Composite biological blocking agent and preparation method and reagent strip

Abstract

The invention discloses a composite biological blocker for immunochromatographic detection, a preparation method thereof and a reagent strip, wherein the blocker includes a blocker buffer, wherein mouse monoclonal antibody IgG-1, mouse monoclonal antibody IgG-2, goat anti-human IgM / RF polyclonal antibody and rabbit polyantibody IgG are diluted in the blocker buffer; BSA is also added to the blocker buffer; wherein the blocker buffer is tris-HCl buffer or phosphate PBS buffer. The blocker can effectively reduce or eliminate the adverse effects of endogenous interfering substances on fluorescent immunochromatographic detection, and prevent the occurrence of false positive and false negative test results; after mixing several different mouse monoclonal antibodies, goat polyantibodies and rabbit polyantibodies, they are used for immunochromatographic detection, which can effectively prevent and control the influence of endogenous interfering substances in the sample on immunochromatographic detection, and improve the accuracy of the detection results of immunochromatographic quantitative detection reagents.

Description

Technical Field

[0001] The invention relates to the technical field of immunochromatographic detection, and in particular to a composite biological blocking agent for immunochromatographic detection, a preparation method thereof and a reagent strip. Background Art

[0002] With the rapid development of Point of Care Test (POCT) technology, hospitals and clinics can also provide people with accurate, sensitive and convenient rapid diagnostic testing services.

[0003] Immunochromatography assay is a new type of immunoassay that appeared in the early 1980s. It is a simple and rapid immunological detection technology based on immunofiltration assay. Its principle is to fix specific antibodies to a certain zone of nitrocellulose membrane. When one end of the dry nitrocellulose is immersed in the sample, the sample will move forward along the membrane due to capillary action. When it moves to the area where the antibody is fixed, the corresponding antigen in the sample will specifically bind to the antibody. The labeled colloidal gold or fluorescent substance can make the area show a certain light under a specific excitation light, thereby realizing specific immunodiagnosis. This technology not only has good sensitivity and specificity, but also is simple to operate and does not require expensive detection equipment, which has enabled immunochromatography technology to develop rapidly in real-time detection.

[0004] However, studies have shown that more than 3% of healthy people have endogenous interference factors in their bodies, mainly including heterophilic antibodies (HA), human anti-animal antibodies (HAAA), rheumatoid factors (RF) and other endogenous interference substances. The proportion is even higher among people who keep pets and have received antibody treatment. Researching effective means to reduce and eliminate endogenous interference is an important research direction to ensure the stability and reliability of immunoassay results.

[0005] Currently, the commercial blockers available on the market mainly include HBR, TRUBlock, MAK33, etc. Blockers are added to the sample pad or the binding pad treatment solution to treat the glass fiber. Blockers can bind to interfering substances, thereby reducing or even eliminating interference. However, because the types and concentrations of interfering substances in the samples are different, and the effects of interfering substances on different test items are also different, commercial blockers cannot completely prevent endogenous factors from interfering with the test results.

[0006] Therefore, it is necessary to develop a blocking agent to prevent endogenous factors from interfering with the test results. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a composite biological blocker for immunochromatographic detection, which contains a variety of animal immunoglobulins with interference such as heterophilic antibodies, anti-animal antibodies, and anti-rheumatoid factors. It has a good blocking effect on complex endogenous interference factors and can effectively reduce the false positive or false negative results of immunochromatographic detection caused by endogenous interfering substances.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is that the composite biological blocker for immunochromatographic detection includes a blocker buffer, in which mouse monoclonal antibody IgG-1, mouse monoclonal antibody IgG-2, goat anti-human IgM / RF polyclonal antibody and rabbit polyclonal IgG are diluted; BSA is also added to the blocker buffer;

[0009] Wherein, the blocking agent buffer is Tris-HCl buffer or phosphate PBS buffer.

[0010] The composite biological blocker for immunochromatographic detection in the above technical scheme can effectively reduce or eliminate the adverse effects of endogenous interfering substances on fluorescent immunochromatographic detection, and prevent the occurrence of false positive and false negative test results; after mixing several different mouse monoclonal antibodies, sheep polyclonal antibodies, and rabbit polyclonal antibodies, they are used for immunochromatographic detection, which can effectively prevent and control the influence of endogenous interfering substances in the sample on immunochromatographic detection, and improve the accuracy of the detection results of immunochromatographic quantitative detection reagents.

