AU2021103920A4 - Protein chip and kit for detecting the SARS-CoV-2 S antigen - Google Patents
Protein chip and kit for detecting the SARS-CoV-2 S antigen Download PDFInfo
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 26
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 26
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 0.000 title claims description 9
- 102000036639 antigens Human genes 0.000 title abstract description 21
- 108091007433 antigens Proteins 0.000 title abstract description 21
- 239000000427 antigen Substances 0.000 title abstract description 19
- 238000001514 detection method Methods 0.000 claims abstract description 82
- 241000711573 Coronaviridae Species 0.000 claims abstract description 28
- 239000013642 negative control Substances 0.000 claims abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 108010061994 Coronavirus Spike Glycoprotein Proteins 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- 238000005507 spraying Methods 0.000 claims description 4
- 101710137302 Surface antigen S Proteins 0.000 abstract description 5
- 241000700605 Viruses Species 0.000 abstract description 5
- 239000013589 supplement Substances 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000002331 protein detection Methods 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 10
- 239000000047 product Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 101710114810 Glycoprotein Proteins 0.000 description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 3
- 101710167605 Spike glycoprotein Proteins 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 244000309467 Human Coronavirus Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
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
-
- 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
- G01N33/5302—Apparatus specially adapted for immunological test procedures
-
- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
-
- 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
- G01N33/68—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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
- B01L3/50857—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates using arrays or bundles of open capillaries for holding samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
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- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
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- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The utility model "a protein chip and kit for detecting novel coronavirus
S antigen" relates to a virus antigen protein detection technology. The protein
chip has at least one sub-detection area, and each sub-detection area is used to
detect one copy Specimens; each sub-detection area is equipped with the
following two detection spots: the first detection spot: formed by attachment
of BSA, which is a negative control spot, and does not show color when the
chip is effective; the second detection: formed by novel Coronavirus antibody,
and the color is positive after detection; the detection spots are separated
from each other. 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.
Drawings
0@ 00 00 00 00
0 0 0 0 0 0 0 0 0 0
,00 0 0 0 0 0 0 0 0
1 00 00 00 00 00
12
11 12
Figure 1
HRP-labeled novel coronavirus antibody
HRP
Novel Coronavirus Antigen
Novel coronavirus BS
BSA
antibody -a
Figure 2
Description
Drawings
0@ 00 00 00 00 00 00 00 00 00
,00 00 00 00 00 1 00 00 00 00 00 12 11 12 Figure 1
HRP-labeled novel coronavirus antibody HRP Novel Coronavirus Antigen
Novel coronavirus BS BSA antibody -a
Figure 2
Protein chip and kit for detecting the SARS-CoV-2 S antigen
Technical Field The invention relates to virus infection diagnosis technology, in particular to protein chips and kits for detecting SARS-CoV-2 S antigen.
Background Coronavirus is related to a variety of diseases of humans and animals, and can cause diseases of the respiratory tract, digestive tract and nervous system of humans and animals. The virus particles are 60-200nm in diameter, spherical or elliptical, and pleomorphic in nature. The virus has an envelope with spike glycoprotein, which is the receptor binding site and the main antigenic site. Compared to by antibody-based testing or nucleic acid-based testing, virus antigen test by detecting the throat swabs of subjects is a kind of earlier-stage active prevention and control, which is an important supplement to current detection system and can meet the realistic need for epidemic prevention and control.
Summary Based on the needs of the above-mentioned field, the invention provides a novel coronavirus S antigen detection kit, which can detect the novel coronavirus S antigen quickly, at low cost. Test results can be issued within minutes, 10 people at one-time test, the average time per person is 4 minutes. One aspect of the invention provides a protein chip for detecting SARS-CoV-2 S antigen protein, which is characterized in that, the protein chip has at least one sub-detection area, and each sub-detection area is used to detect one specimen; each sub-detection area is provided with the following two kinds of detection spots: the first kind of detection spot: formed by coating novel coronavirus antibody, after the detection, the color is positive; the second detection spot: formed by coating BSA, which is a negative control spot; when the chip is effective, it will not show color; all of the detection spots are separated from each other; the novel coronavirus antibody is a monoclonal antibody specific to the S1 subunit of novel coronavirus spike protein, which is used to bind the SI subunit of novel coronavirus spike protein that may exist in tested sample.
Preferably, in the protein chip, characterized in that, in each of sub-detection areas, the first kind of detection spot comes with 2 repeats and are arranged in a row; and the second kind of detection spot has two repeats and be arranged in a column or row.
