Alkaline Phosphatase Protein Chip Kit for Detecting New Coronavirus Antibodies and Its Preparation Method

Abstract

The present invention, "Alkaline Phosphatase Protein Chip Kit for Detecting Novel Coronavirus Antibodies and Its Preparation Method", relates to virus detection technology, and is characterized in that it has at least one protein chip, each protein chip has at least one sub-detection area, and each sub-detection area is used to detect one specimen; the following several detection spots are arranged in each sub-detection area: the first detection spot formed by the attachment of BSA, the second detection spot formed by the attachment of novel coronavirus antigen, and the third detection spot formed by the attachment of human IgG polyclonal antibody or IgM polyclonal antibody; the detection spots are separated from each other; the novel coronavirus antigen is the receptor-binding domain protein of the S1 subunit of the novel coronavirus spike protein or the S1 subunit protein of the novel coronavirus spike 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 an alkaline phosphatase novel coronavirus IgM / IgG antibody detection kit, which can quickly and cheaply detect novel coronavirus antibodies with a small amount of serum (7 microliters). The test results can be issued within 60 minutes, and 10 people can be tested at a time, with an average time of 6 minutes per person. It can be operated manually or on a computer.

[0005] The technical solution of the present invention is as follows:

[0006] A protein chip kit for detecting novel coronavirus antibodies, characterized in that it has at least one protein chip

[0007] Each of the protein chips has at least one sub-detection area, and each sub-detection area is used to detect a sample;

[0008] The following detection spots are set in each sub-detection area:

[0009] The first detection spot formed by BSA attachment

[0010] The second detection spot formed by the attachment of the new coronavirus antigen,

[0011] The third detection plaque is formed by the attachment of human IgG polyclonal antibody or IgM polyclonal antibody.

[0012] The detection spots are separated from each other;

[0013] The novel coronavirus antigen is a receptor binding domain protein of the novel coronavirus spike protein S1 subunit or a novel coronavirus spike protein S1 subunit protein, or a mixture thereof;

[0014] And when the third detection spot is formed by the attachment of polyclonal IgG antibodies on the protein chip, it further includes alkaline phosphatase-labeled anti-human IgG polyclonal antibodies; when the third detection spot is formed by the attachment of polyclonal IgM antibodies on the protein chip, it further includes alkaline phosphatase-labeled anti-human IgM polyclonal antibodies.

[0015] The second detection spot is formed by the attachment of a novel coronavirus antigen with a concentration of 0.1 - 1 mg / ml;

[0016] The third detection spot is formed by the attachment of polyclonal human IgG antibodies or polyclonal IgM antibodies with a concentration of 0.05 - 0.2 mg / ml.

[0017] According to a preferred embodiment, in the kit, the novel coronavirus antigen is a mixture of the receptor-binding domain protein of the spike protein S1 subunit of the novel coronavirus and the spike protein S1 subunit protein of the novel coronavirus at a mass ratio of 1 - 3:1, preferably a mixture at a mass ratio of 1:1. This significantly improves the detection sensitivity and accuracy.

[0018] According to a preferred embodiment, in the kit, on at least one of the protein chips, it includes two sub-detection areas. One sub-detection area has the third detection spot formed by the attachment of polyclonal human IgG antibodies; the other sub-detection area has the third detection spot formed by the attachment of polyclonal IgM antibodies. According to a preferred embodiment, in the kit, it includes two of the protein chips. The third detection spot on one protein chip is formed by the attachment of polyclonal human IgG antibodies; the third detection spot on the other protein chip is formed by the attachment of polyclonal IgM antibodies.

[0019] In the kits of the above two schemes, the IgG antibody and IgM antibody in the serum of suspected patients can be detected simultaneously, improving the detection accuracy.

[0020] 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 column, and the three detection spots are arranged in a rectangular array.

[0021] 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.

[0022] According to a preferred embodiment, in the kit, the interval distance between the detection spots is 0.2 - 1 mm.

[0023] According to a preferred embodiment, the kit further comprises an alkaline phosphatase-labeled anti-human IgG polyclonal antibody, which is used in combination with a sub-detection area having a human IgG polyclonal antibody attached thereto to form the third detection spot, that is, after adding the serum to be tested to the detection spot in the sub-detection area, it is added dropwise to and covers the detection spot.

