CN212459720U - Alkaline phosphatase protein chip kit for detecting novel coronavirus antibody - Google Patents
Alkaline phosphatase protein chip kit for detecting novel coronavirus antibody Download PDFInfo
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
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Abstract
The utility model discloses an alkaline phosphatase protein chip kit for detecting novel coronavirus antibody, which relates to the virus detection technology and is characterized in that the kit is provided with at least one protein chip, each protein chip is provided with at least one sub-detection area, and each sub-detection area is used for detecting a sample; the following detection spots are arranged in each sub-detection area: a first detection spot formed by BSA attachment, a second detection spot formed by novel coronavirus antigen attachment, and a third detection spot formed by human IgG polyclonal antibody or IgM polyclonal antibody attachment; the detection spots are separated from each other; the novel coronavirus antigen is a receptor binding domain protein of a novel coronavirus spike protein S1 subunit or a novel coronavirus spike protein S1 subunit protein, or a mixture thereof.
Description
Technical Field
The utility model relates to a virus infection diagnosis technology, in particular to a protein chip kit for detecting novel coronavirus antibodies.
Background
Coronaviruses are associated with a variety of diseases in humans and animals, and can cause diseases in the respiratory, digestive and nervous systems of humans and animals. The diameter of the virus particle is 60-200nm, and the virus particle is spherical or elliptical and has polymorphism. The virus has an envelope with spinous processes present, and the spike glycoprotein is the receptor binding site and the primary antigenic site.
The virus antibody condition in the body of a suspected patient is detected by serology, the fever patient caused by other pathogens can be eliminated more accurately and quickly with low cost, the clinical diagnosis and screening efficiency is greatly improved, and the defects of high technical requirement and high false negative ratio of nucleic acid detection can be overcome.
SUMMERY OF THE UTILITY MODEL
Based on the demand in above-mentioned field, the utility model provides a novel coronavirus IgM/IgG antibody detect reagent box of alkaline phosphatase can be fast, low-cost, trace serum (7 microlitre) detect novel coronavirus antibody. The detection result can be presented within 60 minutes, 10 persons can be detected at one time, and the average time of each person is 6 minutes. The operation can be carried out by hand or on a machine.
The technical scheme of the utility model as follows:
a protein chip kit for detecting novel coronavirus antibodies, which is characterized by comprising at least one protein chip;
each protein chip is provided with at least one sub-detection area, and each sub-detection area is used for detecting a sample;
the following detection spots are arranged in each sub-detection area:
a first detection spot formed by the attachment of BSA,
a second detection spot formed by attachment of a novel coronavirus antigen,
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 novel coronavirus antigen is a receptor binding domain protein of a novel coronavirus spike protein S1 subunit or a novel coronavirus spike protein S1 subunit protein, or a mixture thereof;
when the third detection spot is formed by the IgG polyclonal antibody attached on the protein chip, the protein chip also comprises an anti-human IgG polyclonal antibody marked by alkaline phosphatase; when the protein chip is provided with the third detection spot formed by the IgM polyclonal antibody, the protein chip also comprises an anti-human IgM polyclonal antibody marked by alkaline phosphatase.
The second detection plaque is formed by attaching a novel coronavirus antigen with the concentration of 0.1-1 mg/ml;
the third detection spot is formed by attaching human IgG polyclonal antibody or IgM polyclonal antibody with the concentration of 0.05-0.2 mg/ml.
According to a preferred embodiment, in the kit, the novel coronavirus antigen is a receptor binding domain protein of a novel coronavirus spike protein S1 subunit and a novel coronavirus spike protein S1 subunit protein of 1 to 3: 1 mass ratio of the mixture, preferably 1:1 mass ratio of the mixture. The detection sensitivity and accuracy are obviously improved.
