CN112881685A - Protein chip and kit for detecting novel coronavirus N antigen and preparation method thereof - Google Patents
Protein chip and kit for detecting novel coronavirus N antigen and preparation method thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention relates to a protein chip, a kit and a preparation method thereof for detecting a novel coronavirus N antigen, relating to a virus antigen protein detection technology, wherein the protein chip is provided with at least one sub-detection area, each sub-detection area is provided with at least one detection spot, and the detection spots are formed by uniformly paving and then drying a solution for detecting an antibody; 1-2.5ng of the detection antibody is attached to each square millimeter of the detection spot, and the detection antibody is a specific antibody of a novel coronavirus N protein; compared with antibody detection, the protein chip can discover infected persons in advance, and is an important supplement of the existing novel coronavirus detection system.
Description
Technical Field
The invention relates to a virus infection diagnosis technology, in particular to a protein chip and a kit for detecting a novel coronavirus N antigen and a preparation method thereof.
Background
The novel coronavirus (2019-nCoV) belongs to a coronavirus of beta genus, and is enveloped, wherein particles are circular or elliptical and have the diameter of 60-140 nm. Has 5 essential genes, respectively for 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 gene to form a nucleocapsid, the E protein is surrounded outside, and the M protein, the S protein and other proteins are embedded in the virus envelope. The S protein enters the cell by 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 conserved and accounts for the largest proportion in the structural protein of the virus, and can be used as a diagnostic antigen.
The novel coronavirus N antigen detection detects virus N antigen conditions in throat swabs or body fluids of subjects, is an early-stage active prevention and control compared with antibody detection or nucleic acid detection, can discover infected persons in advance, is an important supplement of an existing novel coronavirus detection system, and is a practical requirement for epidemic prevention and control.
Disclosure of Invention
Based on the needs in the field, the invention provides a novel coronavirus N antigen detection kit, which can be used for quickly detecting a novel coronavirus N antigen at low cost. The detection result can be presented within 40 minutes, 21 samples are detected at one time, the average time consumption of each sample is less than 2 minutes, the sensitivity reaches picogram level, and the chip is a high-sensitivity chip.
A protein chip for detecting a novel coronavirus N antigen is characterized in that,
the protein chip is provided with at least one sub-detection area, and each sub-detection area is provided with at least one detection spot which is formed by uniformly paving and then drying a solution of a detection antibody;
1-2.5ng of said detection antibody per square millimeter of said detection spot,
the detection antibody is specific for the novel coronavirus N protein.
A protein chip for detecting a virus,
the protein chip is provided with at least one sub-detection area, each sub-detection area is provided with at least one detection spot, and the detection spots are formed by uniformly paving and then drying a solution of a detection antibody of a target virus; 1-2.5ng of the detection antibody is attached to the detection spot per square millimeter.
Preferably, the protein chip is characterized in that the diameter of the detection spot is 3-5 mm.
Preferably, each of the sub-detection regions is a detection spot having an area of 9-25mm2Preferably a circle with a diameter of 4 mm.
Preferably, said protein chip is characterized by having 7-21 of said sub-detection regions.
Preferably, the protein chip is characterized in that a teflon coating is arranged between every two adjacent sub-detection areas and used for separating the detection sub-areas so as to prevent cross contamination among samples.
Preferably, the protein chip is characterized in that the detection antibodies on the detection spots between the sub-detection zones are identical to each other; or
At least two subregions are formed between the detection spots, wherein the detection antibodies on the detection spots are different from each other.
In another aspect of the invention, a kit for detecting a virus is characterized by comprising at least one protein chip as described above, and a secondary HRP-labeled antibody, a 0.05% PBS-Tween solution and an HRP luminescent substrate solution.
In another aspect of the present invention, a method for preparing any one of the protein chips is provided, which comprises the following steps:
(1) a slide glass having at least one sub-detection zone is acquired,
(2) uniformly spraying a spot with the solution of the detection antibody in each sub-detection area, and drying to form the detection spot; 1-2.5ng of the specific antibody per square millimeter of the spot was detected.
