An ELISA kit for detecting total antibodies against novel coronavirus

Abstract

The present invention provides an ELISA kit for detecting total antibodies against novel coronavirus, which comprises a coated plate coated with novel coronavirus antigen, an enzyme conjugate containing HRP-labeled mouse anti-human kappa light chain antibody, a system reaction solution, a negative and positive control solution, and a TMB substrate solution. It can quickly and accurately determine the presence of antibodies against novel coronavirus in a sample. The product uses the indirect method to detect total antibodies, which can reduce the production material cost compared with the sandwich method, and the labeling process is more concise.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection, and particularly to an enzyme-linked immunosorbent assay kit for detecting total antibodies against novel coronavirus. Background Art

[0002] Coronaviruses are a large group of viruses that cause diseases ranging from the common cold to more severe diseases such as Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS-CoV). Common symptoms of infection include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death.

[0003] After being infected with the novel coronavirus, the human immune system will produce corresponding IgA, IgM, and IgG antibodies. Whether the markers exist can be detected through peripheral blood serum, thereby inferring whether the person is infected. Serological tests have become a necessary method for detecting novel coronavirus infection. It is easier to operate, has a short detection time, and a low detection cost. Different Ig types are produced at different times after infection, and the production amounts also vary greatly. A positive serological test indicates the presence of novel coronavirus antibodies, confirming both the possibility of past infection and potential current infection. Total antibodies are more valuable as the first-step primary screening. Currently, there are reagent products for detecting total antibodies by the double-antigen sandwich method, but they have high requirements for the antigen labeling process and are prone to missed detections.

[0004] Natural Ig molecules contain four heterologous polypeptide chains. Among them, the two chains with larger molecular weights are called heavy chains (H), and the two chains with smaller molecular weights are called light chains (L). The amino acid composition and arrangement order of the constant region of the heavy chain are different, and its antigenicity is also different. Accordingly, Ig can be divided into 5 classes, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain respectively. Light chains can be divided into two types, namely kappa (κ) chain and lambda (λ) chain. Accordingly, Ig can be divided into two types, namely κ type and λ type. The types of the two light chains on one Ig molecule are always the same. Different classes of Ig exist in both κ type and λ type. The κ:λ ratio of normal human serum Ig is about 2:1. Summary of the Invention

[0005] To solve the above problems, the present invention provides an enzyme-linked immunosorbent assay kit for detecting total antibodies against novel coronavirus, which can quickly and accurately determine the presence or absence of novel coronavirus antibodies in a sample. The product uses the indirect method to detect total antibodies, which can reduce the production material cost compared with the sandwich method, and the labeling process is more concise.

[0006] The object of the present invention is achieved in the following manner: A kit for the enzyme-linked immunosorbent assay of total antibodies against the novel coronavirus, comprising a coated plate coated with the novel coronavirus antigen, an enzyme conjugate containing an HRP-labeled mouse anti-human kappa light chain antibody, a system reaction solution, a positive and negative control solution, and a TMB substrate solution.

[0007] The system reaction solution includes a buffer, a preservative, casein, bromocresol purple, and Tween 20.

[0008] The buffer is a Tris-NaCl buffer or a PBS buffer, and the preservative is at least one of Proclin 300, NaN3, and Kathon.

[0009] The enzyme conjugate is obtained by mixing an HRP-labeled mouse anti-human kappa light chain antibody and a diluent of the enzyme conjugate in a volume ratio of 1:(4800 - 5200).

[0010] The diluent of the enzyme conjugate contains casein.

[0011] The diluent of the enzyme conjugate further includes a buffer and a preservative. The buffer is a Tris-NaCl buffer or a PBS buffer, and the preservative is at least one of Proclin 300, NaN3, and Kathon.

[0012] The diluent of the enzyme conjugate further includes a protective protein, and the protective protein is at least one of calf serum, bovine serum albumin, and Casein.

[0013] The positive and negative control solution includes a positive control solution and a negative control solution; the negative control solution includes a buffer, a preservative, a protective protein, and nystatin. The buffer is a Tris-NaCl buffer or a PBS buffer, and the preservative is at least one of Proclin 300, NaN3, and Kathon; the protective protein is at least one of calf serum, bovine serum albumin, and Casein; the positive control solution is obtained by mixing inactivated novel coronavirus antibodies and a negative control diluent in a volume ratio of 1:1000.

[0014] The detection kit further includes a washing solution and a termination solution.

