTA;

[0016] 10 vol% calf serum;

[0017] 3% wt bovine serum albumin;

[0018] 3 wt% sodium caseinate;

[0019] 0.05 vol% triton X-100;

[0020] 0.05 - 5 wt% of polymeric human IgG.

[0021] As a further improvement of the above technical solution, the preparation method of the polymeric human IgG is as follows: A 10 mg / ml human IgG solution is incubated in a constant temperature water bath at 60 - 64 °C for 20 - 40 min, and then cooled with ice water.

[0022] The production of polymeric human IgG with a specific degree of polymerization requires precise control of the reaction conditions. The solution concentration, heating temperature, and heating time all need to be well controlled, otherwise, the degree of polymerization will be too low or excessive polymerization will occur. Incubating a 10 mg / ml human IgG solution in a constant temperature water bath at 60 - 64 °C for 20 - 40 min and then cooling can obtain the degree of polymerization expected in the present invention. As the thermal polymerization temperature increases, the degree of the polymerization reaction increases, the degree of polymerization of the product increases, and the binding ability to RF enhances. When the thermal polymerization temperature is 62 °C, the dominant degree of polymerization reaches about 11, and the binding ability to RF is the strongest. However, when the polymerization temperature exceeds 64 °C, excessive polymerization will occur, leading to jumps in detection. When the heating time exceeds 40 minutes, for example, 100 minutes, the degree of polymerization of human IgG may reach 20, and the polymerization system becomes gel-like, unable to play the expected role.

[0023] In a second aspect, the present application provides a kit for detecting IgM antibodies, and the kit includes the buffer for reducing the interference of rheumatoid factor in the IgM antibody detection as described above.

[0024] As a further improvement of the above technical solution, the kit is used for detecting Toxoplasma gondii IgM antibodies.

[0025] In a third aspect, the present application provides a method for using the above kit for detecting IgM antibodies, and the test sample to be detected is diluted with the buffer in a ratio of 1:10.

[0026] As a further improvement of the above technical solution, after diluting the test sample to be detected, the IgM antibodies in the test sample are detected by the capture method.

[0027] As a further improvement of the above technical solution, the method for use includes the following steps:

[0028] (1) Dilute the test sample to be detected with the buffer in a pre-diluted ratio of 1:10;

[0029] (2) Mix 100 ul of the diluted test sample to be detected with 50 ul of magnetic beads coated with anti-human μ chain, incubate at 37 °C for 15 min to form an anti-μ chain - IgM antibody complex, and wash three times with PBS-T buffer (phosphate buffer containing Tween-20, which can provide a relatively stable ionic environment and pH buffering capacity);

[0030] (3) Add 100 ul of acridinium ester-labeled antigen, incubate at 37 °C for 5 min to form a magnetic bead anti-u chain-IgM antibody-acridinium ester antigen complex, and wash three times with PBS-T buffer;

[0031] (4) Add 100 ul each of pre-excitation solution 0.1 M HNO3 and excitation solution 0.25 M NaOH, and record the signal value. Detailed implementation method

[0032] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0033] Example 1

[0034] 1. Preparation of polyclonal human IgG: Keep a 10 mg / ml human IgG solution in a constant temperature water bath at 60 °C for 30 min, and then cool it with ice water.

[0035] Determination of the degree of polymerization: Use a calibrated TSK G-4000 HPLC column to determine the molecular weight of the polymer and calculate the degree of polymerization.

[0036] 2. Prepare the buffer: 50 mM Tris with a pH of 7.2; 0.09 wt% NaCl; 10 mM disodium EDTA; 10 vol% calf serum; 3 wt% bovine serum albumin; 3 wt% sodium caseinate; 0.05 vol% Triton X-100; 0.5 wt% polyclonal human IgG.

[0037] 3. Detection of Toxoplasma IgM antibody by capture method

[0038] Adopt the two-step immunoassay principle to qualitatively determine the IgM antibody of Toxoplasma gondii in human serum / plasma. In the first step, pre-dilute the test sample with the prepared buffer at a ratio of 1:10, then mix 100 ul of the diluted sample with 50 ul of magnetic beads coated with anti-human u chain, incubate at 37 °C for 15 min to form an anti-u chain-IgM antibody complex, and after washing three times with PBS-T; in the second step, add 100 ul of acridinium ester-labeled Toxo p30 antigen and incubate at 37 °C for 5 min to form a magnetic bead anti-u chain-IgM antibody-acridinium ester antigen complex, wash three times with PBS-T, add 100 ul each of pre-excitation solution (0.1 M HNO3) and excitation solution (0.25 M NaOH); record the signal value.