[0011] Preferably, the blocking agent buffer is 10 mM Tris-HCl buffer or 10 mM phosphate PBS buffer.

[0012] Preferably, the pH of the blocking agent buffer is 7.4±0.2.

[0013] Preferably, the mouse monoclonal antibody IgG-1, mouse monoclonal antibody IgG-2, goat anti-human IgM / RF polyclonal antibody and rabbit polyclonal IgG are diluted to 10 mg / mL, 10 mg / mL, 2 mg / mL and 2 mg / mL respectively with the blocking buffer.

[0014] Preferably, the blocking agent buffer is supplemented with BSA to 10 mg / mL.

[0015] Preferably, Proclin 300 is also added to the blocking agent buffer.

[0016] Another technical problem to be solved by the present invention is to provide a method for preparing a composite biological blocking agent for immunochromatographic detection, comprising the following steps:

[0017] S1: preparing a blocking buffer, wherein the blocking buffer is 10 mM Tris-HCl buffer or 10 mM phosphate PBS buffer, and the pH of the blocking buffer is 7.4±0.2;

[0018] S2: dilute mouse monoclonal antibody IgG-1, mouse monoclonal antibody IgG-2, goat anti-human IgM / RF polyclonal antibody and rabbit polyclonal IgG to 10 mg / mL, 10 mg / mL, 2 mg / mL and 2 mg / mL respectively with the blocking buffer in step S1;

[0019] S3: adding BSA to 10 mg / mL in the blocking buffer of step S2;

[0020] S4: Add Proclin 300 to the blocking agent buffer of step S3 to obtain a composite biological blocking agent.

[0021] Another technical problem to be solved by the present invention is to provide a fluorescent immunoassay reagent strip, comprising four parts: a sample pad, a binding pad, a coated nitrocellulose membrane and absorbent paper; wherein the sample pad is treated with a sample pad treatment solution, the sample pad treatment solution comprises a buffer solution with a pH of 8 to 10, a surfactant, trehalose, erythrocyte monoclonal antibody and the aforementioned composite biological blocker of the present invention, and the composite biological blocker is added to the sample pad treatment solution at a volume ratio of 0.5%.

[0022] Preferably, the preparation method of the conjugate pad is as follows: (1) activating fluorescent microspheres and EDC in a phosphate buffer solution; (2) adding detection antibody 1 and chicken IgY to the fluorescent microsphere solution for reaction; (3) performing a blocking reaction on the coupled microsphere-antibody complex, and obtaining an immunofluorescent microsphere marker after centrifugation; (4) coating the immunofluorescent microsphere marker solution of a prepared concentration onto glass fiber, and drying to obtain a conjugate pad.

[0023] Preferably, the preparation method of the nitrocellulose membrane is as follows: the detection antibody II is used to prepare a T line solution, the goat anti-chicken IgY is used to prepare a C line solution, and then the solution is coated on the T detection line and C quality control line areas on the nitrocellulose membrane and dried.

[0024] Preferably, the preparation method of the reagent strip is as follows: starting from the sample application end, the four parts, namely, the sample pad, the conjugate pad, the coated nitrocellulose membrane and the absorbent paper, are sequentially pasted on the bottom plate, and then cut into the required width to form a test strip.

[0025] After the test strip is formed, it is placed in a plastic card shell to form a reagent card. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following is further described in detail with reference to the accompanying drawings and embodiments of the present invention:

[0027] Figure 1 : is a correlation analysis diagram of the test results of the experimental group (adding blockers) in the embodiment and the target value;

[0028] Figure 2 It is a correlation analysis diagram between the detection results of the control group (no blocker added) and the target value in the embodiment. DETAILED DESCRIPTION

[0029] The composite biological blocker for immunochromatographic detection of the present invention comprises a blocker buffer, wherein mouse monoclonal antibody IgG-1, mouse monoclonal antibody IgG-2, goat anti-human IgM / RF polyclonal antibody and rabbit polyclonal IgG are diluted in the blocker buffer; BSA is also added to the blocker buffer, and the BSA is added to 10 mg / mL; Proclin 300 (manufactured by Sigma, USA, item number: 48912-U) is also added to the blocker buffer, which is a high-efficiency biological preservative, and the added amount is one thousandth of the volume ratio, or it may not be added;

[0030] Among them, the blocking agent buffer is Tris-HCl buffer or phosphate PBS buffer; the manufacturer of mouse monoclonal antibody IgG-1, mouse monoclonal antibody IgG-2, goat anti-human IgM / RF polyclonal antibody and rabbit polyclonal IgG is Nanjing Lanxuan Biotechnology Co., Ltd., and the corresponding product numbers are mouse monoclonal antibody IgG-1-5016M, mouse monoclonal antibody IgG-2-5017M, goat anti-human IgM / RF polyclonal antibody-5030P, and rabbit polyclonal IgG-5012N.