According to a preferred embodiment, at least one of said protein chips in said kit comprises two subregions, wherein one of the subregions has said third detection spot formed by the attachment of a human IgG polyclonal antibody; the other of the subregions has the third detection spot formed by the IgM polyclonal antibody attachment. According to a preferred embodiment, the kit comprises two protein chips, wherein the third detection spot on one protein chip is formed by the attachment of a human IgG polyclonal antibody; the third detection spot of the other protein chip is formed by IgM polyclonal antibody attachment.
The kit in the two schemes can simultaneously detect the IgG antibody and the IgM antibody for the serum of a suspected patient, and improve the detection accuracy.
According to a preferred embodiment, in said kit, each detection spot comprises 3 to 6 replicates in each of said sub-detection zones, each detection spot being aligned in a column and the three detection spots being aligned in a rectangular array.
According to a preferred embodiment, in the kit, each of the sub-detection regions has a size of 5 to 10mm in length and 5 to 10mm in width, and the detection spot has a size of 0.5 to 1mm in length and 0.5 to 1mm in width.
According to a preferred embodiment, in the kit, the detection spots are spaced apart by a distance of 0.2 to 1 mm.
According to a preferred embodiment, the kit further comprises an anti-human IgG polyclonal antibody marked by alkaline phosphatase, and the anti-human IgG polyclonal antibody is used for matching with the sub-detection area with the human IgG polyclonal antibody to form the third detection spot, namely the anti-human IgG polyclonal antibody is used for dropwise adding the serum to be detected on the detection spot in the sub-detection area and then dropwise adding the serum to be detected on and covering the detection spot;
the kit also comprises an alkaline phosphatase-labeled anti-human IgM polyclonal antibody which is used for being matched with the sub-detection area with the human IgM polyclonal antibody attached to form the third detection spot for use, namely, after the serum to be detected is dripped on the detection spot in the sub-detection area, the serum to be detected is dripped on and covers the detection spot.
According to a preferred embodiment, the kit further comprises a 0.05% PBS-Tween solution and an alkaline phosphatase chromogenic substrate solution.
The preparation method of any one of the above kits is characterized in that the preparation of the protein chip comprises the following steps:
obtaining a chip substrate which is partitioned to form the sub-detection area;
spotting and fixing BSA, novel coronavirus antigens, human IgG polyclonal antibodies or human IgM polyclonal antibodies in the sub-detection area according to a preset pattern and size to form the first detection spot, the second detection spot and the third detection spot;
the novel coronavirus antigen is a receptor binding domain protein of a novel coronavirus spike protein S1 subunit or a novel coronavirus spike protein S1 subunit protein, or a mixture thereof.
According to a preferred preparation method, 10% BSA, 0.1-1mg/ml of a novel coronavirus antigen, 0.05-0.2mg/ml of a human IgG polyclonal antibody or a human IgM polyclonal antibody are spotted and fixed in the sub-detection region according to a preset pattern and size to form the first, second and third detection spots.
According to a preferred preparation method, 0.5mg/ml of novel coronavirus antigen, 0.1mg/ml of human IgG polyclonal antibody or human IgM polyclonal antibody, 10% BSA are preferred.
According to a preferred preparation method, all detection spots within each sub-detection zone use the same volume of test obtained and are between 1 and 30 nL.
According to a preferred preparation method, the detection spot is formed by multiple non-contact microspotting, preferably 3-10 non-contact microspots, each spotting 300-600 pL.
The utility model provides a simple and easy, economy is quick, the protein chip method of reliable again is practical detects the IgM/IgG antibody in novel coronavirus pneumonia serum, the utility model discloses only need trace serum (7 ul), once detectable many people, issue the testing result within 60 minutes, can qualitatively detect the novel coronavirus IgM/IgG antibody in human serum or the plasma, the testing result is accurate, and sensitivity is high.
The utility model discloses protein chip kit's advantage:
the utility model discloses select new coronavirus antigen, select antigen fragment and the antigen combination scheme that is fit for making trace protein chip, clinical testing data shows that positive serum can be detected to sensitive accuracy.