Preferably, the preparation method is characterized in that the diameter of the detection spot is 3-5 mm, the concentration of the specific antibody solution is 0.8mg/ml, the total amount of the spray points of each detection spot is 30nl, and the detection spot is formed by multiple non-contact micro spray points, wherein each spray point is 300-600 pl.
The invention provides a simple, practical, economic, rapid and practical protein chip method, which can detect 21 parts of novel coronavirus N antigen at a time, can give out a detection result within 40 minutes, can qualitatively and quantitatively detect the novel coronavirus N antigen in a patient body, has accurate result and high sensitivity, can discover an infected person in advance, provides active prevention and control detection at an earlier stage for virus infection, is an important supplement of the existing novel coronavirus detection system, and is a practical requirement for epidemic prevention and control.
The research shows that the detection sensitivity is greatly improved under the condition that the density/dosage of the antibody on the protein chip detection spot is obviously reduced, and the further research shows that the size of the detection spot has substantial influence on the capture of the detection signal, and when the detection spot is increased by about 10 times from 1 square millimeter, such as increased to 9-25 square millimeters, and the density/dosage of the antibody is reduced by 5-10 times, the detection sensitivity of the chip is suddenly improved to picogram level.
The invention has the innovative effect that the sensitivity is greatly improved by changing the preparation process and the quality/density of the antibody on the chip, so the invention is not limited to preparing the protein chip for detecting the novel coronavirus N antigen.
Other advantages of the protein chip and kit that this study improves:
allowing simultaneous detection of multiple samples, or repeated multiple times on the same sample, or samples taken at different time points to obtain dynamic values, or simultaneous detection of samples from different patients, in short, high throughput detection is achieved, overall reducing detection costs and improving detection efficiency.
Drawings
FIG. 1 is a schematic plan view of a protein chip of the present invention;
FIG. 2 is a schematic diagram of the detection principle of the protein chip of the present invention;
FIG. 3 is a schematic diagram showing the detection results of the protein chip of the present invention;
FIG. 4 is a scanned graph of the detection result of the chip in FIG. 3;
FIG. 5 is a signal scanning diagram of the detection result of the chip in FIG. 3;
FIG. 6 is a schematic diagram showing the detection results of the protein chip of the present invention;
FIG. 7 is a scanned graph of the detection result of the chip in FIG. 6;
FIG. 8 is a signal scanning diagram of the detection result of the chip in FIG. 6;
FIG. 9 is a schematic diagram showing the detection results of the protein chip of the present invention;
FIG. 10 is a scanned graph of the detection results of the chip of FIG. 9;
FIG. 11 is a signal scanning diagram of the detection result of the chip in FIG. 9.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments, but the scope of the present invention is not limited thereto. Unless otherwise specified, the procedures used in the following examples are conventional, and all reagents used are commercially available.
As shown in FIG. 1, the present invention provides a protein chip for detecting a novel coronavirus N-antigen, wherein the protein chip has at least one sub-detection area 1, each of which has at least one detection spot formed by uniformly spreading a solution of a detection antibody and then drying;
1-2.5ng of said detection antibody per square millimeter of said detection spot,
the detection antibody is specific for the novel coronavirus N protein.
A protein chip for detecting a virus,
the protein chip is provided with at least one sub-detection area, each sub-detection area is provided with at least one detection spot, and the detection spots are formed by uniformly paving and then drying a solution of a detection antibody of a target virus; 1-2.5ng of the detection antibody is attached to the detection spot per square millimeter.
Preferably, the protein chip is characterized in that the diameter of the detection spot is 3-5 mm.
Preferably, each of the sub-detection regions is a detection spot having an area of 9-25mm2Preferably a circle with a diameter of 4 mm.
Preferably, said protein chip is characterized by having 7-21 of said sub-detection regions.
