AU2021103918A4 - A Protein chip and kit for detecting SARS-CoV-2 N protein and its preparation method - Google Patents
A Protein chip and kit for detecting SARS-CoV-2 N protein and its preparation method Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title description 5
- 238000001514 detection method Methods 0.000 claims abstract description 119
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The present invention "a protein chip and kit for detecting N antigen of novel coronaviruses" of
the present invention relates to a virus antigen protein detection technology, The protein chip
has at least one sub-detection area and each sub-detection area has at least one detection spot.
The detection spot is formed by evenly covering the solution of the detection antibody and then
drying. The detection spot is attached with 1-2.5ng of the detection antibody per square
millimeter, and the detection antibody is a certain antibody of the Novel coronavirus N protein.
Compared with antibody detection, the protein chip of the present invention can detect infected
persons in advance, which is an important supplement to the existing Novel coronavirus
detection system.
Description
EDITORIAL NOTE 2021103918
There are 8 pages of description only.
A Protein chip and kit for detecting SARS-CoV-2 N protein and its preparation method
Technology Field
The present invention relates to virus detection technology, in particular to a protein chip, kit for
SARS-CoV-2 N protein and its preparation method.
Background
The SARS-CoV-2 is a kind of beta-coronaviruses, which has envelope, round or oval particles with
-140nm of diameter. The virus has 5 essential genes, respectively targeting 4 structural proteins
nucleoprotein (N protein), viral envelope (E protein), matrix protein (M protein) and spike protein (S
protein) and RNA-dependent RNA polymerase (RdRp). The N protein wraps the RNA to form a
nucleocapsid, surrounded by the E protein, and the virus envelope contains proteins such as M protein
and S protein. The virus enters host cells by the S protein binding to angiotensin converting enzyme 2
(ACE-2). The N protein plays an important role in the synthesis of viral RNA. Meanwhile, the N protein
is relatively conservative and accounts for the largest proportion of the structural proteins of the virus
and can be used as a diagnostic antigen.
Compared to by antibody-based testing or nucleic acid-based testing, the N protein-based antigen
test by detecting the virus N protein in the throat swabs or body fluids of the subjects is a kind of
earlier-stage active prevention and control. The test can detect infected individuals in advance, which is
an important supplement to current detection system and can meet the realistic need for epidemic
prevention and control.
Summary of the invention
Based on the needs of above-mentioned fields, the present invention provides a detection chip and kit
for the N antigen of novel coronaviruses , which can detect the N antigen of novel coronaviruses rapidly
and at low costs. The test results can be issued within 40 minutes, 21 samples can be tested at a time,
and each sample takes less than 2 minutes on average. The test sensitivity of the detection chip can
reaches picogram level, which mean it is a kind of high-sensitivity chip.
A protein chip for detecting SARS-CoV-2 N protein, which is characterized in that,
the protein chip has at least one sub-detection area, and each sub-detection area has at least one
detection spot, and the detection spot is formed by uniformly coating solution of detection antibody and
then drying;
in the said detection spot, the detection antibody has a density of 1-2.5ng/mm2 ,
the detection antibody is a specific antibody for the SARS-CoV-2 N protein.
Preferably, the protein chip, characterized in that, each sub-detection area is as one detection spot, which
is aregular or irregular shape with an area of 9-25mm2, or acircle with adiameter of 4mm.
Preferably, the protein chip, characterized in that, there are 7-21 sub-detection areas; there is a Teflon
coating between the adjacent sub-detection areas for separating sub-detection areas to prevent
cross-contamination .
A kit for detecting viruses, characterized in that, it comprises at least any one above-said protein chip,
and HRP secondary antibody, 0.05% PBS-Tween solution and HRP chemiluminescent substrates
solution.
A method for preparing any one of above the protein chip, characterized in that, it comprises the
following steps:
(1) obtain a glass slide with at least one sub-detection area,
(2) in each sub-detection area, use the detection antibody solution to spray evenly and then dry to form
the detection spot;
the diameter of the detection spot is 3-5mm, the concentration of the detection antibody solution is
0.8mg/ml, the total volume of detection antibody solution for spraying to each detection spot is 30nl, the
detection spot is formed by multiple non-contact micro spray and each time spraying 300-600pl.
