AU2021103919A4 - Alkaline phosphatase protein chip kit for detecting antibodies of novel coronavirus - Google Patents
Alkaline phosphatase protein chip kit for detecting antibodies of novel coronavirus Download PDFInfo
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- 241000711573 Coronaviridae Species 0.000 title claims abstract description 55
- 102000002260 Alkaline Phosphatase Human genes 0.000 title claims abstract description 21
- 108020004774 Alkaline Phosphatase Proteins 0.000 title claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 147
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 66
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 66
- 239000011248 coating agent Substances 0.000 claims abstract description 35
- 238000000576 coating method Methods 0.000 claims abstract description 35
- 102000036639 antigens Human genes 0.000 claims abstract description 32
- 108091007433 antigens Proteins 0.000 claims abstract description 32
- 229940096437 Protein S Drugs 0.000 claims abstract description 14
- 101710198474 Spike protein Proteins 0.000 claims abstract description 14
- 239000000203 mixture Substances 0.000 claims abstract description 14
- 230000027455 binding Effects 0.000 claims abstract description 13
- 102000005962 receptors Human genes 0.000 claims abstract description 13
- 108020003175 receptors Proteins 0.000 claims abstract description 13
- 108700010904 coronavirus proteins Proteins 0.000 claims abstract description 6
- 239000000427 antigen Substances 0.000 claims description 20
- 108010061994 Coronavirus Spike Glycoprotein Proteins 0.000 claims description 8
- 102000029301 Protein S Human genes 0.000 claims description 6
- 101800000904 Spike protein S1 Proteins 0.000 abstract description 3
- 241000700605 Viruses Species 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 210000002966 serum Anatomy 0.000 description 23
- 238000012360 testing method Methods 0.000 description 22
- 238000002360 preparation method Methods 0.000 description 11
- 238000005507 spraying Methods 0.000 description 8
- 101710145634 Antigen 1 Proteins 0.000 description 7
- 238000000034 method Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 4
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 206010035664 Pneumonia Diseases 0.000 description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 3
- 229920000136 polysorbate Polymers 0.000 description 3
- 239000007921 spray Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 241000282412 Homo Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 150000001413 amino acids Chemical class 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
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 1
- 241001678559 COVID-19 virus Species 0.000 description 1
- 101710114810 Glycoprotein Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 0.000 description 1
- 101710167605 Spike glycoprotein Proteins 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011161 development 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
- 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
- 239000007788 liquid Substances 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
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- 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
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- 244000052769 pathogen Species 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
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
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Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA 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
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
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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
- G01N33/68—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
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- 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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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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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- 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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Abstract
The invention is about a kind of alkaline phosphatase protein chip kit for
detecting antibodies of novel coronavirus, relates to virus detection technology; the kit
has at least one protein chip, and each the protein chips has at least one sub-detection
area, each sub-detection area for detecting one specimen; each sub-detection area is
provided with the following detection spots: the first detection spot formed by the
coating BSA, and the second detection spot formed by the coating the novel
coronavirus antigen protein, the third detection spot formed by coating human IgG
polyclonal antibody or IgM polyclonal antibody; the detection spots are separated
from each other; said novel coronavirus antigen protein is a receptor binding domain
protein of spike protein Sl subunit of the novel coronavirus or spike protein S1
subunit, or a mixture of them.
Drawings
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Description
Drawings
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Alkaline phosphatase protein chip kit for detecting antibodies of novel coronavirus
Technology Field The disclosure relates to a virus infection diagnosis technology, in particular to a protein chip kit for detecting antibodies of novel coronavirus.
Background Coronavirus are associated with a variety of diseases of humans and animals, and can cause diseases of 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. Serological detection of viral antibodies in suspected subjects can more accurately, rapidly and cost-effectively exclude subjects with fever caused by other pathogens, greatly improving clinical diagnosis and screening efficiency, while meet the high technical requirements of nucleic acid testing and cure the high proportion of false negatives.