Preferably, the protein chip is characterized in that a teflon coating is arranged between every two adjacent sub-detection areas and used for separating the detection sub-areas so as to prevent cross contamination among samples.
Preferably, the protein chip is characterized in that the detection antibodies on the detection spots between the sub-detection zones are identical to each other; or
At least two subregions are formed between the detection spots, wherein the detection antibodies on the detection spots are different from each other.
Example 1 protein chip preparation
The reagents used were:
Antigenic protein, product name: SARS-CoV Nucleoprotein/NP Protein (His Tag) from Italy Hokkaia, Cat. No. 40143-V08B.
The PBS formulation comprises 8g of sodium chloride (NaCl), 0.2g of potassium chloride (KCl), 1.44g of disodium hydrogen phosphate (Na2HPO4), 0.24g of potassium dihydrogen phosphate (KH2PO4), pH value is adjusted to 7.4, and constant volume is 1L
PBST formulation: 1L PBS, +1ml Tween-20;
the chip substrate was a diagnostic slide glass purchased from heusotai, and each chip contained 21 detection circular holes (subregions) of 4mm diameter, each detection circular hole being a detection spot.
The antibody 1 with the concentration of 0.8mg/ml is sprayed by adopting a non-contact micro-multiple (3-6 times) spraying point, and the total volume of the spraying points of each detection round hole is 30 nl.
| |
Chip No. 2 | Chip No. 3 | Chip No. 4 | |
| Antibody concentration | 0.6mg/ml | 0.8mg/ml | ||
| Total volume of spray point | 30nl | 30nl | 30nl | 30nl |
In the sub-detection area, the detection spot uniformly covers the detection hole as shown in figure 1.
Example 2 chip Using method
The operation process and principle of the protein chip of the invention comprise the following steps:
1. on the chip No. 1 prepared in Experimental example 1, a test sample (8ul) was added to each of the sub-detection regions, and the sample was stored at room temperature for 20 minutes to allow the novel coronavirus N antigen possibly contained in the sample to bind to the antibody on the chip;
2. washing the chip with 0.05% PBS-Tween for 5s each time for 5 times to remove non-specific binding;
3. antibody 2 (HRP-labeled novel coronavirus antibody, 1:10000 dilution) was added to form an antibody-antigen-horseradish peroxidase-labeled antibody complex on the chip, as shown in fig. 2.
4. Washing the chip with 0.05% PBS-Tween for 5s each time for 5 times to remove non-specific binding;
5. adding horseradish peroxidase luminescent substrate.
The expected results are: the sample contains a novel coronavirus N antigen, and the detection spot displays a luminescent signal; the sample has no new coronavirus N antigen, and the detection spot has no signal.
Example 3. detection of the new coronavirus N antigen using the chip, the procedure was as determined in example 2:
1. a novel coronavirus antigen, purchased from yinqiao, inc, cat No. 40143-V08B, product name: SARS-CoV Nucleoprotein/NP Protein (His Tag). 0.25mg/ml
2. Diluting the novel coronavirus antigen at a ratio of 1:16 to a final concentration of 0.015625mg/ml
3. The diluted antigen protein is added into the detection holes 1-14 and 17-20 of the other chip No. 1 according to the detection scheme shown in FIG. 3. And (3) adding a diluent into a 15 detection hole, adding 1XPBS into a 16 detection hole, and detecting the 21 detection hole as a blank control.
4. After incubation at room temperature for 20 minutes, washing and air drying were carried out, 8ul of antibody 2 (HRP-labeled novel coronavirus antibody, 1: 10000-fold dilution) was added, incubation at room temperature for 20 minutes, washing and air drying were carried out.
5. HRP luminescent substrate was added and scanning photography was performed, and the results are shown in fig. 4 and 5: the 15,16 and 21 test wells gave negative signals, and the other 18 test wells gave positive signals.