Preferably, the protein chip , characterized in that, the diameter of the detection spot is 3-5mm.
Preferably, the protein chip, characterized in that, there are 7-21 sub-detection areas.
Preferably, the protein chip ,characterized in that, between the sub-detection areas, the detection
antibodies on the detection spots therein are completely the same as each other; or
There are at least two sub-detection areas, wherein the detection antibodies on the detection spots
are different from each other.
The present invention provides a simple, economical, rapid, and reliable protein chip method for detecting the N antigen of novel coronavirus, 21 copies can be detected at a time, and the detection result can be issued within 40 minutes, which can qualitatively and quantitatively detect the N antigen of novel coronaviruses in the serum or swad sample of COVID-19 patient's with accurate results and high sensitivity. It can detect infected persons in advance, and provide early-stage active prevention and control testing for virus infection, which is an important supplement to the existing novel coronavirus detection system and a practical need for epidemic prevention and control. In this study, it is discovered by accident that the detection sensitivity is greatly improved when the density/dosage of the antibody on the protein chip detection spot is significantly reduced. In further research, it is found that the area of the detection spot has a substantial influence on the capture of the detection signal. When the detection spot is increased by about 10 times from 1 square millimeter, for example to 9-25 mm2, and the antibody density/dosage is reduced by 5-10 times, the detection sensitivity of the chip suddenly increases to picogram level. The inventive steps of the present invention is that the changes in the preparation process and the quality/density of the antibody on the chip greatly improve the sensitivity. Therefore the present invention is not limited to a protein chip for detecting SARS-CoV-2 N protein. Other advantages of protein chips and kits improved by this study: Multiple samples are allowed to be tested at the same time, or test the same sample multiple times, or test samples taken from one patient at different time points to obtain dynamic values, or samples from different patients can be tested at the same time. In short, high-throughput testing is achieved, which reduces testing costs and improves overall detection efficiency.
Description of the drawings Figure 1. A schematic plan view of the protein chip of the present invention; Figure 2. The schematic diagram of the detection principle of the protein chip of the present invention; Figure 3. The schematic diagram of the detection results of the protein chip of the present invention; Figure 4. The physical scanning diagram of the detection results of the chip in Figure 3; Figure 5. The signal scanning diagram of the detection results of the chip in Figure 3;
Figure 6. The schematic diagram of the detection results of the protein chip of the present invention; Figure 7. The physical scanning diagram of the detection results of the chip in Figure 6; Figure 8. The signal scanning diagram of the detection results of the chip in Figure 6; Figure 9. The schematic diagram of the detection results of the protein chip of the present invention; Figure 10. The physical scanning diagram of the detection results of the chip in Figure 9; Figure 11. The signal scanning diagram of the detection result of the chip in Figure 9.
Detailed description of embodiments
The present invention will be further described in details below with reference to specific embodiments, but the purpose is not to limit the scope of the invention in any ways. If not specified otherwise, the operations used in the following embodiments are all conventional methods, and all reagents used are commercially available. Example 1. Preparation of the protein chip Reagents used: Antibody 1. Product name: SARS-CoV Nucleoprotein / NP Antibody, Mouse Mab, purchased from Sino Biological, Inc., item number 40143-MM05. SARS-CoV-2 Antigen protein, product name: SARS-CoV Nucleoprotein / NP Protein (His Tag), purchased from Sino Biological, Inc., item number 40143-V8B. Antibody 2. Product name: SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (HRP), Rabbit Mab, purchased from Sino Biological, Inc., item number 40143-R040-H PBS formula: sodium chloride (NaCl) 8g, potassium chloride (KCl) 0.2g, disodium hydrogen phosphate (Na2HPO4) 1.44g, potassium dihydrogen phosphate (KH2PO4) 0.24g, adjusted to pH7.4, prepare the volume to IL PBST formula: IL PBS, + 1ml Tween-20; The chip substrate is a diagnostic glass slide, purchased from Citotest Labware Manufacturing Co., Ltd. Each chip contains 21 detection wells (sub-detection area) with a diameter of 4mm, and each detection well is used for one detection spot. Using non-contact printing device to spray several times (3-6 times) for each spot ,wherein the spray concentration and volume is as below table: Chip No. 1 Chip No. 2 Chip No. 3 Chip No. 4
Antibody concentration 0.6mg/ml 0.8mg/ml 0.4mg/ml 1mg/mi The total volume of spray 30nl 30nl 30nl 30nl point In sub-detection area, the detection spot evenly covers the detection wells as shown in Figure 1.