[0039] The results of the chemiluminescence reaction are measured and expressed in relative light units (RLUs). There is a positive relationship between the content of Toxoplasma IgM antibody in the sample and the RLUs value detected by the optical system. Whether there is Toxoplasma IgM antibody in the sample is determined by comparing the chemiluminescence signal of the mixture with the cutoff value (calibrator RLU * 0.1) obtained from the current calibration curve. If the sample RLU ≥ cutoff signal, then the sample is reactive to Toxoplasma IgM antibody.

[0040] Example 2

[0041] 1. Preparation of polyclonal human IgG: A 10 mg / ml human IgG solution is incubated in a constant temperature water bath at 64 °C for 20 min, and then cooled with ice water.

[0042] 2. Preparation of buffer: 50 mM Tris with pH 7.2; 0.1 wt% NaCl; 10 mM disodium EDTA; 12 vol% calf serum; 2 wt% bovine serum albumin; 2 wt% sodium caseinate; 0.05 vol% Triton X-100; 0.05 wt% of polyclonal human IgG.

[0043] Other operations are the same as in Example 1.

[0044] Example 3

[0045] 1. Preparation of polyclonal human IgG: A 10 mg / ml human IgG solution is incubated in a constant temperature water bath at 62 °C for 40 min, and then cooled with ice water.

[0046] 2. Preparation of buffer: 50 mM Tris with pH 7.2; 0.08 wt% NaCl; 10 mM disodium EDTA; 8 vol% calf serum; 4 wt% bovine serum albumin; 4 wt% sodium caseinate; 0.05 vol% Triton X-100; 1.5 wt% of polyclonal human IgG.

[0047] Other operations are the same as in Example 1.

[0048] Example 4

[0049] 1. Preparation of polyclonal human IgG: A 10 mg / ml human IgG solution is incubated in a constant temperature water bath at 60 °C for 40 min, and then cooled with ice water.

[0050] 2. Preparation of buffer: 50 mM Tris with pH 7.2; 0.1 wt% NaCl; 10 mM disodium EDTA; 12 vol% calf serum; 2 wt% bovine serum albumin; 2 wt% sodium caseinate; 0.05 vol% Triton X-100; 5.0 wt% of polyclonal human IgG.

[0051] Other operations are the same as those in Example 1.

[0052] Example 5

[0053] 1. Preparation of polyclonal human IgG: A 10 mg / ml human IgG solution was incubated in a constant temperature water bath at 60 °C for 40 min, and then cooled with ice water.

[0054] 2. Preparation of buffer: 50 mM Tris with pH 7.2; 0.09 wt% NaCl; 10 mM disodium EDTA; 10 vol% calf serum; 3 wt% bovine serum albumin; 3 wt% sodium caseinate; 0.05 vol% triton X-100; 3 wt% polyclonal human IgG.

[0055] Other operations are the same as those in Example 1.

[0056] Comparative Example 1

[0057] 1. Preparation of buffer: 50 mM Tris with pH 7.2; 0.09 wt% NaCl; 10 mM disodium EDTA; 10 vol% calf serum; 3 wt% bovine serum albumin; 3 wt% sodium caseinate; 0.05 vol% triton X-100, without human IgG.

[0058] Other operations are the same as those in Example 1.

[0059] Comparative Example 2

[0060] 1. Preparation of buffer: 50 mM Tris with pH 7.2; 0.09 wt% NaCl; 10 mM disodium EDTA; 10 vol% calf serum; 3 wt% bovine serum albumin; 3 wt% sodium caseinate; 0.05 vol% triton X-100, 1.5 wt% human IgG (not heat-polymerized).

[0061] Other operations are the same as those in Example 1.

[0062] Comparative Example 3

[0063] 1. Preparation of polyclonal human IgG: A 10 mg / ml human IgG solution was incubated in a constant temperature water bath at 68 °C for 30 min, and then cooled with ice water.

[0064] 2. Preparation of buffer: 50 mM Tris with pH 7.2; 0.09 wt% NaCl; 10 mM disodium EDTA; 10 vol% calf serum; 3 wt% bovine serum albumin; 3 wt% sodium caseinate; 0.05 vol% triton X-100; 1.5 wt% polyclonal human IgG.

[0065] Other operations are the same as those in Example 1.

[0066] Comparative Example 4

[0067] 1. Preparation of polyclonal human IgG: A 10 mg / ml human IgG solution was incubated in a constant temperature water bath at 56 °C for 30 min, and then cooled with ice water.

[0068] 2. Preparation of buffer: 50 mM Tris with a pH of 7.2; 0.09 wt% NaCl; 10 mM disodium EDTA; 10 vol% calf serum; 3 wt% bovine serum albumin; 3 wt% sodium caseinate; 0.05 vol% Triton X-100; 1.5 wt% polyclonal human IgG.

[0069] Other operations are the same as those in Example 1.