And secondly, allowing simultaneous detection of multiple samples. Repeating the same sample for multiple times, or samples taken at different time points to obtain dynamic values, or samples from different patients are tested simultaneously; in summary, high throughput detection is achieved. The detection cost is reduced and the detection efficiency is improved as a whole.
Thirdly, the utility model discloses a blood sample and antibody volume that the albumen chip needs all very reduce, need detect serum volume 7.5ul, are used for catching the detection spot of antibody on the albumen chip, and every detection spot only needs protein liquid 1-30 nanoliter, is less than other methods of the same kind far away, greatly reduced the detection cost.
The method utilizes the alkaline phosphatase developing solution to carry out color development judgment on experimental results, does not need a scanning instrument, is simple and visual, has low requirements on personnel technology, and is suitable for large-area use.
Drawings
FIG. 1 is a schematic plan view of a protein chip in the kit of the present invention;
FIG. 2 is a schematic cross-sectional view of three detection spots in a single sub-detection area in a protein chip of the kit of the present invention;
FIG. 3 is a schematic diagram of a single sub-detection zone in a protein chip of the kit of the present invention;
FIG. 4 is a schematic diagram of a protein chip sample in the kit of the present invention;
FIG. 5 is a schematic view of the detection principle of the kit of the present invention;
FIG. 6 is a result diagram of the detection of the serum of healthy human by the protein chip in the kit of the present invention;
FIG. 7 is a result chart of the detection of clinically confirmed serum by the protein chip in the kit of the present invention;
wherein 1-first detection spot; 2-a second detection spot; 3-third detection spot, 4-subdetection zone.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments, but the scope of the invention is not limited thereto. Unless otherwise specified, the procedures used in the following examples are conventional, and all reagents used are commercially available.
A protein chip kit for detecting novel coronavirus antibodies, comprising at least one protein chip, as shown in FIG. 1, each of said protein chips having at least one sub-detection region 4, each sub-detection region 4 being for detecting a specimen; the following detection spots are arranged in each sub-detection area 4: a first detection spot 1 formed by BSA (bovine serum albumin) attachment, a second detection spot 2 formed by novel coronavirus antigen attachment, a third detection spot 3 formed by human IgG polyclonal antibody or IgM polyclonal antibody attachment, wherein the detection spots are separated from each other, as shown in figures 2 and 3; the novel coronavirus antigen is a receptor binding domain protein of a novel coronavirus spike protein S1 subunit or a novel coronavirus spike protein S1 subunit protein, or a mixture thereof;
when the third detection spot is formed by the IgG polyclonal antibody attached on the protein chip, the protein chip also comprises an anti-human IgG polyclonal antibody marked by alkaline phosphatase; when the protein chip is provided with the third detection spot formed by the IgM polyclonal antibody, the protein chip also comprises an anti-human IgM polyclonal antibody marked by alkaline phosphatase.
According to a preferred embodiment, the novel coronavirus antigen on the second plaque 2 in the kit is a receptor binding domain protein of the novel coronavirus spike protein S1 subunit and a receptor binding domain protein of the novel coronavirus spike protein S1 subunit proteins expressed as 1 to 3: 1 mass ratio of the mixture, preferably 1:1 mass ratio of the mixture. Experimental data shows that the chip scheme remarkably improves the detection sensitivity and accuracy compared with a single antigen scheme.
According to a preferred embodiment, at least one of said protein chips in said kit comprises two subregions, wherein one of the subregions has said third detection spot formed by the attachment of a human IgG polyclonal antibody; the other of the subregions has the third detection spot formed by the IgM polyclonal antibody attachment. In this embodiment, taking the chip shown in FIG. 1 as an example, the second detection spot in a part of the sub-detection region 4 is formed by the attachment of a human IgG polyclonal antibody; a part of the second detection spot in the sub-detection region 4 is formed by the attachment of the human IgM polyclonal antibody.
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 a human IgG polyclonal antibody; the third detection spot 3 of the other protein chip is formed by IgM polyclonal antibody attachment.
