Magnetic particle enzyme-linked immunosorbent assay (ELISA) detection method and kit

Abstract

The invention provides a magnetic particle enzyme-linked immunosorbent assay (ELISA) detection method and a kit. The kit comprises a magnetic particle-coated antigen or antibody, an enzyme-labeled antigen or antibody, a chromogenic substrate, a calibrator, a cleaning solution and an eight-row reaction tube device. According to the present invention, the detection of proteins, hormones, small molecule substances, cell factors and the like can be achieved, the strong compatibility with the to-be-detected substances is provided, and the characteristics of prefabrication, short reaction time, high sensitivity, high flux, low economic cost and the like are provided. The method can be applied to the fields of scientific research laboratories, biological medicine and vaccine research and development laboratories, clinical examination laboratories, agriculture and food science laboratories and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a magnetic particle enzyme-linked immunosorbent assay (ELISA) detection method and a kit. Background Art

[0002] ‌ELISA (enzyme-linked immunosorbent assay)‌ is an immunoassay technique based on antigen-antibody reaction, which is widely used in biology, medicine, environmental science and other fields. Its basic principle is to fix the antigen or antibody on a solid phase carrier, and form an antigen-antibody complex by binding the enzyme-labeled antibody or antigen to the target molecule in the sample to be tested. Then the substrate of the enzyme is added, and the enzyme catalyzes the substrate to produce a color change, and the concentration of the target molecule is quantitatively analyzed by measuring the depth of color or fluorescence intensity. ELISA has a wide range of applications: In the field of clinical diagnosis, ELISA is a commonly used tool in clinical diagnosis. It can detect specific antibodies or antigens in blood or other body fluids and is used to diagnose a variety of diseases, such as infectious diseases (such as HIV / AIDS, hepatitis B, hepatitis C, etc.), allergies, autoimmune diseases (such as lupus, rheumatoid arthritis, etc.) and cancer (such as prostate cancer, ovarian cancer, etc.). In the field of agriculture and food science, it is often used to detect toxins and pesticide residues in food, as well as cross-reactive herbicides in environmental samples. In drug discovery, animal research and clinical trials, ELISA can be used to quantify drug and hormone levels to ensure that the drug is at a therapeutic level and is non-toxic.

[0003] At present, ELISA detection technology also has many pain points: antigens or antibodies are coated on 96-well plates. This solid-phase coated material leads to insufficient reaction and low sensitivity, which seriously affects performance; in order to compensate for the insufficient reaction, the reaction time is generally long, and it can take several hours to react; in addition, users need to dilute the concentrated washing solution, calibrator, working antibody 1, and working antibody 2 before starting the experiment, which not only easily leads to batch differences and the introduction of uncertainty, but also greatly wastes the user's time. Summary of the invention

[0004] Purpose of the invention: In view of the deficiencies of the prior art and actual needs, the present invention provides a magnetic microparticle enzyme-linked immunosorbent assay (ELISA) detection method and kit, which adopts high-efficiency, liquid-system magnetic microparticles and enzyme-labeled components, and performs standard prefabrication treatment on calibrators and washing solutions, so as to achieve standard, convenient, efficient and high-sensitivity testing of the analyte, so as to solve the problems of low sensitivity, large batch-to-batch difference, complicated process and long cycle in the prior art.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] The invention provides a magnetic particle enzyme-linked immunosorbent assay (ELISA) detection method and a kit, comprising: magnetic particle-coated antigens or antibodies, enzyme-labeled antigens or antibodies, colorimetric substrates, calibration products, cleaning fluids and an 8-coupling reaction tube device.

[0007] Preferably, the magnetic particles coated with the antigen or antibody can be streptavidin, carboxyl or benzenesulfonyl magnetic beads, the streptavidin magnetic beads are coated with the antigen or antibody through biotin; the carboxyl magnetic beads are coated with the antigen or antibody through EDC reaction; the benzenesulfonyl magnetic beads are coated with the antigen or antibody through covalent bond binding. The concentration is 0.1-5 mg / mL. More preferably, the concentration of the magnetic particles is 0.3 mg / mL.

[0008] Preferably, the enzyme for labeling the antigen or antibody can be alkaline phosphatase (ALP) or horseradish peroxidase (HRP), and the enzyme is coupled to the antigen or antibody by introducing a maleic acid chemical group, and the concentration is 0.1-3 μg / mL. More preferably, the enzyme concentration is 2 μg / mL.

[0009] Preferably, the chromogenic substrate is TMB or pNPP at a concentration of 1-5 mg / mL, more preferably, the concentration of the chromogenic substrate is 2 mg / mL.

[0010] Preferably, the standard corresponding to the substance to be tested having a concentration gradient is freeze-dried and prefabricated, and the concentration range includes the entire linear range of the substance to be tested.

