Protein chip kit for detecting novel coronavirus antibodies and preparation method thereof

Abstract

The present invention "a protein chip kit for detecting new coronavirus antibodies and a preparation method" relates to virus detection technology, and has at least one protein chip, each of which has at least one sub-detection area, and each sub-detection area is used to detect a specimen; each sub-detection area is provided with the following detection spots: a first detection spot formed by the attachment of BSA, a second detection spot formed by the attachment of a new coronavirus antigen, and a third detection spot formed by the attachment of a human IgG polyclonal antibody or an IgM polyclonal antibody; the detection spots are separated from each other; the new coronavirus antigen is a receptor binding domain protein of the new coronavirus spike protein S1 subunit or a new coronavirus spike protein S1 subunit protein, or a mixture thereof.

Description

Technical Field

[0001] The present invention relates to a virus infection diagnosis technology, and in particular to a protein chip kit for detecting novel coronavirus antibodies and a preparation method thereof. Background Art

[0002] Coronavirus is associated with a variety of diseases in humans and animals, and can cause respiratory, digestive and nervous system diseases in humans and animals. The diameter of its virus particles is 60-200nm, spherical or elliptical, and polymorphic. The virus has an envelope with spikes on it. The spike glycoprotein is the receptor binding site and the main antigenic site.

[0003] Serological testing of viral antibodies in suspected patients can more accurately, quickly and cost-effectively exclude patients with fever caused by other pathogens, greatly improving the efficiency of clinical diagnosis and screening. At the same time, it can make up for the defects of nucleic acid testing in terms of high technical requirements and high false negative rate. Summary of the invention

[0004] Based on the needs of the above fields, the present invention provides a novel coronavirus IgM / IgG antibody detection kit, which can detect novel coronavirus antibodies quickly and at low cost (10 yuan / person) with a small amount of serum (7 microliters). The test results can be issued in about 30 minutes, and 10 people can be tested at a time, with an average time of as low as 3 minutes per person. It can be operated manually or on a computer.

[0005] A protein chip kit for detecting novel coronavirus antibodies, characterized in that it comprises at least one protein chip,

[0006] Each of the protein chips has at least one sub-detection area, and each sub-detection area is used to detect a sample;

[0007] The following detection spots are set in each sub-detection area:

[0008] The first detection spot formed by BSA attachment is a negative control spot, and no color is developed when the chip is valid;

[0009] The second detection spot is formed by the attachment of the novel coronavirus antigen, and the color development indicates a positive result;

[0010] The third detection spot formed by the attachment of human IgG polyclonal antibody or IgM polyclonal antibody is a positive control, and the color development indicates that the chip is effective;

[0011] The detection spots are separated from each other;

[0012] The novel coronavirus antigen is the receptor binding domain protein of the novel coronavirus spike protein S1 subunit or the novel coronavirus spike protein S1 subunit protein, or a mixture thereof.

[0013] According to a preferred embodiment, in the kit, the novel coronavirus antigen is a mixture of the receptor binding domain protein of the novel coronavirus spike protein S1 subunit and the novel coronavirus spike protein S1 subunit protein in a mass ratio of 1 to 3:1, preferably a mixture in a mass ratio of 1:1. The detection sensitivity and accuracy are significantly improved.

[0014] According to a preferred embodiment, in the kit, at least one of the protein chips comprises two sub-detection areas, one of which has the third detection spot formed by the attachment of human IgG polyclonal antibodies; the other sub-detection area has the third detection spot formed by the attachment of IgM polyclonal antibodies. According to a preferred embodiment, in the kit, two protein chips are included, one of which has the third detection spot formed by the attachment of human IgG polyclonal antibodies; the other has the third detection spot formed by the attachment of IgM polyclonal antibodies.

[0015] The test kits in the above two schemes can detect IgG antibodies and IgM antibodies in the serum of suspected patients, improve the accuracy of detection, and provide a more comprehensive basis for diagnosis.