[0015] Its principle of action is as follows:

[0016] The novel coronavirus antigen used in this application is a genetically recombinant antigen of the structural region and non-structural region of the novel coronavirus (including N protein, S1 protein, S2 protein, and separately expressed RBD). Using the genetically recombinant antigen of the structural region and non-structural region of the novel coronavirus and horseradish peroxidase-labeled mouse anti-human kappa light chain antibody as the main raw materials, the indirect enzyme-linked immunosorbent assay principle is applied to detect the total antibodies against the novel coronavirus contained in serum or plasma samples. A coated plate is prepared with the novel coronavirus recombinant antigen. After adding the sample to be tested, various antibodies against the novel coronavirus contained in the sample will bind to it. Then, horseradish peroxidase-labeled anti-human kappa light chain antibody is added, and finally, an antigen-antibody-enzyme-labeled antibody complex is formed through the immune reaction. This complex can make the TMB substrate solution develop color, and the intensity of the developed color absorbance is proportional to the content of the total antibodies against the novel coronavirus in the sample. Detailed implementation mode

[0017] The present invention will be specifically described below in combination with specific embodiments. It is necessary to point out here that these embodiments are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the content of the present invention above.

[0018] An enzyme-linked immunosorbent assay kit for detecting total antibodies against the novel coronavirus includes a coated plate coated with the novel coronavirus antigen, an enzyme conjugate containing HRP-labeled mouse anti-human kappa light chain antibody, a system reaction solution, a negative and positive control dilution solution, and a TMB substrate solution.

[0019] The system reaction solution includes a buffer, a preservative, casein, bromocresol purple, and Tween 20.

[0020] The buffer is Tris-NaCl buffer or PBS buffer, and the preservative is at least one of Proclin 300, NaN3, and Kathon.

[0021] The enzyme conjugate is obtained by mixing the HRP-labeled mouse anti-human kappa light chain antibody and the dilution solution of the enzyme conjugate in a volume ratio of 1:(4800 - 5200).

[0022] The dilution solution of the enzyme conjugate contains casein.

[0023] The dilution solution of the enzyme conjugate also includes a buffer and a preservative. The buffer is Tris-NaCl buffer or PBS buffer, and the preservative is at least one of Proclin 300, NaN3, and Kathon.

[0024] The dilution solution of the enzyme conjugate also includes a protective protein, and the protective protein is at least one of calf serum, bovine serum albumin, and Casein.

[0025] The positive and negative control diluents include a positive control diluent and a negative control diluent; the negative control diluent includes a buffer, a preservative, a protective protein, and nystatin. The buffer is Tris-NaCl buffer or PBS buffer, and the preservative is at least one of Proclin 300, NaN3, and Kathon; the protective protein is at least one of calf serum, bovine serum albumin, and Casein; the positive control diluent is obtained by mixing inactivated anti-SARS-CoV-2 antibodies and the negative control diluent at a volume ratio of 1:1000.

[0026] The detection kit also includes a washing solution and a termination solution. The TMB substrate solution, the washing solution, and the termination solution are from conventional materials on the market.

[0027] The present invention will be described in more detail below through specific examples for the understanding of those skilled in the art. Unless otherwise specified, the materials and detection instruments used in the present invention are all conventional material products in this industry, and the detection methods used are conventional methods in this industry. The SARS-CoV-2 antigen used in this application is a gene recombinant antigen of the SARS-CoV-2 structural region and non-structural region (including N protein, S1 protein, S2 protein, and separately expressed RBD), purchased from Nanjing Genscript Biotech.

[0028] Preparation of a qualitative detection kit for total anti-SARS-CoV-2 antibodies

[0029] 1. Preparation of a coated plate coated with SARS-CoV-2 antigen

[0030] ① The SARS-CoV-2 antigen - here, the gene recombinant antigen of SARS-CoV-2 (purchased from Nanjing Genscript Biotech) is added to 0.1M PBS buffer at pH 7.2 at a ratio of N protein:S1 protein:S2 protein:RBD = 2:1:0.5:1, with a total final concentration of 1 μg / mL, and thoroughly mixed at room temperature (18 - 23 °C).

[0031] ② The prepared coating solution is added to a blank ELISA plate (purchased from Xiamen Yunpeng Biotech) at 100 μL / well and placed in a refrigerator at 2 - 8 °C for 16 - 24 hours.

[0032] ③ Discard the coating solution in the ELISA plate, pat dry, and wash the plate 2 times with the washing solution.

[0033] ④ Block within 2 minutes after washing the plate. Add the blocking solution (1% peptone) at 150 μL / well and place in a refrigerator at 2 - 8 °C for 16 - 24 hours.