The kit in the two schemes can detect IgG antibodies and IgM antibodies for blood serum of suspected patients, and the detection accuracy is improved.
According to a preferred embodiment, in said kit, each detection spot comprises 3 to 6 replicates in each of said sub-detection zones, each detection spot being aligned in a column and the three detection spots being aligned in a rectangular array.
According to a preferred embodiment, in the kit, each of the sub-detection regions has a size of 5 to 10mm in length and 5 to 10mm in width, and the detection spot has a size of 0.5 to 1mm in length and 0.5 to 1mm in width.
According to a preferred embodiment, in the kit, the detection spots are spaced apart by a distance of 0.2 to 1 mm.
According to a preferred embodiment, the kit further comprises an anti-human IgG polyclonal antibody marked by alkaline phosphatase, and the anti-human IgG polyclonal antibody is used for matching with the sub-detection area with the human IgG polyclonal antibody to form the third detection spot, namely the anti-human IgG polyclonal antibody is used for dropwise adding the serum to be detected on the detection spot in the sub-detection area and then dropwise adding the serum to be detected on and covering the detection spot;
the kit also comprises an alkaline phosphatase-labeled anti-human IgM polyclonal antibody which is used for being matched with the sub-detection area with the human IgM polyclonal antibody attached to form the third detection spot for use, namely, after the serum to be detected is dripped on the detection spot in the sub-detection area, the serum to be detected is dripped on and covers the detection spot.
According to a preferred embodiment, the kit further comprises a 0.05% PBS-Tween solution and an alkaline phosphatase chromogenic substrate solution.
The utility model discloses still provide the preparation method of above-mentioned any kit, its characterized in that, the preparation of protein chip includes following step:
obtaining a chip substrate which is partitioned to form the sub-detection area;
spotting and fixing BSA, novel coronavirus antigens, human IgG polyclonal antibodies or human IgM polyclonal antibodies in the sub-detection area according to a preset pattern and size to form the first detection spot, the second detection spot and the third detection spot;
the novel coronavirus antigen is a receptor binding domain protein of a novel coronavirus spike protein S1 subunit or a novel coronavirus spike protein S1 subunit protein, or a mixture thereof.
According to a preferred preparation method, 10% BSA, 0.1-1mg/ml of a novel coronavirus antigen, 0.05-0.2mg/ml of a human IgG polyclonal antibody or a human IgM polyclonal antibody are spotted and fixed in the sub-detection region according to a preset pattern and size to form the first, second and third detection spots.
According to a preferred preparation method, 0.5mg/ml of novel coronavirus antigen, 0.1mg/ml of human IgG polyclonal antibody or human IgM polyclonal antibody, 10% BSA are preferred.
According to a preferred preparation method, all detection spots within each sub-detection zone use the same volume of test obtained and are between 1 and 30 nL.
According to a preferred preparation method, the detection spot is formed by multiple non-contact microspotting, preferably 3-10 non-contact microspots, each spotting 300-600 pL.
Example 1 protein chip preparation
The reagents used were:
10%BSA
Human IgG at a concentration of 0.1mg/ml, purchased from Biotechnology engineering (Shanghai) Ltd;
human IgM, R & D Systems, Inc at a concentration of 0.1 mg/ml.
Alkaline phosphatase-labeled anti-human IgG purchased from Biotechnology engineering (Shanghai) Inc.;
alkaline phosphatase-labeled anti-human IgM available from Biotechnology engineering (Shanghai) Ltd;
PBS formulation including 8g sodium chloride (NaCl), 0.2g potassium chloride (KCl) and disodium hydrogen phosphate (Na)2HPO4)1.44g, potassium dihydrogen phosphate (KH)2PO4) 0.24g, adjusting the pH value to 7.4, and fixing the volume to 1L;
PBST formulation: 1L PBS, + 1ml Tween-20;
the chip substrate is an aldehyde-based chip purchased from Baiao corporation of Shanghai, each chip comprises 10 detection grids, and each grid is used for detecting one part of serum and 10 parts of serum at a time.