Summary Based on the needs of the above-mentioned field, the invention provides an alkaline phosphatase novel coronavirus IgM/IgG antibody detection kit, which can detect novel coronavirus antibodies quickly, at low cost, and with a small amount of serum (7 microliters). The test results can be issued in 60 minutes, and the test samples for 10 people at one time, which takes an average of 6 minutes per person. It can be operated manually or on the machine. The technical solution is as follows:
A protein chip kit for detecting antibodies of novel coronavirus, characterized in that, it has at least one protein chip, said protein chip has at least one sub-detection area, each sub-detection area for detecting one specimen; Each sub-detection area is provided with the following detection spots: The first detection spot formed by the coating BSA, The second detection spot formed by the coating the novel coronavirus antigen protein, and the third detection spot formed by coating human IgG polyclonal antibody or IgM polyclonal antibody; The detection spots are separated from each other; said novel coronavirus antigen protein is a receptor binding domain protein of spike protein Sl subunit of the novel coronavirus or spike protein S1 subunit, or a mixture thereof.
Preferably, the protein chip kit, characterized in that: When the protein chip has the third detection spot formed by coating IgG polyclonal antibody, the kit contains alkaline phosphatase-labeled anti-human IgG polyclonal antibody; When the protein chip has the third detection spot formed by coating IgM polyclonal antibody, the kit contains alkaline phosphatase-labeled anti-human IgM polyclonal antibody. Preferably, the protein chip kit according to claim 1, characterized in that: said second detection spot formed by coating novel coronavirus antigen at a concentration of 0.1-1 mg/ml; said third detection spot formed by coating human IgG polyclonal antibody or IgM polyclonal antibody at a concentration of 0.05-0.2 mg/ml.
According to a preferred embodiment, in said kit, said novel coronavirus antigen is a mixture of a receptor binding domain protein of novel coronavirus spike protein Sl subunit and novel coronavirus spike protein S1 subunit protein in a mass ratio of 1 to 3:1, Preferably a mixture of 1:1 mass ratio. said novel coronavirus antigen protein is mixture of the receptor binding domain protein of spike protein Sl subunit of the novel coronavirus and the spike protein S1 subunit with weight ratio 1 ~3:1; preferably a mixture of 1:1 mass ratio.
According to a preferred embodiment, in said kit, at least one of said protein chips contains two kinds of sub-detection areas, wherein one kind of sub-detection area has the third detection spot formed by coating human IgG polyclonal antibody; the other kind of sub-detection zone has said third detection spot formed by coating IgM polyclonal antibody; or According to a preferred embodiment, in said kit, it contains two kinds of said protein chips, wherein one kind of the protein chip has said third detection spot formed by coating human IgG polyclonal antibody; and the other kind of protein chip has said third detection spot formed by coating IgM polyclonal antibody.
The kits in above two schemes allow simultaneous detection of IgG antibodies and IgM antibodies in the sera of suspected patients, improving the accuracy of detection. According to a preferred embodiment, in said kit, in each said sub-detection zone, each kind of detection spot contains 3-6 repeats, each detection spot is arranged in a row, and the three detection spots are arranged in a rectangle Array. According to a preferred embodiment, in said kit, the size of said each sub-detection zone is 5-10 mm in length and 5-10 mm in width, and the size of the detection spot is 0.5-1 mm in length and 0.5-1 mm in width. According to a preferred embodiment, in said kit, the separation distance between said detection spots is 0.2-1 mm. According to a preferred embodiment, said kit further contains an alkaline phosphatase-labeled anti-human IgG polyclonal antibody for attaching to human IgG polyclonal antibody to form the sub-detection area of the third detection spot used in conjunction, that is, after the serum to be tested is dripped on said detection spot in the sub-detection zone, it is dripped onto and covered said detection spot; further comprising an alkaline phosphatase-labeled anti-human IgM polyclonal antibody for use in conjunction with the sub-detection zone having the human IgM polyclonal antibody attached to form said third detection spot, that is, after the serum to be tested is dripped on said detection spot In the sub-detection zone, it is dripped onto and covered said detection spot.