[0034] ⑤ Discard the blocking solution of the ELISA plate, and then place the ELISA plate in a drying oven (humidity ≤ 30%) for 4 - 8 hours.

[0035] Preparation is completed.

[0036] 2. Preparation of the system reaction solution of the present invention

[0037] In the first step, prepare the Tris-NaCl buffer solution by mixing the raw materials in Table 1 below to form a Tris-NaCl aqueous solution;

[0038] Table 1

[0039]

[0040] Then adjust the pH value of the Tris-NaCl aqueous solution to 6.0 with 6M hydrochloric acid to prepare the Tris-NaCl buffer solution.

[0041] In the second step, prepare the system reaction solution

[0042] Prepare the system reaction solution by mixing the raw materials in Table 2 below.

[0043] Table 2

[0044]

[0045] 3. Preparation of the enzyme conjugate of the present invention

[0046] In the first step, prepare the Tris-NaCl buffer solution by mixing the raw materials in Table 3 below to form a Tris-NaCl aqueous solution;

[0047] Table 3

[0048]

[0049] Then adjust the pH value of the Tris-NaCl aqueous solution to 7.4 with 6M hydrochloric acid to prepare the Tris-NaCl buffer solution.

[0050] In the second step, prepare the diluent of the enzyme conjugate

[0051] Prepare the diluent of the enzyme conjugate by mixing the raw materials in Table 4 below.

[0052] Table 4

[0053]

[0054] After preparation, the diluent of the enzyme conjugate and the HRP-labeled mouse anti-human κ-chain antibody (purchased from Luoyang Bio-Tong Experimental Materials Center) are mixed in a volume ratio of 5000:1: to form the enzyme conjugate.

[0055] 4. Preparation of the positive and negative control solutions of the present invention

[0056] In the first step, prepare the HEPES buffer solution by mixing the raw materials in Table 5 below to form a HEPES solution;

[0057] Table 5

[0058]

[0059] Then, adjust the pH value of the HEPES solution to 7.2 with 1 M sodium hydroxide to prepare the HEPES buffer solution.

[0060] In the second step, prepare the negative and positive control solutions. Mix the raw materials in Table 6 below to prepare the negative control solution;

[0061] Table 6

[0062]

[0063] Dilute the inactivated COVID-19 antibody, specifically the recombinant human antibody against COVID-19 (purchased from Nanjing Genscript Biotech, product number A02039) with the negative control solution at a volume ratio of 1:1000, and mix well to prepare the positive control solution. The negative control solution and the positive control solution must be present in each experiment to ensure the validity of the experiment.

[0064] 5. Other general reagent components - TMB substrate solution, termination solution, and washing solution are purchased from Zhengzhou Dano Biotech.

[0065] 6. Test procedure

[0066] (1) [Main components] are as shown in Table 7 below

[0067] Table 7

[0068]

[0069] (2) [Testing method]

[0070] * Preparation before experiment

[0071] 1. Restore all reagents and samples to room temperature (18 - 25 °C) for at least 30 minutes.

[0072] 2. Set the incubator or water bath to the reaction temperature of 37 °C.

[0073] 3. Dilute the washing solution 20 times with purified water, shake well, and set aside for later use.

[0074] * Experimental steps

[0075] 1. Sampling: Take out the numbered coated wells on the coated plate. Except for the first well numbered A1 as the blank control well, add 100 µL of the system reaction solution to the remaining wells in sequence using a micropipette. Then, add 10 µL of the negative control solution to the coated wells numbered B1 and C1 respectively, add 10 µL of the positive control solution to the coated wells numbered D1 and E1 respectively, and add 10 µL of the test sample to the remaining coated wells. After adding the test sample, the color of the system reaction solution will change from green to blue. Gently shake and mix well for 30 seconds.

[0076] 2. Incubation: Place the coated plate after sampling in an environment at 37 °C for 30 minutes.

[0077] 3. Washing: Discard the liquid in the coated wells, fill each coated well with the diluted washing solution, let it stand for 20 seconds, then discard it. Repeat this washing process 5 times, making sure to discard all the liquid each time. Finally, pat dry on absorbent paper.

[0078] 4. Sampling: Add 100 µL of the enzyme conjugate to each well except the blank control well. Then repeat steps 2 and 3.

[0079] 5. Color development: Add 100 µL of the substrate solution to each coated well and react in the dark at room temperature of 18 - 23 °C for 10 minutes.