TABLE 1 chip fabrication protocol
Each spot reagent was spotted in 1 column with four replicate spots per column as shown in FIG. 1.
Wherein the sample application adopts non-contact trace sample application, the sample application amount of each detection spot is 30nL, the sample application amount is 600pL each time, and the sample application is completed by 5 times.
The spot volumes of each detection spot are the same.
Example 2 determination of detection method
The operation flow and principle of the protein chip of the utility model are shown in fig. 5, and the steps are as follows:
1. on the prepared chip, adding one patient serum (7 ul) into each sub-detection zone 4, and storing at room temperature for 15 minutes to combine the novel coronavirus antibody in the serum with the novel coronavirus antigen on the chip;
2. washing the chip with 0.05% PBS-Tween for 5s each time for 5 times, and cleaning non-specific binding;
3. when the second detection spot is formed by the attachment of the human IgG polyclonal antibody, adding the anti-human IgG marked by alkaline phosphatase; when the second detection spot is formed by the adhesion of the human IgM polyclonal antibody, adding alkaline phosphatase-labeled anti-human IgM; storing for 1525 minutes at room temperature, forming a novel coronavirus antigen-novel coronavirus antibody-alkaline phosphatase labeled anti-human IgG (or a novel coronavirus antigen-novel coronavirus antibody-alkaline phosphatase labeled anti-human IgM complex) on the chip, a human IgG-alkaline phosphatase labeled anti-human IgG complex (or a human IgM-alkaline phosphatase labeled anti-human IgM complex), washing the chip with 0.05% PBS-Tween for 5 seconds each time, washing for 5 times, and washing for non-specific binding;
4. adding alkaline phosphatase chromogenic substrate NBT/BCIP.
The expected results are: for positive sera, the first test spot did not develop a color, the second test spot developed a purplish blue color, and the third test spot developed a purplish blue color. As shown in FIG. 6, the detection wells 1-10 are all the detection results of normal human serum, which are negative results, the BSA detection spot and the novel coronavirus antigen detection spot do not develop color, and the human IgG/IgM detection spot shows purple blue.
Experimental example, the chip of scheme 3 was used to detect clinical serum of new coronavirus, using the procedure determined in example 2:
clinical serum is from the serum of 9 novel patients with coronavirus pneumonia collected in Zhejiang hospital.
The results are shown in FIG. 7, the detection well 10 is healthy serum, and the human IgG/IgM detection spot shows bluish purple, which is a negative result;
the 1 st to 9 th detection holes are novel coronavirus pneumonia patient serum, and the result shows two rows of purplish blue detection spots, namely the novel coronavirus antigen detection spots and the human IgG/IgM detection spots show purplish blue, and the detection result is positive.
Chip detection of other schemes obtains the same qualitative result.
Claims (8)
1. An alkaline phosphatase protein chip kit for detecting novel coronavirus antibody, which is characterized by comprising at least one protein chip
Each protein chip is provided with at least one sub-detection area, and each sub-detection area is used for detecting a sample;
the following detection spots are arranged in each sub-detection area:
first detection spot formed by BSA attachment
A second detection spot formed by attachment of a novel coronavirus antigen,
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 novel coronavirus antigen is a receptor binding domain protein of a novel coronavirus spike protein S1 subunit or a novel coronavirus spike protein S1 subunit protein, or a mixture thereof;
when the third detection spot is formed by the IgG polyclonal antibody attached on the protein chip, the protein chip also comprises an anti-human IgG polyclonal antibody marked by alkaline phosphatase;
when the third detection spot is formed by the IgM polyclonal antibody attached on the protein chip, the third detection spot also comprises an anti-human IgM polyclonal antibody marked by alkaline phosphatase.
2. The protein chip kit according to claim 1,
the second detection plaque is formed by attaching a novel coronavirus antigen with the concentration of 0.1-1 mg/ml;
the third detection spot is formed by attaching human IgG polyclonal antibody or IgM polyclonal antibody with the concentration of 0.05-0.2 mg/ml.
