CN114113586A - Novel coronavirus antibody detection test strip and application thereof - Google Patents
Novel coronavirus antibody detection test strip and application thereof Download PDFInfo
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- CN114113586A CN114113586A CN202010866730.5A CN202010866730A CN114113586A CN 114113586 A CN114113586 A CN 114113586A CN 202010866730 A CN202010866730 A CN 202010866730A CN 114113586 A CN114113586 A CN 114113586A
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- G—PHYSICS
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- 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
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- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
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Abstract
The invention relates to a novel coronavirus antibody detection test strip and application thereof, belonging to the technical field of virus antibody detection. The test strip comprises a bottom plate, a sample pad, a gold-labeled combination pad, a blood filtering membrane, a detection line, a quality control line, a nitrocellulose membrane and an absorption pad; the sample pad, the gold-labeled combination pad, the blood filtering membrane, the nitrocellulose membrane and the absorption pad are sequentially stuck on the bottom plate from left to right, the sample pad is connected with the gold-labeled combination pad, the gold-labeled combination pad is connected with the blood filtering membrane, the blood filtering membrane is connected with the nitrocellulose membrane, and the nitrocellulose membrane is connected with the absorption pad; a detection line and a quality control line are sequentially distributed on the nitrocellulose membrane from left to right; the test strip adopts a sandwich method, detects the novel coronavirus antibody by the novel coronavirus antigen coated on the nitrocellulose membrane and the novel coronavirus antigen coated on the gold-labeled binding pad by utilizing the principle of colloidal gold immunochromatography, and is quick and accurate.
Description
Technical Field
The invention relates to the technical field of virus antibody detection, in particular to a novel coronavirus antibody detection test strip and application thereof.
Background
The International Committee for viral Taxonomy (International Committee on Taxonomy of virues, ICTV) announced that the formal classification of the novel coronavirus (2019-nCoV) was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on day 11, 2.2020. The World Health Organization (WHO) announces on the same day that the official name of the disease caused by this virus is COVID-19. Coronaviruses are a large family of viruses known to cause the common cold and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS).
After the body is infected with the novel virus for the first time, the immune system of the body can carry out immune defense on the virus and generate a specific antibody. IgM antibodies appear generally at 1-2 weeks, and IgG antibodies are produced at about 4 weeks or so. The use of the reagent for detecting the antibody of the feline new coronavirus provides serological evidence for clinic and can be used together with a nucleic acid detection method, thereby providing more comprehensive and accurate information for diagnosing the new coronavirus.
The antibody test paper strip can also assist the development of a novel feline coronavirus (SARS-CoV-2) vaccine, and is used for detecting the condition of the antibody generated in the body of the cat after the vaccination and the effect of the vaccine.
Disclosure of Invention
The invention provides a novel coronavirus antibody detection test strip and application thereof, and aims to solve the problems that the detection steps are complicated and time-consuming, a professional is required to operate and the test strip mainly depends on a laboratory in the existing novel feline coronavirus antibody detection technology. The invention adopts a sandwich method, detects the novel coronavirus total antibody in a sample by a novel coronavirus antigen coated on a nitrocellulose membrane and a novel coronavirus antigen coated on a gold-labeled binding pad by utilizing the principle of immunochromatography, and has quick and accurate detection method.
According to a first aspect of the present invention, there is provided a novel coronavirus antibody detection test strip, which comprises a base plate, a sample pad, a gold-labeled conjugate pad, a hemofiltration membrane, a detection line, a quality control line, a nitrocellulose membrane, and an absorption pad; the sample pad, the gold-labeled combination pad, the blood filtering membrane, the nitrocellulose membrane and the absorption pad are sequentially stuck on the bottom plate from left to right, the sample pad is connected with the gold-labeled combination pad), the gold-labeled combination pad is connected with the blood filtering membrane, the blood filtering membrane is connected with the nitrocellulose membrane, and the nitrocellulose membrane is connected with the absorption pad;
detection lines and quality control lines are sequentially distributed on the nitrocellulose membrane from left to right;
the gold-labeled binding pad is coated with a conjugate marker of a novel coronavirus recombinant antigen containing an Fc label and colloidal gold, and the amino acid of the Fc label is shown as SEQ ID NO. 1;
the detection line on the nitrocellulose membrane is coated with a novel coronavirus recombinant antigen; the quality control line on the nitrocellulose membrane is coated with an anti-mouse IgG-Fc fragment antibody secondary antibody, wherein the amino acid of the Fc fragment is shown in SEQ ID NO 1;
the novel coronavirus recombinant antigen coated by the gold-labeled binding pad and coupled with the colloidal gold and the novel coronavirus recombinant antigen coated by the detection line contain the same antigenic determinant;
the conjugate of the novel coronavirus recombinant antigen containing the Fc label and coated by the gold-labeled binding pad and the colloidal gold is used for specifically binding with a novel coronavirus antibody in a sample to be detected, and the novel coronavirus recombinant antigen coated by the detection line is used for specifically binding with the novel coronavirus antibody in the sample to be detected;
the conjugate of the novel coronavirus recombinant antigen containing the Fc tag and the colloidal gold coated by the gold-labeled binding pad is used for specifically binding with an anti-mouse IgG-Fc fragment antibody secondary antibody coated by a quality control line.