[0031] The blocking agent buffer is 10 mM Tris-HCl buffer or 10 mM phosphate PBS buffer, with a pH of 7.4±0.2.

[0032] The mouse monoclonal antibody IgG-1, mouse monoclonal antibody IgG-2, goat anti-human IgM / RF polyclonal antibody and rabbit polyclonal IgG were diluted to 10 mg / mL, 10 mg / mL, 2 mg / mL and 2 mg / mL respectively with the blocking buffer.

[0033] The method for preparing the composite biological blocking agent for immunochromatographic detection of the present invention comprises the following steps:

[0034] S1: preparing a blocking buffer, wherein the blocking buffer is 10 mM Tris-HCl buffer or 10 mM phosphate PBS buffer, and the pH of the blocking buffer is 7.4±0.2;

[0035] S2: dilute mouse monoclonal antibody IgG-1, mouse monoclonal antibody IgG-2, goat anti-human IgM / RF polyclonal antibody and rabbit polyclonal IgG to 10 mg / mL, 10 mg / mL, 2 mg / mL and 2 mg / mL respectively with the blocking buffer in step S1;

[0036] S3: adding BSA to 10 mg / mL in the blocking buffer of step S2;

[0037] S4: Add Proclin 300 to the blocking agent buffer of step S3 to obtain a composite biological blocking agent.

[0038] The fluorescent immunoassay reagent strip of the present invention comprises four parts: a sample pad, a binding pad, a coated nitrocellulose membrane and absorbent paper; wherein the sample pad is treated with a sample pad treatment solution, the sample pad treatment solution comprises a buffer solution with a pH of 8 to 10, a surfactant, trehalose, an erythrocyte monoclonal antibody and the composite biological blocker according to any one of claims 1 to 5, and the composite biological blocker is added to the sample pad treatment solution at a volume ratio of 0.5%.

[0039] The preparation method of the conjugate pad is as follows: (1) activating fluorescent microspheres and EDC in a phosphate buffer solution; (2) adding a detection antibody and chicken IgY to the fluorescent microsphere solution for reaction; (3) performing a blocking reaction on the coupled microsphere-antibody complex, and obtaining an immunofluorescent microsphere marker after centrifugation; and (4) coating the immunofluorescent microsphere marker solution with a prepared concentration onto glass fiber and drying to obtain a conjugate pad.

[0040] The preparation method of the nitrocellulose membrane is as follows: the detection antibody II is used to prepare a T line solution, the goat anti-chicken IgY is used to prepare a C line solution, and then the solution is coated on the T detection line and C quality control line areas on the nitrocellulose membrane, and then dried.

[0041] The preparation method of the reagent strip is as follows: starting from the sample application end, the four parts, namely, the sample pad, the binding pad, the coated nitrocellulose membrane and the absorbent paper, are sequentially pasted on the bottom plate, and then cut into the required width to form a test strip.

[0042] The composite biological blocker of the present invention comprises a variety of animal immunoglobulins interfering with heterophilic antibodies, anti-animal antibodies, anti-rheumatoid factors, etc. It has a good blocking effect on complex endogenous interfering factors and can effectively reduce the false positive or false negative results of immunochromatographic detection caused by endogenous interfering substances.

[0043] Performance analysis of composite biological blocking reagents, taking the immunochromatographic detection of creatine kinase isoenzyme (CKMB) as an example:

[0044] 1) Used for quantitative detection of CKMB by fluorescent immunochromatography.

[0045] 2) Six human serum samples that have been confirmed to have obvious endogenous interference (experimental samples, numbered 1 to 6) and two human serum samples that have been confirmed to have no obvious endogenous interference (control samples, numbered 7 to 8) were selected, and the sample pads were treated in the above manner. The experimental group was a sample pad with a blocker added, and the control group was a sample pad without a blocker added. Fluorescent immunoassay test strips were prepared, and the test experiment was performed according to the instructions. The results are shown in Table 1 below:

[0046]

[0047] Table 1

[0048] The test results of the experimental group and the control group were used as the horizontal axis, and the target value was used as the vertical axis. The correlation analysis between the test results of the experimental group and the control group and the target value was performed, such as Figure 1 and Figure 2 shown.