Preferably, in the protein chip, characterized in that, in each sub-detection area, the first and second kinds of detection spot have different shapes. Preferably, in the protein chip, characterized in that, each the sub-detection area has dimensions of 5~10 mm in length and 5~10 mm in width, and the detection spots have dimensions of 0.5~1 mm in length and 0.5 ~1 mm in width; in each of the sub-detection areas, the distance between the adjacent detection spots is 0.2-1 mm; all detection spots in each sub-detection area are formed by the same volume of coated reagent, which is 80-120nl; the detection spots are formed by multiple non-contact micro spraying, each spraying 300-600pl.
In another aspect, present invention provides a kit for detecting SARS-CoV-2 S antigen protein, characterized in that, it comprises at least one protein chip abovesaid and an HRP-labeled monoclonal antibody specific to the S1 subunit of novel coronavirus spike protein. The invention provides a simple, economical, rapid, and reliable protein chip method for detecting the novel coronavirus novel coronavirus S antigen. It can detect multiple people at one time, and the test results can be issued within 40 minutes, which can be qualitatively and quantitatively detect novel coronavirus S antigen in patients with accurate results and high sensitivity, which can detect infected persons in advance and provide early-stage active prevention and control methods for virus infection, this is an useful supplement to current detection system and can meet the realistic need for epidemic prevention and control.
Advantages of the protein chip and kit The present invention selects novel coronavirus antigens and chooses antigen fragments and antigen combination schemes that is suitable for making microprotein chips. The clinical test data show that positive serum can be detected sensitively and accurately. Allow multiple samples to be tested at the same time. The same sample can be repeated several times, or using samples taken 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 can be achieved, reducing the overall cost of testing and improving the efficiency of testing. The protein chip of the present invention requires very little sample volume and detection antibody volume. Each detection spot used to capture antigen on the protein chip only needs 100 nanoliters of antibody solution, which is far lower than other similar methods and greatly reduces detection cost. The chip has a high sensitivity by using horseradish peroxidase substrate for luminescence to determine the experimental results.
Description of the drawings Figure 1. Schematic flat view the protein chip; wherein, 1-sub-detection area, 11- the first kind of detection spot, 12- the second kind of detection spot. Figure 2. Schematic view of the detection principle of the protein chip; Figure 3. Schematic view (top) and physical scan (bottom) of the detection results of the u protein chip.
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 protein chip
Reagents used: 10%BSA Antibody 1. Product name: Anti-SARS-CoV-2 spike glycoprotein antibody
[H6]-Chimeric ELISA, 1mg/ml, produced by ABCAM company, item No. ab272854 new product; Antigen protein, product name: Recombinant human coronavirus SARS-CoV-2 Spike; Glycoprotein S1 (Active), produced by ABCAM company, item No.ab273068 new product; Antibody 24 :Product name: Anti-SARS-CoV-2 spike glycoprotein antibody
[H6]-Chimeric (HRP) ELISA, concentration lmg/ml, produced by ABCAM company, item No. ab273169;
PBS formula: sodium chloride (NaCI) 8g, potassium chloride (KCl) 0.2g, disodium hydrogen phosphate (Na2HPO4) 1.44g, potassium dihydrogen phosphate (KH2PO4) 0.24g, adjusted pH 7.4, contained inl.0L PBST formula: IL PBS + Iml Tween-20; The chip substrate is an aldehyde-based chip, produced by Shanghai Baiao
Company. Each chip contains 10 detection squares, each square detects one
serum, and 10 serums are tested at one time. The first kind of detection spot 11(BSA control spot): the spotting reagent is
The second kind of detection spots 12 (antigen detection spots) : the spotting
reagent is 0.8 mg/ml antibody 1".
In a sub-detection area 1, each kind of spotting reagent is spotted in 1
column, with 2 repeated detection spots in each column, as shown in Figure 1.
The spotting uses non-contact micro spotting device to spot several times(3-6
times) for each spot. The total amount of spotting reagent for each detection spot
is 100nl. In some chips, the shape of the first kind of detection spot and the second
kind of detection spot are different. For example, the first detection kind of spot
is a circle, and the second kind of detection plate is a triangle or a quadrilateral,
or a specific letter shape.