[0016] According to a preferred embodiment, in the kit, in each of the sub-detection areas, each detection spot contains 3-6 repetitions, each detection spot is arranged in a row, and the three detection spots are arranged in a rectangular array.

[0017] According to a preferred embodiment, in the kit, the size of each sub-detection area is 5-10 mm in length and 5-10 mm in width, and the size of the detection spot is 0.5-1 mm in length and 0.5-1 mm in width.

[0018] According to a preferred embodiment, in the kit, the spacing distance between the detection spots is 0.2-1 mm.

[0019] According to a preferred embodiment, the kit further comprises an HRP-labeled anti-human IgG polyclonal antibody for use in conjunction with the sub-detection area to which the human IgG polyclonal antibody is attached to form the third detection spot, i.e., after the serum to be tested is dripped onto the detection spot in the sub-detection area, it is dripped onto and covers the detection spot;

[0020] It also contains HRP-labeled anti-human IgM polyclonal antibodies for use in conjunction with the sub-detection area with human IgM polyclonal antibodies attached to form the third detection spot, that is, after the serum to be tested is dripped on the detection spot in the sub-detection area, it is dripped onto and covers the detection spot.

[0021] According to a preferred embodiment, the kit further comprises 0.05% PBS-Tween solution and HRP color development substrate solution.

[0022] The method for preparing any of the above-mentioned kits is characterized in that the preparation of the protein chip comprises the following steps:

[0023] Obtaining a chip substrate partitioned to form the sub-detection areas;

[0024] BSA, novel coronavirus antigen, human IgG polyclonal antibody or human IgM polyclonal antibody are spotted and fixed in the sub-detection area according to a preset pattern and size to form the first, second and third detection spots;

[0025] The novel coronavirus antigen is the receptor binding domain protein of the novel coronavirus spike protein S1 subunit or the novel coronavirus spike protein S1 subunit protein, or a mixture thereof.

[0026] According to a preferred preparation method, 10% BSA, 0.1-1 mg / ml novel coronavirus antigen, and 0.05-0.2 mg / ml human IgG polyclonal antibody or human IgM polyclonal antibody are spotted and fixed in the sub-detection area according to a preset pattern and size to form the first, second, and third detection spots.

[0027] According to a preferred preparation method, 0.5 mg / ml new coronavirus antigen, 0.1 mg / ml human IgG polyclonal antibody or human IgM polyclonal antibody, and 10% BSA are preferred.

[0028] According to a preferred preparation method, all detection spots in each sub-detection area use the same test volume, which is between 1-30 nL.

[0029] According to a preferred preparation method, the detection spot is formed by multiple non-contact micro-spotting, preferably by 3-10 non-contact micro-spotting, each spotting 300-600 pL.

[0030] The present invention provides a simple, easy, economical, rapid, and reliable protein chip method for detecting IgM / IgG antibodies in novel coronavirus pneumonia serum. The present invention only requires a trace amount of serum (7ul), can detect multiple people at a time, and can issue test results in about 30 minutes. It can qualitatively detect novel coronavirus IgM / IgG antibodies in human serum or plasma, and the test results are accurate and highly sensitive.

[0031] Advantages of the protein chip kit of the present invention:

[0032] 1. The present invention selects coronavirus antigens, antigen fragments suitable for making micro-protein chips and antigen combination schemes. Clinical test data show that positive serum can be detected sensitively and accurately.

[0033] 1. Allows multiple samples to be tested simultaneously. Repeat the same sample multiple times, or samples taken at different time points to obtain dynamic values, or samples from different patients are tested simultaneously; in short, high-throughput testing is achieved. Overall, the testing cost is reduced and the testing efficiency is improved.

[0034] 3. The protein chip of the present invention requires significantly less blood sample and antibody, requiring only 7.5ul of serum to be tested. Each detection spot on the protein chip used to capture antibodies requires only 1-30 nanoliters of protein liquid, which is far lower than other similar methods, greatly reducing the testing cost.