Experimental example 4. detection of the N antigen of a novel coronavirus using the chip, the procedure was as defined in example 2:
1. a novel coronavirus antigen, purchased from yinqiao, inc, cat No. 40143-V08B, product name: SARS-CoV Nucleoprotein/NP Protein (His Tag). 0.25mg/ml
2. The novel coronavirus antigens were diluted at the following concentration fold ratios, 1: 16; 1: 32, a first step of removing the first layer; 1: 64; 1: 128; 1: 256; 1: 512; 1: 1024; 1: 2048; 1: 4096
3. According to the detection schematic diagram of FIG. 6, the diluted antigen protein is added into the detection holes 1-9 and 10 of the chip No. 1, respectively, and then the diluent is added into the detection holes 11 and 1XPBS is added into the detection holes 12, and the detection holes are blank control detection.
4. After incubation at room temperature for 20 minutes, washing and air drying were carried out, 8ul of antibody 2 (HRP-labeled novel coronavirus antibody, 1: 10000-fold dilution) was added, incubation at room temperature for 20 minutes, washing and air drying were carried out.
5. HRP luminescent substrate was added and scanning photography was performed.
The results are shown in FIG. 7 and FIG. 8: 1-9 detection holes, and the detection signals are gradually weakened along with the reduction of the antigen concentration; the 10, 11 and 12 test wells gave negative signals.
Experimental example 5. detection of novel coronavirus antigens using the chip, the procedure was as determined in example 2:
1. a novel coronavirus antigen, purchased from yinqiao, inc, cat No. 40143-V08B, product name: SARS-CoV Nucleoprotein/NP Protein (His Tag). 0.25mg/ml
2. The novel coronavirus antigens were diluted at the following concentration fold 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 diluted antigen protein is sequentially added into the detection holes 1-20 of the other chip No. 1 according to the detection sequence number of the detection schematic diagram of FIG. 9, and the detection hole 21 is blank control detection.
4. After incubation at room temperature for 20 minutes, washing and air drying were carried out, 8ul of antibody 2 (HRP-labeled novel coronavirus antibody, 1: 10000-fold dilution) was added, incubation at room temperature for 20 minutes, washing and air drying were carried out.
5. HRP luminescent substrate was added and scanning photography was performed.
The results are shown in FIGS. 10 and 11:
the detection signal intensity of the detection hole 1-7 is obviously different from that of the blank control, and the antigen 1:96000 is added into the detection hole 7 with the final concentration of 1.3 ng/ml;
the detection signal is also present in the 8-15 detection wells relative to the detection signal of the blank, the detection signal is lower than 1-7, and the antigen is added into the 15 detection wells with the dilution ratio of 1:24576000, and the final concentration is 5.08 pg/ml.
Parallel tests are carried out on the chip No. 2, the chip No. 3 and the chip No. 4, and the comprehensive results show that the chip kit can detect the picogram-level virus antigen in the sample.
Claims (10)
1. A protein chip for detecting a novel coronavirus N antigen is characterized in that,
the protein chip is provided with at least one sub-detection area, and each sub-detection area is provided with at least one detection spot which is formed by uniformly paving and then drying a solution of a detection antibody;
1-2.5ng of said detection antibody per square millimeter of said detection spot,
the detection antibody is specific for the novel coronavirus N protein.
2. A protein chip for detecting a virus,
the protein chip is provided with at least one sub-detection area, each sub-detection area is provided with at least one detection spot, and the detection spots are formed by uniformly paving and then drying a solution of a detection antibody of a target virus;
1-2.5ng of the detection antibody is attached to the detection spot per square millimeter.
3. The protein chip according to claim 1 or 2, wherein the diameter of the detection spot is 3 to 5 mm.
4. The protein chip according to claim 1 or 2, wherein each of the sub-detection regions is a detection spot having an area of 9-25mm2Preferably a circle with a diameter of 4 mm.