According to a preferred embodiment, said kit further contains 0.05% PBS-Tween solution and alkaline phosphatase substrate solution.
The method for preparing any one of said kits described above, characterized in that, the preparation of said protein chip includes the following steps:
Obtaining a chip substrate with divided regions as the sub-detection;
Spraying the BSA, novel coronavirus antigen, human IgG polyclonal antibody or human IgM polyclonal antibody in the sub-detection areas according to the preset pattern and size to form said first, second and third detections spot;
said novel coronavirus antigen protein is a receptor binding domain protein of spike protein SI subunit of the novel coronavirus or spike protein SI subunit, or a mixture thereof.
According to a preferred preparation method, 10% BSA, 0.1-1mg/ml novel coronavirus antigen, 0.05-0.2mg/ml human IgG polyclonal antibody or human IgM polyclonal antibody are spotted and fixed on the preset pattern and size to form said first, second and third detection spots.
According to a preferred preparation method, 0.5 mg/ml novel coronavirus antigen, 0.1 mg/ml human IgG polyclonal antibody or human IgM polyclonal antibody, and 10% BSA are preferred. According to a preferred preparation method, all detection spots in each sub-detection area use the same volume of the obtained test, and it is between 1-30 nL. According to a preferred preparation method, said detection spots are formed by multiple non-contact micro-spots, preferably 3-10 non-contact micro-spots, with 300-600 pL per spot. The invention provides a simple, economical, rapid, and reliable protein chip method for detecting IgM/IgG antibodies in serum of novel coronavirus pneumonia. which only needs a trace amount of serum (7ul), can detect multiple samples at one time, and provides results within 60 minutes. which can qualitatively detect IgM/IgG antibodies of novel coronavirus in human serum or plasma, with accurate and sensitive detection results.
Advantages of this utility protein microarray kit. 1. 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 showed that positive serum can be detected sensitively and accurately. 2. 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. 3. The amount of blood samples and antibodies required for the protein chip of the invention are greatly reduced. The amount of serum that needs to be detected is 7-7.5ul. The detection spots on the protein chip are used to capture antibodies. each detection spot only needs 10-30 nanoliters of protein liquid, it is much lower than other similar methods, greatly reducing the cost of testing.
4. The utility model uses alkaline phosphatase coloring solution to judge the results of color development without scanning instrument, it is simple and visualized, does not require high technical personnel, and is suitable for large-area use.
Description of the drawings
Figure 1. Schematic flat view of the protein chip in the kit of present invention; Figure 2. Schematic cross-sectional view of three detection spots in a single sub-detection area in a protein chip in the kit of present invention; Figure 3. Schematic view of a single sub-detection area in the protein chip in the kit of present invention; Figure 4. Schematic view of the protein chip sample in the kit of present invention; Figure 5. Schematic view of detection principle of the kit of present invention; Figure 6. The result of detecting healthy human serum with protein chip in the kit of present invention; Figure 7. The view of the results of the protein chip detection of clinically confirmed serum in the kit of present invention; wherein, 1-the first detection spot; 2-the second detection spot; 3-the third detection spot, 4-sub-detection area.
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.
A protein chip kit for detecting antibodies of novel coronavirus, characterized in that, it has at least one protein chip, as shown in FIG. 1,each said protein chip has at least one sub-detection area 4, each sub-detection area 4 for detecting one specimen; each sub-detection area is provided with the following detection spots: the first detection spot 1 formed by the coating BSA, the second detection spot 2 formed by the coating the novel coronavirus antigen protein, and the third detection spot 3 formed by coating human IgG polyclonal antibody or IgM polyclonal antibody; the detection spots are separated from each other; said novel coronavirus antigen protein is a receptor binding domain protein of spike protein S Isubunit of the novel coronavirus or spike protein S Isubunit, or a mixture thereof.