[0011] Preferably, the cleaning solution contains 50 mmol / L phosphate buffer, 1% BSA, 1% Tween 20, and 0.1% PC-300.

[0012] Preferably, the 8-row reaction tube device contains a PP tube for independent reaction, and 8 tubes are connected in a row as a group; and also includes a base for placing the 8-row reaction tubes.

[0013] In the present invention, the enzyme for labeling the antigen or antibody may be alkaline phosphatase (ALP) or horseradish peroxidase (HRP); the enzyme may be naturally extracted or obtained through various recombinant technologies.

[0014] The present invention adopts high-efficiency, liquid system magnetic particles and enzyme labeling components, and performs standard prefabrication treatment on calibration products and washing solutions, which can realize standard, convenient, efficient and high-sensitivity testing of the objects to be tested, so as to solve the problems of low sensitivity, large batch difference, complicated process and long cycle in the prior art.

[0015] The present invention also provides a magnetic microparticle enzyme-linked immunosorbent assay (ELISA) detection method and a kit operation method, including:

[0016] (1) Add the test substance and magnetic particles to the reaction tube, shake to mix, and incubate at 37°C for 15 minutes.

[0017] (2) Use cleaning solution to clean three times, either by centrifugation or magnetic absorption.

[0018] (3) After washing, add the colorimetric substrate, shake to mix, and incubate at 37°C for 5-15 minutes.

[0019] (4) After centrifugation or magnetic collection, transfer the colored supernatant to the detection plate.

[0020] (5) Read the results using an ELISA reader.

[0021] Preferably, in step (1), the amount of the substance to be tested added is 100 μL.

[0022] Preferably, in step (2), the centrifugal magnetic centrifuge is set to a centrifugal parameter of 7000 rpm and centrifuged for 2 min.

[0023] Preferably, in step (4), the centrifugal magnetic centrifuge is set to a centrifugal parameter of 7000 rpm and centrifuged for 5 min.

[0024] The beneficial effects of the present invention are:

[0025] (1) All components of the present invention are prefabricated, the components are stable, the storage conditions are loose, the performance is stable, and the batch-to-batch difference is small.

[0026] (2) The present invention uses high-efficiency, liquid-system magnetic particles and enzymes with a large reaction area, which can achieve standard, convenient, efficient and high-sensitivity testing of the analyte, solving the problems of low sensitivity, large batch-to-batch differences, complicated processes and long cycles in the prior art.

[0027] (3) The present invention adopts an 8-row reaction tube device with flexible specifications and design, which is more suitable for the usage scenario. Users can purchase the required quantity according to their needs, which is more economical and environmentally friendly and avoids reagent waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The reaction ratio and sensitivity of the ELISA test kit of the present invention were compared with those of a traditional ELISA test kit of an imported manufacturer. DETAILED DESCRIPTION

[0029] In order to more clearly illustrate the present invention, the present invention is further described in detail below in conjunction with embodiments. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0030] The present invention provides a magnetic microparticle enzyme-linked immunosorbent assay (ELISA) detection method and a kit preparation method. The operation steps take Aβ1-42 detection as an example and include the following steps:

[0031] (1) Take Aβ1-42 coated antibody, dilute it to 1 mg / mL, use NHS-Bio to couple Aβ1-42 coated antibody, incubate with streptavidin magnetic beads overnight with shaking, wash the magnetic beads three times with magnetic bead dilution solution, and dilute the magnetic beads to 0.3 mg / mL for use.

[0032] (2) Take the Aβ1-42 labeled antibody, dilute it to 1 mg / mL, and then perform maleylation treatment. Then, incubate it with activated alkaline phosphatase overnight, desalt it, and dilute it to 2 μg / mL for use.

[0033] (3) Weigh 20 mg of pNPP, dissolve it in 10 mL of substrate buffer, mix well, filter and set aside.

[0034] (4) Weigh 100 mL of 50 mmol / L phosphate buffer, add 1 g BSA, 1 g Tween 20, and 0.1 g PC-300, respectively, dissolve and mix well, filter and set aside.

[0035] (5) Add the test substance and magnetic particles to the reaction tube, shake to mix, and incubate at 37°C for 15 minutes.

[0036] (6) Wash three times with cleaning solution; after washing, add the color developing substrate, shake and mix, and incubate at 37°C for 5 minutes.

[0037] (7) After centrifugation or magnetic collection, transfer the colored supernatant to the detection plate and read the result using an ELISA reader.

[0038] Aβ1-42 detection kit was prepared, the reaction ratio and sensitivity of the calibrator were tested, and compared with a traditional ELISA detection kit from an imported manufacturer. The results are as follows Figure 1 As shown in Table 1, it can be seen from the figure that the reagent invention has higher sensitivity and reaction ratio.