Example 2. How to use the chip
The operating process and principle are shown as below steps:
1.Add samples (1ul) for each sub-detection area and store it at room
temperature for 15 minutes, so that the virus antigens that possibly exist in the
sample may be combined with the antibodies on the chip;
2.Wash the chip with 0.05% PBS-Tween 5 times, 5 seconds each time, to
remove non-specific binding;
3.Add antibody 24 (HRP-labeled novel coronavirus antibody, 1:1000 dilution concentration: lug/ml) to form an antibody-antigen-HRP labeled antibody complex on the chip, as shown in Figure 2.
4.Wash the chip with 0.05% PBS-Tween 5 times, 5 seconds each time, to remove non-specific binding;
5. Add HRP substrate.
Expected result: For the samples containing the novel coronavirus antigen, the first detection spot does not show luminous signal, and the second detection spot shows a luminous signal; for samples without novel coronavirus antigen, both the first detection spot and the second detection spot have no signal.
Example 3. The chip is used to detect novel coronavirus antigen by steps in Example 2:
1. Novel coronavirus antigen, purchased from ABCAM company, item No. ab273068, product name: Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein SI (Active), 0.2mg/ml.
2. Diluting the novel coronavirus antigen by a ratio of 1:100.
3. The above-mentioned diluted antigen protein is added to detection wells No.2-5 and No.6-9 of the chip produced in Example 1, wells No.1 and 10 are blank control.
4. After being incubated at room temperature for 25 minutes, washed and dried, and added 15ul antibody 24 (HRP-labeled new coronavirus antibody, 1:100 dilution), incubated at room temperature for another 25 minutes, washed and dried again.
5. Adding HRP luminescent substrate, scan and perform scanning photography, the results as shown in Figure 3: wells No.2-5 and wells No. 6-9 novel coronavirus antigen detection wells show positive signals; 1 and 10 are blank control detection holes without signal; 2, 3, 4, 8 , 9 Detection background signal appears in the detection wells.
Claims (1)
- Claims 1. A protein chip for detecting SARS-CoV-2 S antigen protein, which is characterized in that, the protein chip has at least one sub-detection area, and each sub-detection area is used to detect one specimen; each sub-detection area is provided with the following two kinds of detection spots: the first kind of detection spot: formed by coating novel coronavirus antibody, after the detection, the color is positive; the second detection spot: formed by coating BSA, which is a negative control spot; when the chip is effective, it will not show color; all of the detection spots are separated from each other; the novel coronavirus antibody is a monoclonal antibody specific to the S1 subunit of novel coronavirus spike protein, which is used to bind the SI subunit of novel coronavirus spike protein that may exist in tested sample. 2.The protein chip according to claim 1, characterized in that, in each of sub-detection areas, the first kind of detection spot comes with 2 repeats and are arranged in a row; and the second kind of detection spot has two repeats and be arranged in a column or row.3.The protein chip according to claim 1, characterized in that, in each sub-detection area, the first and second kinds of detection spot have different shapes. 4.The protein chip according to claim 1, characterized in that, each the sub-detection area has dimensions of 5~10 mm in length and 5~10 mm in width, and the detection spots have dimensions of 0.5~1 mm in length and 0.5 ~1 mm in width; in each of the sub-detection areas, the distance between the adjacent detection spots is 0.2-1 mm; all detection spots in each sub-detection area are formed by the same volume of coated reagent, which is 80-120nl; the detection spots are formed by multiple non-contact micro spraying, each spraying 300-600pl.5.A kit for detecting SARS-CoV-2 S antigen protein, characterized in that, it comprises at least one protein chip according to any one of claims 1 to 4, and an HRP-labeled monoclonal antibody specific to the S1 subunit of novel coronavirus spike protein.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021103920A AU2021103920A4 (en) | 2021-07-07 | 2021-07-07 | Protein chip and kit for detecting the SARS-CoV-2 S antigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021103920A AU2021103920A4 (en) | 2021-07-07 | 2021-07-07 | Protein chip and kit for detecting the SARS-CoV-2 S antigen |
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| Publication Number | Publication Date |
|---|---|
| AU2021103920A4 true AU2021103920A4 (en) | 2021-10-21 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2021103920A Ceased AU2021103920A4 (en) | 2021-07-07 | 2021-07-07 | Protein chip and kit for detecting the SARS-CoV-2 S antigen |
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| Country | Link |
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| AU (1) | AU2021103920A4 (en) |
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2021
- 2021-07-07 AU AU2021103920A patent/AU2021103920A4/en not_active Ceased
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