According to a preferred embodiment, the protein chip kit, characterized in that: when the protein chip has the third detection spot formed by coating IgG polyclonal antibody, the kit contains alkaline phosphatase-labeled anti-human IgG polyclonal antibody; when the protein chip has the third detection spot formed by coating IgM polyclonal antibody, the kit contains alkaline phosphatase-labeled anti-human IgM polyclonal antibody.
According to a preferred embodiment, in said kit, the second detection spot 2 of said novel coronavirus antigen is a mixture of receptor binding domain protein of novel coronavirus spike protein Sl subunit and novel coronavirus spike protein S1 subunit protein in a mass ratio of 1 to 3:1, preferably in a mass ratio of 1:1. The experimental data found that such type of chip solution significantly improved the detection sensitivity and accuracy rate than single antigen solution. According to a preferred embodiment, in said kit, at least one of said protein chips contains two sub-detection zones, one of which has said third detection spot formed by coating human IgG polyclonal antibody; another sub-detection zone has said third detection spot formed by coating IgM polyclonal antibodies. In this scheme, taking the chip shown in Figure 1 as an example, a part of the second detection spot in sub-detection zone 4 is formed by coating of human IgG polyclonal antibodies; a part of second detection spot in sub-detection zone 4 is formed by attachment of human IgM polyclonal antibody. According to a preferred embodiment, said kit includes two types of protein chips, wherein a third detection spot 3 on one protein chip is formed by coating human IgG polyclonal antibodies; and said third detection spot 3 on the other protein chip is formed by coating IgM polyclonal antibody. The kits in above two schemes can detect IgG antibodies and IgM antibodies in the serum of suspected patients to improve the accuracy of detection. According to a preferred embodiment, in said kit, in each sub-detection zone, each detection spot contains 3-6 repeats, each detection spot is arranged in a row, and the three detection spots are arranged in a rectangle Array. According to a preferred embodiment, in said kit, the size of each said sub-detection zone is 5-10 mm in length and 5-10 mm in width, and the size of detection spot is 0.5-1 mm in length and 0.5-1 mm in width. According to a preferred embodiment, in said kit, the separation distance between said detection spots is 0.2-1 mm.
According to a preferred embodiment, said kit further comprising an alkaline phosphatase-labeled anti-human IgG polyclonal antibody for use with a sub-detection zone having a human IgG polyclonal antibody coated to form said third detection spot, in the sense, that is, it is used to drop the serum to be tested on the detection spot in said sub-detection zone, and then drop it onto and cover the detection spot; It also comprising an alkaline phosphatase-labeled anti-human IgM polyclonal antibody for use with a sub-detection zone having a human IgM polyclonal antibody coated to form said third detection spot, that is, it is used to drop the serum to be tested on the detection spot in said sub-detection zone, and then drop it onto and cover the detection spot. The invention also provides a method for preparing any one of above kits, characterized in that the preparation of the protein chip includes the following steps: Obtaining a chip substrate with divided regions as the sub-detection; Spraying the BSA, novel coronavirus antigen, human IgG polyclonal antibody or human IgM polyclonal antibody in the sub-detection areas according to the preset pattern and size to form said first, second and third detections spot; Said novel coronavirus antigen protein is a receptor binding domain protein of spike protein S Isubunit of the novel coronavirus or spike protein S Isubunit, or a mixture thereof. According to a preferred preparation method, 10% BSA, 0.1-1mg/ml novel coronavirus antigen, 0.05-0.2mg/ml human IgG polyclonal antibody or human IgM polyclonal antibody are spotted and fixed on preset pattern and size to form said first, second and third detection spots. According to a preferred preparation method, 0.5 mg/ml novel coronavirus antigen, 0.1 mg/ml human IgG polyclonal antibody or human IgM polyclonal antibody, and 10% BSA are preferred. According to a preferred preparation method, all detection spots in each sub-detection area use the same volume of the obtained test, and it is between 10-30nL. According to a preferred preparation method, said detection spots are formed by multiple non-contact micro spray, preferably 3-10 times of non-contact micro spray and each time spraying 300-600pl.