3. The protein chip kit according to claim 1,
the novel coronavirus antigen is a receptor binding domain protein of a novel coronavirus spike protein S1 subunit and a novel coronavirus spike protein S1 subunit protein, wherein the ratio of the receptor binding domain protein to the novel coronavirus spike protein S1 subunit is 1-3: 1 mass ratio of the mixture.
4. The protein chip kit of claim 1, wherein at least one of said protein chips comprises two subregions, wherein one of the subregions has said third detection spot formed by the attachment of a human IgG polyclonal antibody; the other of the subregions has the third detection spot formed by the IgM polyclonal antibody attachment.
5. The protein chip kit according to claim 1, comprising two kinds of said protein chips, wherein a third detection spot on one of the protein chips is formed by attachment of a human IgG polyclonal antibody; the third detection spot of the other protein chip is formed by IgM polyclonal antibody attachment.
6. The kit of claim 1, wherein within each of said sub-detection zones, each detection spot comprises 3-6 repeats, each detection spot is aligned in a column, and the three detection spots are aligned in a rectangular array;
each of the sub-detection regions is 5-10 mm long and 5-10 mm wide, and the detection spots are 0.5-1 mm long and 0.5-1 mm wide.
7. The kit according to claim 1, wherein the detection spots are spaced apart by a distance of 0.2 to 1 mm.
8. The kit of any one of claims 1 to 7, further comprising a 0.05% PBS-Tween solution and an alkaline phosphatase chromogenic substrate solution.
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| CN202010095501 | 2020-02-17 | ||
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| CN202010206827.3A Pending CN111679083A (en) | 2020-02-17 | 2020-03-23 | Alkaline phosphatase protein chip kit for detecting novel coronavirus antibody and preparation method thereof |
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| CN112964874B (en) * | 2021-02-22 | 2022-11-01 | 深圳市亚辉龙生物科技股份有限公司 | SARS-CoV-2 detecting reagent kit based on indirect method |
| CN113267623A (en) * | 2021-03-29 | 2021-08-17 | 深圳市赛尔生物技术有限公司 | Kit for simultaneously detecting multiple pathogens such as new coronavirus and preparation method thereof |
| CN113252892B (en) * | 2021-06-22 | 2021-11-02 | 北京市肝病研究所 | Probe and kit for improving detection sensitivity of pathogenic microorganism antigen |
| CN113512113B (en) * | 2021-08-03 | 2024-07-05 | 浙江大学医学院附属第一医院 | Humanized broad-spectrum high-neutralization activity anti-novel coronavirus monoclonal antibody and application thereof |
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| CN1453590A (en) * | 2003-06-18 | 2003-11-05 | 广州万孚生物技术有限公司 | Reagent kit for LgG immunoblotting diagnosis of SARS coronavirus antibody |
| CN2735340Y (en) * | 2003-12-25 | 2005-10-19 | 上海生物芯片有限公司 | Preparation of SARS antibody screening polypeptide chip and detecting kit |
| WO2007053165A2 (en) * | 2005-01-11 | 2007-05-10 | Yale University | Virus protein microarray and uses therefor |
| EP3058370A4 (en) * | 2013-10-14 | 2017-08-30 | The University of North Carolina at Chapel Hill | Methods and compositions for coronavirus diagnostics and therapeutics |
| CN103823058B (en) * | 2014-02-27 | 2016-02-03 | 首都医科大学附属北京佑安医院 | The chemiluminescence protein chip method of Antigens albumen and kit in serum |
| CN104678103A (en) * | 2014-08-05 | 2015-06-03 | 首都医科大学附属北京佑安医院 | Chemical luminescent protein chip, kit and detection method for detecting fucose index of seroglycoid |
| CN107727864A (en) * | 2016-07-01 | 2018-02-23 | 首都医科大学附属北京佑安医院 | The protein chip of abnormal decarboxyprothrombin, kit and preparation method thereof in a kind of detection serum |
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