[0024] It further comprises an alkaline phosphatase-labeled anti-human IgM polyclonal antibody, which is used in combination with a sub-detection area having a human IgM polyclonal antibody attached thereto to form the third detection spot, that is, after adding the serum to be tested to the detection spot in the sub-detection area, it is added dropwise to and covers the detection spot.

[0025] According to a preferred embodiment, the kit further comprises a 0.05% PBS-Tween solution and an alkaline phosphatase chromogenic substrate solution.

[0026] The preparation method of any of the above-mentioned kits is characterized in that the preparation of the protein chip comprises the following steps:

[0027] Obtain a chip substrate with the sub-detection area set by zoning;

[0028] According to a preset pattern and size, BSA, novel coronavirus antigen, 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;

[0029] The novel coronavirus antigen is the receptor-binding domain protein of the spike protein S1 subunit of the novel coronavirus or the spike protein S1 subunit protein of the novel coronavirus, or a mixture thereof.

[0030] According to a preferred preparation method, 10% BSA, 0.1 - 1 mg / ml novel coronavirus antigen, 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.

[0031] According to a preferred preparation method, 0.5 mg / ml novel coronavirus antigen, 0.1 mg / ml human IgG polyclonal antibody or human IgM polyclonal antibody, and 10% BSA are preferred.

[0032] According to a preferred preparation method, all detection spots in each sub-detection area are spotted with the same volume of the test sample, and the volume is between 1 - 30 nL.

[0033] According to a preferred preparation method, the detection spots are formed by multiple non-contact micro-sampling, preferably by 3 - 10 times of non-contact micro-sampling, and 300 - 600 pL is sampled each time.

[0034] The present invention provides a simple, feasible, economical, rapid, practical and reliable protein chip method for detecting IgM / IgG antibodies in the serum of patients infected with novel coronavirus. The present invention only requires a small amount of serum (7 μL), can detect multiple samples at one time, provides the detection results within 60 minutes, can qualitatively detect the novel coronavirus IgM / IgG antibodies in human serum or plasma, and has accurate detection results and high sensitivity.

[0035] Advantages of the protein chip kit of the present invention:

[0036] First, the present invention selects the antigens of novel coronavirus, selects the antigen fragments and antigen combination schemes suitable for making micro protein chips, and the clinical detection data shows that positive serum can be detected sensitively and accurately.

[0037] Second, it allows multiple samples to be detected simultaneously. Repeat the same sample multiple times, or samples taken at different time points to obtain dynamic values, or samples from different patients are detected simultaneously; in short, high-throughput detection is achieved. Overall, the detection cost is reduced and the detection efficiency is improved.

[0038] Third, the protein chip of the present invention requires much less blood sample and antibody. 7.5 μL of serum is required for detection, and for the detection spots on the protein chip used to capture antibodies, only 1 - 30 nanoliters of protein liquid is required for each detection spot, which is much lower than other similar methods, greatly reducing the detection cost.

[0039] Fourth, the present invention uses an alkaline phosphatase chromogenic solution to develop color to judge the experimental results, does not require a scanning instrument, is simple and visual, has low requirements for personnel technology, and is suitable for large-scale use. Description of the Drawings

[0040] Figure 1 . Schematic plan view of the protein chip in the kit of the present invention;

[0041] Figure 2 . Schematic cross-sectional view of three detection spots in a single sub-detection area of the protein chip in the kit of the present invention;

[0042] Figure 3 . Schematic view of a single sub-detection area of the protein chip in the kit of the present invention;

[0043] Figure 4 . Schematic view of the protein chip sample in the kit of the present invention;

[0044] Figure 5 . Schematic view of the detection principle of the kit of the present invention;

[0045] Figure 6 . Results graph of detecting healthy human serum with the protein chip in the kit of the present invention;

[0046] Figure 7 . Results graph of detecting clinically diagnosed serum with the protein chip in the kit of the present invention;

[0047] Wherein 1 - the first detection spot; 2 - the second detection spot; 3 - the third detection spot, 4 - sub - detection area. Detailed implementation manners

[0048] The present invention will be further described in detail below in conjunction with the detailed implementation manners, but the scope of the present invention is not limited. Unless otherwise specified, the operations used in the following examples are conventional methods, and the reagents used can be obtained commercially.