[0070] Comparative Example 5

[0071] 1. Preparation of polyclonal human IgG: A 10 mg / ml human IgG solution was incubated in a constant temperature water bath at 60 °C for 120 min, and then cooled with ice water.

[0072] 2. Preparation of buffer: 50 mM Tris with a pH of 7.2; 0.09 wt% NaCl; 10 mM disodium EDTA; 10 vol% calf serum; 3 wt% bovine serum albumin; 3 wt% sodium caseinate; 0.05 vol% Triton X-100; 1.5 wt% polyclonal human IgG.

[0073] Other operations are the same as those in Example 1.

[0074] 15 samples were from rheumatoid arthritis positive patients with high RF concentrations, 120 - 460 IU / mI (numbered RF1 - 15). 15 samples were from healthy physical examination subjects (numbered normal 1 - 15). All the testers had 2.0 mL of venous blood drawn on an empty stomach in the early morning, centrifuged at 3500 r / min, and the serum was separated and stored at -20 °C until the same batch of tests. The test results of Examples 1 - 5 are shown in Table 1 below.

[0075] Table 1

[0076]

[0077]

[0078] The test results of Comparative Examples 1 - 5 are shown in Table 2 below.

[0079] Table 2

[0080]

[0081]

[0082]

[0083] It can be seen from the detection results of Examples 1-5 and Comparative Examples 1-5 that for the same samples from 15 rheumatoid arthritis positive patients, no false positive occurs when detected after dilution with the buffers of Examples 1-5, while relatively high false positives occur after dilution with the buffers of Comparative Examples 1-5.

[0084] The above examples are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the above examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. A buffer for reducing the interference of rheumatoid factor in IgM antibody capture assay, characterized in that: The buffer solution contains 0.05 - 5% (by mass) of polyclonal human IgG, wherein the degree of polymerization of the polyclonal human IgG is 8 - 14.

2. The buffer for reducing the interference of rheumatoid factor in the IgM antibody capture assay according to claim 1, wherein: The buffer solution also contains 8 - 12% (by volume) of calf serum and 2 - 4% (by mass) of bovine serum albumin.

3. The buffer for reducing the interference of rheumatoid factor in the IgM antibody capture assay according to claim 1, characterized in that: The buffer solution also contains 0.08 - 0.1% (by weight) of NaCl and 2 - 4% (by weight) of sodium caseinate.

4. The buffer for reducing the interference of rheumatoid factor in the IgM antibody capture assay according to claim 1, wherein: The buffer solution also contains 10 mM of disodium EDTA and 0.05% (by volume) of Triton X - 100.

5. The buffer for reducing the interference of rheumatoid factor in the IgM antibody capture assay according to claim 1, characterized in that: The specific composition of the buffer solution is as follows: 50 mM Tris with a pH of 7.2; 0.09 wt% NaCl; 10 mM disodium EDTA; 10 vol% calf serum; 3% wt bovine serum albumin; 3 wt% sodium caseinate; 0.05 vol% Triton X - 100; 0.05 - 5 wt% of polyclonal human IgG.

6. The buffer for reducing the interference of rheumatoid factor in the IgM antibody capture assay according to any one of claims 1-5, characterized in that: The preparation method of the polyclonal human IgG is as follows: A 10 mg / ml human IgG solution is incubated in a constant temperature water bath at 60 - 64 °C for 20 - 40 min, and then cooled with ice water.

7. A kit for detecting IgM antibodies by the capture method, characterized in that: It includes the buffer solution for reducing the interference of rheumatoid factor in IgM antibody detection as described in any one of claims 1 - 6.

8. A kit for detecting IgM antibodies by the capture method as described in claim 7, characterized in that: The kit is used for detecting Toxoplasma gondii IgM antibody.

9. A method for using a kit for detecting IgM antibodies by the capture method as described in claim 7, characterized in that: The test sample is diluted with the buffer solution at a ratio of 1:

10.

10. A method for using a kit for detecting IgM antibodies by the capture method as described in claim 9, characterized in that: It includes the following steps: (1) The test sample is pre - diluted with the buffer solution at a ratio of 1:

10. (2) 100 ul of the diluted test sample is mixed with 50 ul of magnetic beads coated with anti - human μ chain, and incubated at 37 °C for 15 min to form an anti - μ chain - IgM antibody complex, and then rinsed three times with PBS - T. (3) 100 ul of acridinium ester - labeled antigen is added and incubated at 37 °C for 5 min to form a magnetic bead anti - μ chain - IgM antibody - acridinium ester antigen complex, and then washed three times with PBS - T. (4) 100 ul each of pre - excitation solution 0.1 M HNO3 and excitation solution 0.25 M NaOH are added, and the signal value is recorded.

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