Example 1. Preparation of protein chip
Reagents used: 10%BSA
Antigen 1": The receptor binding domain protein of the novel coronavirus spike protein Si subunit; product name: 2019-nCoV Spike Protein (RBD, Fc Tag), containing 457 amino acids, molecular weight 51.5kDa, produced by Sino Biological, Inc., item No. 40592-VO5H.
Antigen 2": Novel Coronavirus Spike Protein S Subunit Protein; product name: SARS-CoV-2 (2019-nCoV) Spike Protein (S5 Subunit, His Tag), containing 681 amino acids, molecular weight 76.5 kDa, produced by Sino Biological, Inc., item No. 40591-V08B1. Human IgG, 0.1mg/ml, produced by Sangon Biotech (Shanghai) Co., Ltd; Human IgM , 0.1 mg/ml, product of R&D Systems, Inc. Anti-human IgG labeled with alkaline phosphatase, produced by Sangon Biotech (Shanghai) Co., Ltd. Anti-human IgM labeled with alkaline phosphatase, produced by Sangon Biotech (Shanghai) Co., Ltd.; PBS solution: sodium chloride (NaCl) 8g, potassium chloride (KCl) 0.2g, disodium hydrogen phosphate (Na2HPO4) 1.44g, potassium dihydrogen phosphate (KH 2 PO 4) 0.24g, adjusted to pH 7.4 contained inl.OL PBST formula: IL PBS, + 1ml 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 serum samples are tested at one time.
Table 1. Chip designs Sub-detection First Second detection spot Third detection spot area program detection spot reagent reagent reagent Chip 1 10%BSA 0.5mg/ml antigenI 0.1mg/ml human IgG Chip 2 10%BSA 0.5mg/ml antigen2 0.1mg/ml humanIgG Chip 3 10%BSA 0.5mg/ml 0.1mg/ml humanIgG ( antigen1:antigen2=1:1) Chip 4 10%BSA 0.5mg/ml 0.1mg/ml humanIgG ( antigen1:antigen2=1:3) Chip 5 10%BSA 0.5mg/ml 0.1mg/ml humanIgG ( antigen1":antigen2=3:1) Chip 6 10%BSA 0.5mg/ml antigen 0.1mg/ml humanIgM Chip 7 10%BSA 0.5mg/ml antigen2" 0.1mg/ml humanIgM
Chip 8 10%BSA 0.5mg/ml 0.1mg/ml humanIgM ( antigen1:antigen2"=1:1) Chip 9 10%BSA 0.5mg/ml 0.1mg/ml humanIgM ( antigen1":antigen2"=1:3
) Chip 10 10%BSA 0.5mg/ml 0.1mg/ml humanIgM ( antigen1":antigen2=3:1) Each kind of detection spot was sprayed into a column with including four repeated detection spots, as shown in Figure 1. The spraying is done by non-contact micro spray device, the total spraying volume for each spot is 30nL, and each spraying is 600pL, and spraying for each spot need 5 times. The spraying volume of each spot is the same.