[0035] 4. The invention uses HRP colorimetric solution to develop color and judge the experimental results. It does not require scanning equipment, is simple and visual, has low technical requirements for personnel, and is suitable for large-scale use. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 . A schematic diagram of a protein chip in the kit of the present invention;

[0037] Figure 2 . A schematic cross-sectional view of three detection spots in a single sub-detection area of ​​a protein chip in the kit of the present invention;

[0038] Figure 3 . Schematic diagram of the detection principle of the kit of the present invention;

[0039] Figure 4 . The result of the protein chip in the kit of the present invention detecting the serum of healthy people;

[0040] Figure 5 . The result diagram of the protein chip in the kit of the present invention detecting clinically confirmed serum;

[0041] Among them, 1-the first detection spot; 2-the second detection spot; 3-the third detection spot, 4-the sub-detection area. DETAILED DESCRIPTION

[0042] The present invention will be further described in detail below in conjunction with specific embodiments, but the scope of the present invention is not limited thereto. Unless otherwise specified, the operations used in the following examples are all conventional methods, and the reagents used can all be commercially available.

[0043] A protein chip kit for detecting antibodies against novel coronavirus, characterized in that it has at least one protein chip, such as Figure 1As shown, each of the protein chips has at least one sub-detection area 4, and each sub-detection area 4 is used to detect a specimen; each sub-detection area 4 is provided with the following detection spots: a first detection spot 1 formed by the attachment of BSA, a second detection spot 2 formed by the attachment of a novel coronavirus antigen, and a third detection spot 3 formed by the attachment of a human IgG polyclonal antibody or an IgM polyclonal antibody, and the detection spots are separated from each other, as shown in FIG. Figure 2 As shown; the new coronavirus antigen is the receptor binding domain protein of the new coronavirus spike protein S1 subunit or the new coronavirus spike protein S1 subunit protein, or a mixture thereof.

[0044] According to a preferred embodiment, in the kit, the novel coronavirus antigen on the second detection spot 2 is a mixture of the novel coronavirus antigen, the receptor binding domain protein of the novel coronavirus spike protein S1 subunit and the novel coronavirus spike protein S1 subunit protein, in a mass ratio of 1 to 3:1, preferably a mixture in a mass ratio of 1:1. Experimental data found that this type of chip solution significantly improved the detection sensitivity and accuracy compared with the single antigen solution.

[0045] According to a preferred embodiment, in the kit, at least one of the protein chips comprises two sub-detection areas, one of which has the third detection spot formed by the attachment of human IgG polyclonal antibodies; and the other sub-detection area has the third detection spot formed by the attachment of IgM polyclonal antibodies. Figure 1 Taking the chip shown as an example, the second detection spot in a part of the sub-detection area 4 is formed by the attachment of human IgG polyclonal antibody; the second detection spot in a part of the sub-detection area 4 is formed by the attachment of human IgM polyclonal antibody.

[0046] According to a preferred embodiment, the kit comprises two protein chips, wherein the third detection spot 3 on one protein chip is formed by the attachment of human IgG polyclonal antibodies; and the third detection spot 3 on the other protein chip is formed by the attachment of IgM polyclonal antibodies.

[0047] The test kits in the above two schemes can detect IgG antibodies and IgM antibodies in the serum of suspected patients, thereby improving the accuracy of detection.

[0048] According to a preferred embodiment, in the kit, in each of the sub-detection areas, each detection spot contains 3-6 repetitions, each detection spot is arranged in a row, and the three detection spots are arranged in a rectangular array.

[0049] According to a preferred embodiment, in the kit, the size of each sub-detection area is 5-10 mm in length and 5-10 mm in width, and the size of the detection spot is 0.5-1 mm in length and 0.5-1 mm in width.