[0049] From Table 1, Figure 1 and Figure 2 It can be seen that the test results of the experimental group with added blockers have a small deviation from the target value and a good correlation; while the test results of the control group without added blockers for samples numbered 1 to 6 have a large deviation from the target value, and the overall correlation is very poor. The composite biological blocker of the present invention can effectively reduce or eliminate the adverse effects of endogenous interfering substances on CKMB fluorescent immunochromatographic detection, prevent the occurrence of false positive and false negative test results, and ensure the accuracy of the test results.

[0050] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by 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, such as forming a test strip into a plastic card shell to form a reagent card. These changes and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A fluorescent immunoassay reagent strip, characterized in that: The invention comprises four parts: a sample pad, a binding pad, a coated nitrocellulose membrane and absorbent paper; wherein the sample pad is treated with a sample pad treatment solution, the sample pad treatment solution comprises a buffer solution with a pH of 8 to 10, a surfactant, trehalose, an erythrocyte monoclonal antibody and a composite biological blocker, and the composite biological blocker is added to the sample pad treatment solution at a volume ratio of 0.5%; The preparation method of the conjugate pad is as follows: (1) activating fluorescent microspheres and EDC in a phosphate buffer solution; (2) adding a detection antibody and chicken IgY to the fluorescent microsphere solution for reaction; (3) performing a blocking reaction on the coupled microsphere-antibody complex, and obtaining an immunofluorescent microsphere marker after centrifugation; (4) coating the immunofluorescent microsphere marker solution with a prepared concentration onto glass fiber, and drying to obtain a conjugate pad; The composite biological blocking agent comprises a blocking agent buffer, in which mouse monoclonal antibody IgG-1, mouse monoclonal antibody IgG-2, goat anti-human IgM / RF polyclonal antibody and rabbit polyclonal IgG are diluted; BSA is also added to the blocking agent buffer; The mouse monoclonal antibody IgG-1, mouse monoclonal antibody IgG-2, goat anti-human IgM / RF polyclonal antibody and rabbit polyclonal IgG were diluted to 10 mg / mL, 10 mg / mL, 2 mg / mL and 2 mg / mL respectively with the blocking buffer; Wherein, the blocking agent buffer is Tris-HCl buffer or phosphate PBS buffer.

2. The fluorescent immunoassay reagent strip according to claim 1, characterized in that: The blocking agent buffer is 10 mM Tris-HCl buffer or 10 mM phosphate PBS buffer.

3. The fluorescent immunoassay reagent strip according to claim 2, characterized in that: The pH of the blocking buffer was 7.4 ± 0.

2.

4. The fluorescent immunoassay reagent strip according to any one of claims 1 to 3, wherein BSA is added to the blocking buffer to a value of 10 mg / mL.

5. The fluorescent immunoassay reagent strip according to claim 4, wherein Proclin 300 is further added to the blocking agent buffer.

6. A method for preparing a composite biological blocking agent for immunochromatographic detection, characterized in that: The following steps are involved: S1: preparing a blocking buffer, wherein the blocking buffer is 10 mM Tris-HCl buffer or 10 mM phosphate PBS buffer, and the pH of the blocking buffer is 7.4±0.2; S2: dilute mouse monoclonal antibody IgG-1, mouse monoclonal antibody IgG-2, goat anti-human IgM / RF polyclonal antibody and rabbit polyclonal IgG to 10 mg / mL, 10 mg / mL, 2 mg / mL and 2 mg / mL respectively with the blocking buffer in step S1; S3: adding BSA to 10 mg / mL in the blocking buffer of step S2; S4: Add Proclin 300 to the blocking agent buffer of step S3 to obtain a composite biological blocking agent.

7. The fluorescent immunoassay reagent strip according to claim 1, characterized in that: The preparation method of the nitrocellulose membrane is as follows: the detection antibody II is used to prepare a T line solution, the goat anti-chicken IgY is used to prepare a C line solution, and then the solution is coated on the T detection line and C quality control line areas on the nitrocellulose membrane, and then dried.

8. The fluorescent immunoassay reagent strip according to claim 1, characterized in that: The preparation method of the test strip is as follows: Starting from the sample adding end, there are four parts, namely the sample pad, the binding pad, the coated nitrocellulose membrane and the absorbent paper. They are pasted on the bottom plate in sequence and then cut into the required width to form a test strip.

Images