Example 2. Determination of detection method The operating process and principle are shown in Figure 5, and the steps are as follows:
Step 1. On one of the prepared chips, add a serum sample of one subject (7ul) to each sub-detection area and keep at room temperature for 15 minutes to allow the novel coronavirus antibody in the serum combine with the novel coronavirus antigen on the chip; Step 2. Wash the chip with 0.05% PBS-Tween for 5 times, and 5 seconds each time, to remove non-specific binding; Step 3.When the second detection spot is formed by human IgG polyclonal antibody attachment, add alkaline phosphatase-labeled anti-human IgG; when the second detection spot is formed by human IgM polyclonal antibody attachment, add alkaline phosphatase-labeled anti-human IgM; kept at room temperature at 15-25 minutes to form a novel coronavirus antigen-novel coronavirus antibody-alkaline phosphatase-labeled anti-human IgG (or novel coronavirus antigen-novel coronavirus antibody-alkaline phosphatase-labeled anti-human IgM complex), and human IgG-alkaline phosphatase-labeled anti-human IgG complex (or human IgM-alkaline phosphatase-labeled anti-human IgM complex) on the chip, and the chip was washed with 0.05% PBS-Tween for 5 times, and 5 seconds each time, to remove non-specific binding; Step 4.Add alkaline phosphatase substrate NBT/BCIP. Expected result: for positive serum sample, the first detection spot does not show color, the second detection spot shows purple blue, and the third detection spot shows purple blue. As shown in Figure 6, the test wells 1-10 are test results of all normal serum samples, which are negative. The BSA test spot and the novel coronavirus antigen test spot do not show color, and the human IgG/IgM test spot shows purple blue.
Experimental example, using the chip 3 for the detection of novel coronaviruses clinical serum by the steps in the method in the Example 2. The clinical serum samples were obtained from 9 subjects diagnosed Novel Coronavirus Pneumonia hospitalized by Zhejiang Hospital. The result is shown in Figure 7. Test well No.10 is the healthy serum, and the human IgG/IgM test spot shows a purple blue color, which is a negative result; Test wells No. 1-9 are the serum of subjects with novel coronavirus pneumonia. The results shows two rows of purple-blue test spots, namely, the novel coronavirus antigen test spot and the human IgG/IgM test spot shows purple-blue, which is a positive result. The same qualitative results were obtained for other schemes of chip testing.
Claims (5)
- Claims 1.A protein chip kit for detecting antibodies of novel coronavirus, characterized in that, it has at least one protein chip, Each said protein chip has at least one sub-detection area, each sub-detection area for detecting one specimen; Each sub-detection area is provided with the following detection spots: The first detection spot formed by the coating BSA, The second detection spot formed by the coating the novel coronavirus antigen protein,and The third detection spot formed by coating human IgG polyclonal antibody or IgM polyclonal antibody; The detection spots are separated from each other; Said novel coronavirus antigen protein is a receptor binding domain protein of spike protein S Isubunit of the novel coronavirus or spike protein S Isubunit, or a mixture thereof.
- 2.The protein chip kit according to claim 1, characterized in that: When the protein chip has the third detection spot formed by coating IgG polyclonal antibody, the kit contains alkaline phosphatase-labeled anti-human IgG polyclonal antibody; When the protein chip has the third detection spot formed by coating IgM polyclonal antibody, the kit contains alkaline phosphatase-labeled anti-human IgM polyclonal antibody.
- 3. The protein chip kit according to claim 1, characterized in that: Said second detection spot formed by coating novel coronavirus antigen at a concentration of 0.1-1 mg/ml; Said third detection spot formed by coating human IgG polyclonal antibody or IgM polyclonal antibody at a concentration of 0.05-0.2 mg/ml.
- 4.The protein chip kit according to claim 1, characterized in that: Said novel coronavirus antigen is a mixture of a receptor binding domain protein of novel coronavirus spike protein Si subunit and novel coronavirus spike protein SI subunit protein in a mass ratio of 1 to 3:1, Preferably a mixture of 1:1 mass ratio. Said novel coronavirus antigen protein is mixture of the receptor binding domain protein of spike protein Si subunit of the novel coronavirus and the spike protein SI subunit with weight ratio 1~3:1
- 5.The protein chip kit according to claim 1, characterized in that, at least one of said protein chips contains two kinds of sub-detection areas, wherein one kind of sub-detection area has the third detection spot formed by coating human IgG polyclonal antibody; the other kind of sub-detection area has said third detection spot formed by coating IgM polyclonal antibody; or Contains two kinds of said protein chips, wherein one kind of the protein chip has said third detection spot formed by coating human IgG polyclonal antibody; and the other kind of protein chip has said third detection spot formed by coating IgM polyclonal antibody.
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