[0049] A protein chip kit for detecting novel coronavirus antibodies, characterized in that it has at least one protein chip, as Figure 1 shown. Each said protein chip has at least one sub - detection area 4, and each sub - detection area 4 is used to detect one specimen; the following several detection spots are arranged in each sub - detection area 4: the first detection spot 1 formed by the attachment of BSA, the second detection spot 2 formed by the attachment of novel coronavirus antigen, the third detection spot 3 formed by the attachment of human IgG polyclonal antibody or IgM polyclonal antibody, and the detection spots are separated from each other, as Figure 2 , shown in 3; the said novel coronavirus antigen is the receptor - binding domain protein of the S1 subunit of the novel coronavirus spike protein or the S1 subunit protein of the novel coronavirus spike protein, or a mixture thereof;

[0050] And when the third detection spot is formed by the attachment of IgG polyclonal antibody on the protein chip, it further includes an alkaline phosphatase - labeled anti - human IgG polyclonal antibody; when the third detection spot is formed by the attachment of IgM polyclonal antibody on the protein chip, it further includes an alkaline phosphatase - labeled anti - human IgM polyclonal antibody.

[0051] According to a preferred embodiment, in the said kit, the novel coronavirus antigen on the second detection spot 2 is a mixture of the receptor - binding domain protein of the S1 subunit of the novel coronavirus and the S1 subunit protein of the novel coronavirus at a mass ratio of 1 - 3:1, preferably a mixture at a mass ratio of 1:1. Experimental data shows that this kind of chip scheme significantly improves the detection sensitivity and accuracy compared with the single - antigen scheme.

[0052] According to a preferred embodiment, in the kit, on at least one of the protein chips, there are two sub-detection regions. One of the sub-detection regions has the third detection spot formed by the attachment of polyclonal antibodies of human IgG; the other sub-detection region has the third detection spot formed by the attachment of polyclonal antibodies of IgM. In this scheme, taking the chip shown by Figure 1 as an example, the second detection spots in a part of the sub-detection region 4 are formed by the attachment of polyclonal antibodies of human IgG; the second detection spots in a part of the sub-detection region 4 are formed by the attachment of polyclonal antibodies of human IgM.

[0053] According to a preferred embodiment, in the kit, there are two of the protein chips. The third detection spot 3 on one of the protein chips is formed by the attachment of polyclonal antibodies of human IgG; the third detection spot 3 on the other protein chip is formed by the attachment of polyclonal antibodies of IgM.

[0054] In the kits of the above two schemes, IgG antibodies and IgM antibodies in the serum of suspected patients can be detected, improving the detection accuracy.

[0055] According to a preferred embodiment, in the kit, in each of the sub-detection regions, each detection spot contains 3 - 6 repetitions, each detection spot is arranged in a column, and the three detection spots are arranged in a rectangular array.

[0056] According to a preferred embodiment, in the kit, the size of each of the sub-detection regions 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.

[0057] According to a preferred embodiment, in the kit, the spacing distance between the detection spots is 0.2 - 1 mm.

[0058] According to a preferred embodiment, in the kit, it also contains alkaline phosphatase-labeled anti-human IgG polyclonal antibodies, which are used in cooperation with the sub-detection region having the third detection spot formed by the attachment of polyclonal antibodies of human IgG, that is, after adding the serum to be tested to the detection spots in the sub-detection region, it is added to and covers the detection spots;

[0059] It also contains alkaline phosphatase-labeled anti-human IgM polyclonal antibodies, which are used in cooperation with the sub-detection region having the third detection spot formed by the attachment of polyclonal antibodies of human IgM, that is, after adding the serum to be tested to the detection spots in the sub-detection region, it is added to and covers the detection spots.

[0060] According to a preferred embodiment, in the kit, it also contains 0.05% PBS-Tween solution and alkaline phosphatase chromogenic substrate solution.

[0061] 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:

[0062] Obtaining a chip substrate with the sub-detection areas set by partitioning;

[0063] Spotting and immobilizing BSA, novel coronavirus antigen, human IgG polyclonal antibody or human IgM polyclonal antibody in the sub-detection areas according to a preset pattern and size to form the first, second, and third detection spots;

[0064] The novel coronavirus antigen is the receptor-binding domain protein of the S1 subunit of the novel coronavirus spike protein or the S1 subunit protein of the novel coronavirus spike protein, or a mixture thereof.

[0065] According to a preferred preparation method, 10% BSA, 0.1-1 mg / ml novel coronavirus antigen, 0.05-0.2 mg / ml human IgG polyclonal antibody or human IgM polyclonal antibody are spotted and immobilized in the sub-detection areas according to a preset pattern and size to form the first, second, and third detection spots.