[0050] According to a preferred embodiment, in the kit, the spacing distance between the detection spots is 0.2-1 mm.

[0051] According to a preferred embodiment, the kit further comprises an HRP-labeled anti-human IgG polyclonal antibody for use in conjunction with the sub-detection area to which the human IgG polyclonal antibody is attached to form the third detection spot, i.e., after the serum to be tested is dripped onto the detection spot in the sub-detection area, it is dripped onto and covers the detection spot;

[0052] It also contains HRP-labeled anti-human IgM polyclonal antibodies for use in conjunction with the sub-detection area with human IgM polyclonal antibodies attached to form the third detection spot, that is, after the serum to be tested is dripped on the detection spot in the sub-detection area, it is dripped onto and covers the detection spot.

[0053] According to a preferred embodiment, the kit further comprises 0.05% PBS-Tween solution and HRP color development substrate solution.

[0054] The present invention also provides a method for preparing any of the above-mentioned kits, characterized in that the preparation of the protein chip comprises the following steps:

[0055] Obtaining a chip substrate partitioned to form the sub-detection areas;

[0056] BSA, novel coronavirus antigen, human IgG polyclonal antibody or human IgM polyclonal antibody are spotted and fixed in the sub-detection area according to a preset pattern and size to form the first, second and third detection spots;

[0057] The novel coronavirus antigen is the receptor binding domain protein of the novel coronavirus spike protein S1 subunit or the novel coronavirus spike protein S1 subunit protein, or a mixture thereof.

[0058] According to a preferred preparation method, 10% BSA, 0.1-1 mg / ml novel coronavirus antigen, and 0.05-0.2 mg / ml human IgG polyclonal antibody or human IgM polyclonal antibody are spotted and fixed in the sub-detection area according to a preset pattern and size to form the first, second, and third detection spots.

[0059] According to a preferred preparation method, 0.5 mg / ml new coronavirus antigen, 0.1 mg / ml human IgG polyclonal antibody or human IgM polyclonal antibody, and 10% BSA are preferred.

[0060] According to a preferred preparation method, all detection spots in each sub-detection area use the same test volume, which is between 1-30 nL.

[0061] According to a preferred preparation method, the detection spot is formed by multiple non-contact micro-spotting, preferably by 3-10 non-contact micro-spotting, each spotting 300-600 pL.

[0062] Example 1 Protein chip preparation

[0063] Reagents used:

[0064] 10% BSA

[0065] Antigen 1: Receptor binding domain protein of the S1 subunit of the novel coronavirus spike protein: Product name: 2019-nCoVSpike Protein (RBD, Fc Tag), containing 457 amino acids, molecular weight 51.5 kDa, purchased from Beijing Sino Biological Technology Co., Ltd. Item No. 40592-V05H .

[0066] Antigen 2: Novel coronavirus spike protein S1 subunit protein: Product name: SARS-CoV-2 (2019-nCoV) Spike Protein (S1 Subunit, His Tag), containing 681 amino acids, molecular weight 76.5kDa, purchased from Beijing Sino Biological Technology Co., Ltd. Item No. 40591-V08B1 .

[0067] Human IgG at a concentration of 0.1 mg / ml was purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0068] Human IgM at a concentration of 0.1 mg / ml, R&D Systems, Inc.

[0069] HRP-labeled anti-human IgG was purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0070] HRP-labeled anti-human IgM was purchased from Beijing Sino Biological Technology Co., Ltd.;

[0071] PBS formula: sodium chloride (NaCl) 8g, potassium chloride (KCl) 0.2g, disodium hydrogen phosphate (Na2HPO4) 1.44g, potassium dihydrogen phosphate (KH2PO4) 0.24g, adjust pH to 7.4, constant volume 1L

[0072] PBST formula: 1L PBS, + 1ml Tween-20;

[0073] The chip substrate is an aldehyde-based chip purchased from Shanghai Bio-Tech Co., Ltd. Each chip contains 10 test grids, each grid detects one serum, and 10 serum samples are tested at a time.