[0039] Table 1. Comparison of reaction ratio and sensitivity using the present invention.

[0040]

[0041] The reaction ratio and sensitivity show that the present invention has obvious sensitivity and reaction ratio advantages.

[0042] We also verified the other properties of the magnetic particle enzyme-linked immunosorbent assay (ELISA) and the kit.

[0043] The intra-batch precision and inter-batch precision experiments were compared using the ELISA test kit of the present invention and a traditional ELISA test kit of an imported manufacturer. The test results are shown in Tables 2 and 3.

[0044] Table 2. Comparison of intra-batch precision of the ELISA test kit of the present invention and that of a traditional ELISA test kit of an imported manufacturer.

[0045]

[0046] Table 3. Comparison of batch precision between the ELISA test kit of the present invention and a traditional ELISA test kit of an imported manufacturer.

[0047]

[0048] The comparison of intra-batch precision and inter-batch precision experiments shows that the present invention has obvious precision advantages compared with a certain imported traditional ELISA kit. The present invention also has obvious price advantages and cycle advantages compared with imports.

[0049] The experimental time of the present invention was compared with that of a traditional ELISA detection kit of an imported manufacturer, and the results are shown in Table 4.

[0050] Table 4. Comparison of experimental time.

[0051]

[0052] Time comparison shows that the prefabricated components and the efficient magnetic particle liquid system of the present invention greatly shorten the test time and improve the work efficiency of the user.

[0053] The stability of the magnetic microparticle enzyme-linked immunosorbent assay (ELISA) method and kit was verified, and the test results are shown in Table 5.

[0054] Table 5. Shelf stability verification results of the kit of the present invention.

[0055]

[0056] The experiment shows that the reaction ratio and sensitivity of the test kit are not significantly changed when stored at 2-8°C for 0, 3, 6, 9, and 12 months, indicating that the storage conditions of the present invention are loose and the performance is stable. In the specific application of the laboratory, it is convenient for laboratory storage and stocking, and the performance is stable within the validity period of 12 months.

[0057] The preferred embodiments of the present invention disclosed above are only examples to help explain the present invention more clearly, rather than limiting the implementation methods of the present invention. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A magnetic particle enzyme-linked immunosorbent assay (ELISA) detection method and kit, comprising: Magnetic particle-coated antigen or antibody, enzyme-labeled antigen or antibody, colorimetric substrate, calibration substance, cleaning solution and 8-coupling reaction tube device.

2. A magnetic particle enzyme-linked immunosorbent assay (ELISA) detection method and kit according to claim 1, characterized in that: The antigen or antibody coated with the magnetic microparticles contains streptavidin, carboxyl or benzenesulfonyl magnetic beads, and the magnetic beads are coupled to the antibody or antigen of the substance to be tested by a coating method, and the concentration of the magnetic beads is 0.1-5 mg / mL.

3. A magnetic microparticle enzyme-linked immunosorbent assay (ELISA) detection method and kit according to claim 1, characterized in that: The enzyme-labeled antigen or antibody contains alkaline phosphatase (ALP) or horseradish peroxidase (HRP), and the enzyme is coupled with the antibody or antigen of the substance to be detected by a labeling method, and the enzyme concentration is 0.1-3 μg / mL.

4. A magnetic particle enzyme-linked immunosorbent assay (ELISA) detection method and kit according to claim 1, characterized in that: The chromogenic substrate contains TMB or pNPP at a concentration of 1-5 mg / mL.

5. A magnetic particle enzyme-linked immunosorbent assay (ELISA) detection method and kit according to claim 1, characterized in that: The calibrator is a standard substance corresponding to the substance to be tested with a concentration gradient.

6. A magnetic particle enzyme-linked immunosorbent assay (ELISA) detection method and kit according to claim 1, characterized in that: The cleaning solution contains 50 mmol / L phosphate buffer, 1% BSA, 1% Tween 20, and 0.1% PC300.

7. A magnetic particle enzyme-linked immunosorbent assay (ELISA) detection method and kit according to claim 1, characterized in that: The 8-row reaction tube device contains a PP tube for independent reaction, and 8 tubes are connected in a row as a group; and also includes a base for placing the 8-row reaction tubes.

8. A magnetic microparticle enzyme-linked immunosorbent assay (ELISA) detection method and kit according to claim 2, characterized in that: Streptavidin magnetic beads are coated with antigens or antibodies through biotin; carboxyl magnetic beads are coated with antigens or antibodies through EDC reaction; and benzenesulfonyl magnetic beads are coated with antigens or antibodies through covalent bonds.

9. A magnetic microparticle enzyme-linked immunosorbent assay (ELISA) detection method and kit according to claim 3, characterized in that: The enzyme is coupled to the antigen or antibody by introducing a maleyl chemical group.

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