[0066] According to a preferred preparation method, 0.5 mg / ml novel coronavirus antigen, 0.1 mg / ml human IgG polyclonal antibody or human IgM polyclonal antibody, and 10% BSA are preferred.

[0067] According to a preferred preparation method, all the detection spots in each sub-detection area use the same volume of the test sample, and the volume is between 1-30 nL.

[0068] According to a preferred preparation method, the detection spots are formed by multiple non-contact micro-spotting, preferably formed by 3-10 times of non-contact micro-spotting, and each spotting is 300-600 pL.

[0069] Example 1 Preparation of Protein Chip

[0070] Reagents used:

[0071] 10% BSA

[0072] Antigen 1: Receptor-binding domain protein of the S1 subunit of the novel coronavirus spike protein: Product name: 2019-nCoV Spike Protein (RBD, Fc Tag), containing 457 amino acids, molecular weight 51.5 kDa, purchased from Beijing Sino Biological Inc., Item No. 40592-V05H .

[0073] Antigen 2: Spike protein S1 subunit of novel coronavirus: Product name: SARS-CoV-2 (2019-nCoV) Spike Protein (S1 Subunit, His Tag), containing 681 amino acids, with a molecular weight of 76.5 kDa, purchased from Sino Biological Inc., Item No. 40591-V08B1 .

[0074] Human IgG with a concentration of 0.1 mg / ml, purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0075] Human IgM with a concentration of 0.1 mg / ml, from R&D Systems, Inc.

[0076] Alkaline phosphatase-labeled anti-human IgG, purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0077] Alkaline phosphatase-labeled anti-human IgM, purchased from Sangon Biotech (Shanghai) Co., Ltd.;

[0078] PBS formulation: Sodium chloride (NaCl) 8 g, potassium chloride (KCl) 0.2 g, disodium hydrogen phosphate (Na2HPO4) 1.44 g, potassium dihydrogen phosphate (KH2PO4) 0.24 g, adjust pH to 7.4, make up the volume to 1 L

[0079] PBST formulation: 1 L of PBS + 1 ml of Tween-20;

[0080] The chip substrate is an aldehyde chip, purchased from Shanghai Bioaotest Biotechnology Co., Ltd. Each chip contains 10 detection squares, and each square detects one serum sample, so 10 serum samples can be detected at a time.

[0081] Table 1. Chip production scheme

[0082]

[0083] Each detection spot reagent is spotted into 1 column, and there are four repeated detection spots in each column, such as Figure 1 .

[0084] Among them, non-contact micro-spotting is used for spotting. The spotting volume of each detection spot is 30 nL, and 600 pL is spotted each time. The spotting is completed in 5 times.

[0085] The spotting volume of each detection spot is the same.

[0086] Example 2. Determination of the detection method

[0087] The operation process and principle of the protein chip of the present invention are as shown in Figure 5 and the steps are as follows:

[0088] 1. On the prepared chip, add a portion of patient serum (7 μl) to each sub-detection area 4, and store it at room temperature for 15 minutes to allow the novel coronavirus antibody in the serum to bind to the novel coronavirus antigen on the chip;

[0089] 2. Wash the chip with 0.05% PBS-Tween, 5 s each time, for 5 times to wash away non-specific binding;

[0090] 3. When the second detection spot is formed by the attachment of human IgG polyclonal antibody, add alkaline phosphatase-labeled anti-human IgG; when the second detection spot is formed by the attachment of human IgM polyclonal antibody, add alkaline phosphatase-labeled anti-human IgM; store at room temperature for 15 - 25 minutes to form a novel coronavirus antigen - novel coronavirus antibody - alkaline phosphatase-labeled anti-human IgG (or novel coronavirus antigen - novel coronavirus antibody - alkaline phosphatase-labeled anti-human IgM complex) and human IgG - alkaline phosphatase-labeled anti-human IgG complex (or human IgM - alkaline phosphatase-labeled anti-human IgM complex) on the chip, and then wash the chip with 0.05% PBS-Tween, 5 s each time, for 5 times to wash away non-specific binding;

[0091] 4. Add alkaline phosphatase chromogenic substrate NBT / BCIP.