Preferably, the anti-mouse IgG-Fc fragment antibody secondary antibody is a rabbit anti-mouse IgG-Fc fragment antibody secondary antibody.
Preferably, the novel coronavirus recombinant antigen is a novel coronavirus recombinant S protein.
According to another aspect of the present invention there is provided the use of any one of the test strips for the detection of novel coronavirus antibodies.
Preferably, the method comprises the following steps:
s1: dropwise adding a sample to be detected on a sample pad, gradually carrying out chromatography on the sample to be detected to a gold-labeled binding pad, a blood filtering membrane, a nitrocellulose membrane and an absorption pad after 5-15 min, and judging the condition of the novel coronavirus antibody in the sample to be detected according to a color development result;
s2: if purple red strips appear in the detection line and the quality control line, the new coronavirus antibody in the sample to be detected is positive;
if a mauve strip only appears in the quality control line and no mauve strip exists in the detection line, the new coronavirus antibody in the sample to be detected is negative;
if a mauve strip appears in the detection line and no mauve strip exists in the quality control line, the detection card is invalid; and if no mauve strip exists in the detection line and the quality control line, the detection card is invalid.
Preferably, the sample to be tested is diluted with phosphate buffer solution and dropped onto the sample pad.
Preferably, the pH value of the sample to be detected is 6.5-8.0.
Generally, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:
(1) the invention adopts a sandwich method, detects the novel coronavirus total antibody in the blood of animals such as cats and the like by a novel coronavirus antigen coated on a nitrocellulose membrane and a novel coronavirus antigen coated on a gold-labeled binding pad by utilizing the principle of immunochromatography, and has quick and accurate detection method.
(2) The test strip adopts a highly specific antibody-antigen reaction and immunochromatographic analysis technology, and the novel coronavirus antibody in a sample is specifically combined with the novel coronavirus antigen on the conjugate of the novel coronavirus antigen and the colloidal gold; detecting specific binding of an in-line coated novel coronavirus antigen to a novel coronavirus antibody in the sample; the quality control line is coated with rabbit anti-mouse IgG-Fc fragment antibody, and the Fc label of the novel coronavirus recombinant antigen on the conjugate marker of the novel coronavirus recombinant antigen and the colloidal gold is specifically combined.
(3) The Fc label of the novel coronavirus recombinant antigen on the coupling marker of the colloidal gold is only combined with the rabbit anti-mouse IgG-Fc fragment antibody coated on the quality control line, and cannot be combined with the novel coronavirus antigen on the detection line, so that the novel coronavirus recombinant antigen has high specificity.
(4) The test strip can realize the on-site rapid detection of novel coronavirus antibodies of animals such as cats, is not limited to people any more, and has higher practical value and popularization value.
(5) Preferably, the anti-mouse IgG-Fc fragment antibody secondary antibody is a rabbit anti-mouse IgG-Fc fragment, and the production process is simple and easy to obtain.
(6) Preferably, the novel coronavirus recombinant antigen is novel coronavirus recombinant S protein, and the novel coronavirus recombinant S protein is good in specificity, free of biological activity and safe.