Example 2: The method of using the chip
The operation process and principle of the protein chip of the present invention, the steps are as follows: On the chip No. 1 prepared in the experimental Example 1, add a test sample (8ul) to each sub-detection area and keep it at room temperature for 20 minutes, so that the N antigen of the novel
coronaviruses that the sample may contain combines with the antibody on the chip ;
2.Wash the chip with 0.05% PBS-Tween for 5 times, and 5 seconds each time, to remove non-specific binding;
3.Add antibody 2 (HRP-labeled novel coronavirus antibody, diluted in a ratio 1:10000) to form an
antibody-antigen-horseradish peroxidase-labeled antibody complex on the chip, as shown in Figure 2.
4.Wash the chip with 0.05% PBS-Tween for 5 times, and 5 seconds each time, to remove
non-specific binding;
5.Add horseradish peroxidase luminescent substrate.
Expected result: If the sample contains the N Protein, the detection spot will show a luminous signal; if there is no the N Protein in the sample, the detection spot has no signal.
Example 3. The chip is used to detect SARS-CoV-2 N protein by the steps in the method in the Example 2
1 . SARS-CoV-2 Antigen protein, purchased from Sino Biological, Inc., item number 40143-V8B,
product name: SARS-CoV Nucleoprotein / NP Protein (His Tag). 0.25mg/ml.
2 . Dilute the SARS-CoV-2 Antigen protein in a ratio of 1:16 to make it has a final concentration
of 0.015625mg/ml.
3.The above-mentioned diluted antigen protein is added to the detection wells 1-14 and 17-20 of another chip No. 1 according to the assay schematic diagram in FIG. 3. Add the diluent to detection well , add 1XPBS to detection well 16 , and detection well 21 is for blank control test.
4 . After incubating for 20 minutes at room temperature, the chip is washed and dried, and then be
added 8ul antibody 2 (HRP-labeled antibody, 1:10000 dilution) and incubated at room temperature for another 20 minutes, then washed and dried again.
5 .Add HRP luminescent substrate, scan and take pictures, the results are shown in Figures 4 and 5:
detection wells 15, 16 and 21 have no signals (negative signals), and the other 18 detection wells have positive signals.
Example 4. The chip is used to detect SARS-CoV-2 Nprotein by the steps in the method in the Example 2 1.SARS-CoV-2 Antigen protein, purchased from Sino Biological, Inc., item number 40143-V8B, product name: SARS-CoV Nucleoprotein / NP Protein (His Tag). 0.25mg/ml. 2.Diluting the antigen protein according to the following concentration ratios, 1:16; 1:32; 1:64; 1:128; 1:256; 1:512; 1:1024; 1:2048; 1:4096. 3.According to the assay schematic diagram of Fig. 6, the above-mentioned diluted antigen protein is added to the detection wells 1-9 of another chip No. 1, detection well 10 is added with diluent, detection well 11 is added with 1XPBS, and detection well 12 is for blank control. 4.After incubating for 20 minutes at room temperature, the chip is washed and dried, and then be added 8ul antibody 2 (HRP-labeled antibody, 1:10000 dilution) and incubated at room temperature for another 20 minutes, then washed and dried again. 5.Add HRP luminescent substrate, scan and take pictures. The results are shown as Figure 7 and Figure 8: the detection wells 1-9, with the antigen concentration decreases, the detection signal gradually weakens; detection wells 10, 11 and 12 are negative signals.