[0074] Table 1. Chip production plan

[0075]

[0076] Each test spot is repeated four times and arranged in a row, such as Figure 1 .

[0077] The spotting was performed by non-contact micro-spotting, with a spotting volume of 30 nL for each detection spot, 600 pL for each spotting, and the spotting was completed by 5 times.

[0078] The spot volume of each detection spot is the same.

[0079] Example 2. Determination of detection method

[0080] The operation process and principle of the protein chip of the present invention are as follows: Figure 3 As shown, the steps are as follows:

[0081] 1. On the prepared chip, add one portion of patient serum (7ul) to each sub-detection area 4 and store at room temperature for 25 minutes to allow the novel coronavirus antibodies in the serum to bind to the novel coronavirus antigens on the chip;

[0082] 2. Wash the chip with 0.05% PBS-Tween for 5 seconds each time, 5 times to remove non-specific binding;

[0083] 3. When the second detection spot is formed by the attachment of human IgG polyclonal antibody, add HRP-labeled anti-human IgG; when the second detection spot is formed by the attachment of human IgM polyclonal antibody, add HRP-labeled anti-human IgM; store at room temperature for more than 10 minutes, preferably 15 minutes, to form a novel coronavirus antigen-novel coronavirus antibody-HRP-labeled anti-human IgG (or novel coronavirus antigen-novel coronavirus antibody-HRP-labeled anti-human IgM complex) and a human IgG-HRP-labeled anti-human IgG complex (or human IgM-HRP-labeled anti-human IgM complex) on the chip, wash the chip with 0.05% PBS-Tween, 5 seconds each time, wash 5 times, and clean non-specific binding;

[0084] 4. Add HRP color development substrate.

[0085] Expected results: For positive serum, the first test spot is not colored, the second test spot is tan, and the third test spot is tan. Figure 4 As shown, detection wells 1-2 and 4-10 are the test results of normal human serum, which are negative results. The BSA test spot and the new coronavirus antigen test spot have no color, and the human IgG / IgM test spot appears brown; detection well 3 is set as an unsampled detection well to verify that the detection wells are spaced from each other and will not be affected by other detection wells.

[0086] Experimental example: using the chip of scheme 3 to detect clinical serum of new coronavirus, the steps adopt the method determined in example 2:

[0087] The clinical serum came from the serum of 7 patients admitted to Zhejiang Hospital who were clinically diagnosed with new coronavirus pneumonia.

[0088] After 30 minutes of reaction, the results are as follows Figure 5 As shown, test wells 3, 4, and 5 are healthy serum, and the human IgG / IgM test spots are brown, indicating negative results;

[0089] Detection wells 1, 2, 6, 7, 9, and 10 are samples of sera from six patients with novel coronavirus pneumonia diluted by one half.

[0090] The results showed two rows of brown test spots, namely the new coronavirus antigen test spot and the human IgG / IgM test spot, which showed brown, indicating a positive result. Test well 8 was the serum of a patient with new coronavirus pneumonia, and only the human IgG / IgM test spot showed brown, showing a negative result. Among the patients diagnosed by clinical tests, there are indeed a small number of patients who do not produce antibodies in their bodies, which is a normal phenomenon.

[0091] The chips of schemes 4, 5, 8-10 in Table 1 tested the same batch of serum and obtained basically consistent results. However, the chips of schemes 1, 2, 6, and 7, i.e., single antigen detection spots, showed weak color development.

[0092] Limited by the sample size, the detection rate of the kit of the present invention is 85%; if the sample size is increased, it is expected to reach more than 90%.