[0092] Expected results: For positive serum, the first detection spot does not show color, the second detection spot shows purple-blue, and the third detection spot shows purple-blue. As Figure 6 shown, the detection holes 1 - 10 are all the test results of normal human serum, belonging to negative results. The BSA detection spot and the novel coronavirus antigen detection spot do not show color, and the human IgG / IgM detection spot shows purple-blue.

[0093] Experimental example: Use the chip of Scheme 3 to detect the clinical serum of novel coronavirus, and the steps are carried out by the method determined in Example 2:

[0094] The clinical serum is from the serum of 9 patients infected with novel coronavirus admitted to Zhejiang Hospital.

[0095] The results are as Figure 7 shown. The detection hole 10 is healthy serum, and the human IgG / IgM detection spot shows purple-blue, which is a negative result;

[0096] The detection holes 1 - 9 are the serum of patients infected with novel coronavirus. The results show two rows of purple-blue detection spots, that is, the novel coronavirus antigen detection spot and the human IgG / IgM detection spot show purple-blue, which is a positive result.

[0097] The chips of other schemes also obtain the same qualitative results.

Claims

1. A protein chip kit for detecting novel coronavirus antibodies, characterized in that, Having at least one protein chip; Each of the protein chips has at least one sub-detection area, and each sub-detection area is used to detect a specimen; the following types of detection spots are arranged in each sub-detection area: The first detection spot formed by the attachment of BSA; The second detection spot formed by the attachment of the novel coronavirus antigen; The third detection spot formed by the attachment of the polyclonal antibody of human IgG labeled with alkaline phosphatase or the polyclonal antibody of IgM labeled with alkaline phosphatase; The detection spots are separated from each other; The novel coronavirus antigen is the receptor-binding domain protein of the S1 subunit of the novel coronavirus spike protein or the S1 subunit protein of the novel coronavirus spike protein, or a mixture thereof.

2. The protein chip kit according to claim 1, characterized in that, At least one of the protein chips contains two sub-detection areas: One of the sub-detection areas has the third detection spot formed by the attachment of the polyclonal antibody of human IgG; The other sub-detection area has the third detection spot formed by the attachment of the polyclonal antibody of IgM; When the third detection spot formed by the attachment of the polyclonal antibody of human IgG is present on the protein chip, the polyclonal antibody of anti-human IgG labeled with alkaline phosphatase is added; When the third detection spot formed by the attachment of the polyclonal antibody of IgM is present on the protein chip, the polyclonal antibody of anti-human IgM labeled with alkaline phosphatase is added; The concentrations of the polyclonal antibody of human IgG and the polyclonal antibody of IgM are 0.05 - 0.2 mg / ml.

3. The protein chip kit according to claim 1, characterized in that, The second detection spot is formed by the attachment of the novel coronavirus antigen with a concentration of 0.1 - 1 mg / ml.

4. The protein chip kit according to claim 1, characterized in that, The novel coronavirus antigen is a mixture of the receptor-binding domain protein of the S1 subunit of the novel coronavirus spike protein and the S1 subunit protein of the novel coronavirus spike protein in a mass ratio of 1 - 3:

1.

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

6. The kit according to claim 1, characterized in that, In each of the sub-detection areas, there are 3 - 6 of each type of detection spot, the same detection spots are arranged in a column, and the three detection spots are arranged in a rectangular array; 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, The spacing distance between the detection spots is 0.2 - 1 mm.

8. The kit according to any one of claims 1-7, characterized in that, It also contains a 0.05% PBS-Tween solution and an alkaline phosphatase chromogenic substrate solution.

9. A preparation method of the kit according to any one of claims 1 to 8, characterized in that, The preparation of the protein chip includes the following steps: Obtaining a chip substrate with the sub-detection areas set by zoning; Spotting and fixing BSA, the novel coronavirus antigen, the polyclonal antibody of human IgG or the polyclonal antibody of human IgM in the sub-detection areas according to a preset pattern and size to form the first, second, and third detection spots; The novel coronavirus antigen is the receptor-binding domain protein of the S1 subunit of the novel coronavirus spike protein or the S1 subunit protein of the novel coronavirus spike protein, or a mixture thereof.

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

11. The preparation method according to claim 10, characterized in that, All the detection spots in each sub-detection area use the same volume of the test sample, and the volume is between 1 - 30 nL; The detection spots are formed by 3 - 10 times of non-contact micro-spotting, and each time the spotting volume is 300 - 600 pL.

Images