Example 5. The chip is used to detect SARS-CoV-2 N protein by the steps in the method in the Example 2
1.SARS-CoV-2 Antigen protein, purchased from Sino Biological, Inc., item number 40143-V8B, product name: SARS-CoV Nucleoprotein / NP Protein (His Tag). 0.25mg/ml. 2.Diluting the antigen protein according to the following concentration ratios,
1 : 1500 ; 1 : 3000 ;1 : 6000 ; 1:12000; 1:24000;
1:48000; 1:96000; 1:192000; 1:384000; 1:768000; 1:1536000; 1:3072000; 1:6144000; 1:12288000; 1:24576000; 1:49152000;1:98304000;1:196608000;1:393216000;1:786432000. 3. The above-mentioned diluted antigen protein is added to detection wells 1-20 of another chip No. 1 in sequence according to the sequence number of assay schematic in Figure 9, and the 21st detection well is for blank control. 4.After incubating for 20 minutes at room temperature, the chip is washed and dried, and then be added 8ul antibody 2 (HRP-labeled antibody, 1:10000 dilution) and incubated at room temperature for another 20 minutes, then washed and dried again. 5.Add HRP luminescent substrate, scan and take pictures. The results are shown in Figure 10 and Figure 11: There is a significant difference between detection signal intensity of the 1-7 detection wells and that of the blank control. Add antigen in a ratio of 1:96000 to the detection well No. 7, and the final concentration is 1.3ng/ml; Comparing to the detection signals of the blank control, detection signals also appeared in detection wells 8-15, but the detection signals are weaker than those of detection wellsl-7. The dilution ratio of antigen added to detection well15 is 1:24576000, and the final concentration is 5.08pg/ml.
Parallel tests were performed on Chip No. 2, Chip No. 3, and Chip No. 4, and the comprehensive results
showed that the chip kit of the present invention can detect picogram level virus antigens in samples.
EDITORIAL NOTE 2021103918
There are 2 pages of claims only.
Claims (5)
1.A protein chip for detecting SARS-CoV-2 N protein, which is characterized in that,
the protein chip has at least one sub-detection area, and each sub-detection area has
at least one detection spot, and the detection spot is formed by uniformly coating solution
of detection antibody and then drying;
in the said detection spot, the detection antibody has a density of 1-2.5ng/mm2
, the detection antibody is a specific antibody for the SARS-CoV-2 N protein.
2.The protein chip according to claim 1, characterized in that, each sub-detection area is 2 as one detection spot, which is a regular or irregular shape with an area of 9-25mm2, or a
circle with a diameter of 4mm.
3.The protein chip according to claim 1 or 2, characterized in that, there are 7-21
sub-detection areas; there is a Teflon coating between the adjacent sub-detection areas for
separating sub-detection areas to prevent cross-contamination .
4. A kit for detecting viruses, characterized in that, it comprises at least any one protein
chip according to claims 1-3, and HRP secondary antibody, 0.05% PBS-Tween solution
and HRP chemiluminescent substrates solution.
5. The method for preparing the protein chip according to any of the claims 1-3,
characterized in that, it comprises the following steps:
(1) obtain a glass slide with at least one sub-detection area,
(2) in each sub-detection area, use the detection antibody solution to spray evenly and
then dry to form the detection spot; the diameter of the detection spot is 3-5mm, the concentration of the detection antibody solution is 0.8mg/ml, the total volume of detection antibody solution for spraying to each detection spot is 30nl, the detection spot is formed by multiple non-contact micro spray and each time spraying 300-600pl
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|---|---|---|---|---|
| KR20000071894A (en) * | 1999-08-19 | 2000-12-05 | 김선영 | Multipurpose diagnostic systems using protein chips |
| CN101726586A (en) * | 2008-10-14 | 2010-06-09 | 上海裕隆生物科技有限公司 | Lupus erythematosus detection protein chip and kit thereof |
| 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 |
| 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 |
| CN107748261A (en) * | 2017-06-30 | 2018-03-02 | 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 |
| CN112034174B (en) * | 2020-03-20 | 2024-03-29 | 中国人民解放军军事科学院军事医学研究院 | Polypeptide chip and application thereof in virus detection |
-
2021
- 2021-01-21 CN CN202110079358.8A patent/CN112881685B/en active Active
- 2021-07-07 AU AU2021103918A patent/AU2021103918A4/en not_active Ceased
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| Publication number | Publication date |
|---|---|
| CN112881685B (en) | 2023-02-17 |
| CN112881685A (en) | 2021-06-01 |
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