[0080] 6. Termination: Add 50 µL of the termination solution to each coated well to terminate the reaction.

[0081] (3) Result calculation

[0082] (1). Detection with an ELISA reader: Select an ELISA reader with a wavelength of 450 nm and a reference wavelength of 630 nm. Measure the OD value of each well within 10 minutes after terminating the reaction.

[0083] (2). Calculation:

[0084] ① Calculate the average value `C NC of the OD values of the negative control solution (Negative Control, NC) added; if the OD value of the positive control solution added > 0.8, it indicates that the entire experimental data is valid and can be used. Otherwise, discard this data.

[0085] ② Calculate the cutoff value using the following formula:

[0086] Positive determination value = 1.1 * (`C NC + 0.18)

[0087] Negative determination value = 0.9 * (`C NC + 0.18)

[0088] Compare the OD value of the test sample added with the following table to determine the result of the sample. The result judgment refers to Table 8 below:

[0089] Table 8

[0090]

[0091] 7. Clinical performance evaluation of the kit of the present invention and advantages of the invention

[0092] The kit of the present invention and the total antibody detection kit for novel coronavirus (enzyme-linked immunosorbent assay) produced by Wantai Biological Co., Ltd. were used to parallelly examine 874 samples of the physical examination population, and the test results were analyzed. The test results are shown in Table 9 below:

[0093] Table 9

[0094]

[0095] For parallel comparative detection of 58 samples confirmed to be positive for novel coronavirus, the results are shown in Table 10 below:

[0096] Table 10

[0097]

[0098] The present invention supplements the method for qualitatively detecting the total antibody of novel coronavirus, makes up for the defects of timeliness and cost of the existing detection methods on the market, and at the same time maintains good detection effects. The design of color change after adding samples in the system diluent further improves the detection efficiency and reduces the operation error rate.

[0099] The above are only the preferred embodiments of the present invention, but the protection scope of the present invention is not limited thereto. It should be pointed out that for those skilled in the art and any person familiar with the technical field, without departing from the overall concept of the present invention, equivalent substitutions or changes made according to the technical solution and inventive concept of the present invention, as well as several changes and improvements made, should also be regarded as the protection scope of the present invention.

Claims

1. A kit for enzyme-linked immunosorbent assay of total antibodies against novel coronavirus, characterized in that: It includes a coated plate coated with the antigen of the novel coronavirus, an enzyme conjugate containing an HRP-labeled mouse anti-human kappa light chain antibody, a system reaction solution, a positive and negative control solution, and a TMB substrate solution; the antigen of the novel coronavirus is a recombinant antigen of the novel coronavirus in the ratio of N protein:S1 protein:S2 protein:RBD = 2:1:0.5:

1. The enzyme conjugate is obtained by mixing an HRP-labeled mouse anti-human kappa light chain antibody and a diluent of the enzyme conjugate in a volume ratio of 1:(4800 - 5200). The diluent of the enzyme conjugate contains casein; the positive and negative control solution includes a positive control solution and a negative control solution; the negative control solution includes a buffer, a preservative, a protective protein, and nystatin. The buffer is Tris-NaCl buffer or PBS buffer, and the preservative is at least one of Proclin 300, NaN3, and Kathon; the protective protein is at least one of calf serum, bovine serum albumin, and Casein; the positive control solution is obtained by mixing inactivated novel coronavirus antibodies and a negative control diluent in a volume ratio of 1:1000; the system reaction solution includes a buffer, a preservative, casein, bromocresol purple, and Tween 20.

2. The kit for enzyme-linked immunosorbent assay of total antibodies against novel coronavirus according to claim 1, characterized in that: The buffer is Tris-NaCl buffer or PBS buffer, and the preservative is at least one of Proclin 300, NaN3, and Kathon.

3. The kit for enzyme-linked immunosorbent assay of total antibodies against novel coronavirus according to claim 1, characterized in that: The diluent of the enzyme conjugate also includes a buffer and a preservative. The buffer is Tris-NaCl buffer or PBS buffer, and the preservative is at least one of Proclin 300, NaN3, and Kathon.

4. The kit for enzyme-linked immunosorbent assay of total antibodies against novel coronavirus according to claim 1, characterized in that: The diluent of the enzyme conjugate also includes a protective protein. The protective protein is at least one of calf serum, bovine serum albumin, and Casein.

5. The kit for enzyme-linked immunosorbent assay of total antibodies against novel coronavirus according to claim 1, characterized in that: The detection kit also includes a washing solution and a termination solution.

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