Claims

1. A protein chip kit for detecting novel coronavirus antibodies, characterized in that: There is at least one protein chip, each of which has at least one sub-detection area, and each sub-detection area is used to detect a sample; The following detection spots are set in each sub-detection area: The first detection spot formed by BSA attachment is a negative control spot, and no color is developed when the chip is valid; The second detection spot is formed by the attachment of the novel coronavirus antigen, and the color development indicates a positive result; The third detection spot formed by the attachment of human IgG polyclonal antibody or IgM polyclonal antibody is a positive control, and the color development indicates that the chip is effective; The detection spots are separated from each other, and the interval between the detection spots is 0.2 to 1 mm; The novel coronavirus antigen is: a receptor binding domain protein of the novel coronavirus spike protein S1 subunit, a novel coronavirus spike protein S1 subunit protein, or a mixture thereof in a mass ratio of 1 to 3:1; The receptor binding domain protein of the novel coronavirus spike protein S1 subunit comprises at least 457 amino acids; The novel coronavirus spike protein S1 subunit protein contains at least 681 amino acids; The kit also contains HRP-labeled anti-human IgG polyclonal antibodies for use in conjunction with the sub-detection area to which the human IgG polyclonal antibodies are attached to form the third detection spot, i.e., for dripping the test serum onto the detection spot in the sub-detection area and then dripping onto and covering the detection spot; The kit also contains HRP-labeled anti-human IgM polyclonal antibodies for use in conjunction with the sub-detection area to which the human IgM polyclonal antibodies are attached to form the third detection spot, i.e., after the serum to be tested is dripped on the detection spot in the sub-detection area, it is dripped onto and covers the detection spot.

2. The protein chip kit according to claim 1, characterized in that: The new coronavirus antigen is: a mixture of the receptor binding domain protein of the new coronavirus spike protein S1 subunit and the new coronavirus spike protein S1 subunit protein in a 1:1 mass ratio.

3. The protein chip kit according to claim 1, characterized in that: At least one of the protein chips comprises two sub-detection areas, one of which has the third detection spot formed by the attachment of human IgG polyclonal antibodies; and the other sub-detection area has the third detection spot formed by the attachment of IgM polyclonal antibodies.

4. The protein chip kit according to claim 1, characterized in that It comprises two protein chips, wherein the third detection spot on one protein chip is formed by the attachment of human IgG polyclonal antibody; and the third detection spot on the other protein chip is formed by the attachment of IgM polyclonal antibody.

5. The kit according to claim 1, characterized in that In each of the sub-detection areas, 3 to 6 detection spots of each type are provided, the same detection spots are arranged in a row, and the three detection spots are arranged in a rectangular array.

6. The kit according to claim 1, characterized in that The size of each sub-detection area is 5-10 mm in length and 5-10 mm in width, and the size of the detection spot is 0.5-1 mm in length and 0.5-1 mm in width.

7. The kit according to claim 1, characterized in that It also contains 0.05% PBS-Tween solution and HRP color development substrate solution.

8. The method for preparing the kit according to any one of claims 1 to 7, characterized in that: The preparation of the protein chip comprises the following steps: Obtaining a chip substrate partitioned to form the sub-detection areas; BSA, new coronavirus antigen, human IgG polyclonal antibody or human IgM polyclonal antibody are spotted and fixed in the sub-detection area according to a preset pattern and size to form the first, second and third detection spots; the new coronavirus antigen is the receptor binding domain protein of the new coronavirus spike protein S1 subunit or the new coronavirus spike protein S1 subunit protein, or a mixture thereof.

9. The preparation method according to claim 8, characterized in that: According to the preset pattern and size, 10% BSA, 0.1-1 mg / ml new coronavirus antigen, and 0.05-0.2 mg / ml human IgG polyclonal antibody or human IgM polyclonal antibody are spotted and fixed in the sub-detection area to form the first, second, and third detection spots.

10. The preparation method according to claim 8 or 9, characterized in that: All detection spots in each sub-detection area used the same test volume, which was between 1 and 30 nL. The detection spot is formed by 3-10 non-contact micro-spotting, with 300-600 pL spotted each time.

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