5. The protein chip according to any one of claims 1 to 4, wherein there are 7 to 21 of said sub-detection zones.
6. The protein chip of any one of claims 1 to 5, wherein a Teflon coating is disposed between adjacent ones of the detection subregions to space the detection subregions apart from each other to prevent cross-contamination between samples.
7. The protein chip according to any one of claims 1 to 6, wherein the detection antibodies on the detection spots are identical to each other between the subregions; or
At least two subregions are formed between the detection spots, wherein the detection antibodies on the detection spots are different from each other.
8. A kit for detecting a virus, comprising at least the protein chip of any one of claims 1 to 7, and HRP-labeled secondary antibody, 0.05% PBS-Tween solution and HRP luminescent substrate solution.
9. The method for preparing a protein chip according to any one of claims 1 to 7, comprising the steps of:
(1) a slide glass having at least one sub-detection zone is acquired,
(2) uniformly spraying a spot with the solution of the detection antibody in each sub-detection area, and drying to form the detection spot; 1-2.5ng of the specific antibody per square millimeter of the spot was detected.
10. The production method according to claim 9, wherein the diameter of the detection spot is 3 to 5mm, the concentration of the specific antibody solution is 0.8mg/ml, the total amount of the spots sprayed per detection spot is 30nl, and the detection spot is formed by multiple non-contact microspots of 300-600pl per spot.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110079358.8A CN112881685B (en) | 2021-01-21 | 2021-01-21 | Protein chip and kit for detecting novel coronavirus N antigen and preparation method thereof |
| AU2021103918A AU2021103918A4 (en) | 2021-01-21 | 2021-07-07 | A Protein chip and kit for detecting SARS-CoV-2 N protein and its preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110079358.8A CN112881685B (en) | 2021-01-21 | 2021-01-21 | Protein chip and kit for detecting novel coronavirus N antigen and preparation method thereof |
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| CN112881685A true CN112881685A (en) | 2021-06-01 |
| CN112881685B CN112881685B (en) | 2023-02-17 |
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| CN202110079358.8A Active CN112881685B (en) | 2021-01-21 | 2021-01-21 | Protein chip and kit for detecting novel coronavirus N antigen and preparation method thereof |
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| AU (1) | AU2021103918A4 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023284686A1 (en) * | 2021-07-12 | 2023-01-19 | 西湖大学 | Biosensing detection method and system for binding antibodies |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001014425A1 (en) * | 1999-08-19 | 2001-03-01 | Diachip Limited | 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 |
| CN103823058A (en) * | 2014-02-27 | 2014-05-28 | 首都医科大学附属北京佑安医院 | Method for measuring antigen protein in serum by chemiluminescence protein chips and kit |
| 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 |
| US20190204325A1 (en) * | 2016-07-01 | 2019-07-04 | Beijing Youan Hospital, Capital Medical University | Protein Chip, Kit and Preparation Method thereof for Detecting Abnormal Decarboxy Prothrombin in Serum |
| CN112034174A (en) * | 2020-03-20 | 2020-12-04 | 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
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001014425A1 (en) * | 1999-08-19 | 2001-03-01 | Diachip Limited | 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 |
| CN103823058A (en) * | 2014-02-27 | 2014-05-28 | 首都医科大学附属北京佑安医院 | Method for measuring antigen protein in serum by chemiluminescence protein chips and kit |
| US20190204325A1 (en) * | 2016-07-01 | 2019-07-04 | Beijing Youan Hospital, Capital Medical University | Protein Chip, Kit and Preparation Method thereof for Detecting Abnormal Decarboxy Prothrombin in 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 |
| CN112034174A (en) * | 2020-03-20 | 2020-12-04 | 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 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023284686A1 (en) * | 2021-07-12 | 2023-01-19 | 西湖大学 | Biosensing detection method and system for binding antibodies |
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| Publication number | Publication date |
|---|---|
| AU2021103918A4 (en) | 2021-09-09 |
| CN112881685B (en) | 2023-02-17 |
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