Drawings
FIG. 1 is a schematic structural diagram of a novel coronavirus antibody detection test strip, wherein the test strip comprises a base plate 1, a sample pad 2, a gold-labeled binding pad 3, a hemofiltration membrane 4, a detection line 5, a quality control line 6, a nitrocellulose membrane 7 and an absorption pad 8.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The invention relates to a novel coronavirus antibody detection test strip, which comprises a bottom plate 1, a sample pad 2, a gold-labeled binding pad 3, a hemofiltration membrane 4, a detection line 5, a quality control line 6, a nitrocellulose membrane 7 and an absorption pad 8, wherein the bottom plate is provided with a sample pad; the sample pad 2, the gold-labeled combination pad 3, the blood filtering membrane 4, the nitrocellulose membrane 7 and the absorption pad 8 are sequentially pasted on the bottom plate 1 from left to right, the sample pad 2 is connected with the gold-labeled combination pad 3, the gold-labeled combination pad 3 is connected with the blood filtering membrane 4, the blood filtering membrane 4 is connected with the nitrocellulose membrane 7, and the nitrocellulose membrane 7 is connected with the absorption pad 8;
a detection line 5 and a quality control line 6 are sequentially distributed on the nitrocellulose membrane 7 from left to right;
the gold-labeled binding pad 3 is coated with a conjugate marker of a novel coronavirus recombinant antigen containing an Fc label and colloidal gold, and the amino acid of the Fc label is shown as SEQ ID NO. 1;
a detection line 5 on the nitrocellulose membrane 7 is coated with a novel coronavirus recombinant antigen; the quality control line 6 on the nitrocellulose membrane 7 is coated with an anti-mouse IgG-Fc fragment antibody secondary antibody, wherein the amino acid of the Fc fragment is shown as SEQ ID NO. 1;
the novel coronavirus recombinant antigen coated by the gold-labeled binding pad 3 and coupled with the colloidal gold and the novel coronavirus recombinant antigen coated by the detection line 5 contain the same antigenic determinant;
the conjugate of the novel coronavirus recombinant antigen containing the Fc label and coated by the gold-labeled binding pad 3 and the colloidal gold is used for specifically binding with a novel coronavirus antibody in a sample to be detected, and the novel coronavirus recombinant antigen coated by the detection line 5 is used for specifically binding with the novel coronavirus antibody in the sample to be detected;
the conjugate of the novel coronavirus recombinant antigen containing the Fc tag and coated by the gold-labeled binding pad 3 and the colloidal gold is used for specifically binding with an anti-mouse IgG-Fc fragment antibody secondary antibody coated by the quality control line 6.
In some embodiments, the anti-mouse IgG-Fc fragment antibody secondary antibody is a rabbit anti-mouse IgG-Fc fragment antibody secondary antibody.
In some embodiments, the novel coronavirus recombinant antigen is a novel coronavirus recombinant S protein.
Example 1: preparation of test strip components
1. Preparation of labeled substance
1.1 preparation of colloidal gold labels
Preparing a colloidal gold solution by adopting a trisodium citrate reduction method, adjusting the pH value of the colloidal gold solution to 9.5 by using 0.2mol/L potassium carbonate solution, uniformly mixing the purified novel coronavirus recombinant antigen containing the Fc tag with the colloidal gold solution, adding 10% sodium chloride solution, uniformly mixing, standing at room temperature for 2 hours to obtain a coupling marker of the novel coronavirus antigen and the colloidal gold, and storing at 4 ℃ for later use.
2. Assembly of test strips
(1) Preparing a clean tray, putting the clean tray into a sample pad, pouring a sample pad treatment solution, soaking for 10min, putting the sample pad into an oven, drying at 37 ℃ for 24h +/-4 h, sealing an aluminum foil bag to obtain a sample pad 2, and storing at room temperature for later use.
(2) And (3) spraying the conjugate marker of the novel coronavirus antigen and the colloidal gold on the bonding pad by using a gold spraying instrument, drying in an oven at 37 ℃ for 2h, sealing by using an aluminum foil bag to obtain a gold-labeled bonding pad 3, and storing at room temperature for later use.
(3) A film scribing instrument is adopted to respectively scribe rabbit anti-mouse IgG-Fc fragment antibody-6 # and novel coronavirus antigen-5 # on a quality control line (namely a C line) 6 and a detection line (namely a T line) 5 on a nitrocellulose membrane 7, drying is carried out for 24h +/-4 h in an oven at 37 ℃, an aluminum foil bag is sealed, the nitrocellulose membrane 7 with the detection line 5 (coated with the novel coronavirus antigen-5 #) and the quality control line 6 (coated with the rabbit anti-mouse IgG-Fc fragment antibody-6 #) is obtained, and the nitrocellulose membrane 7 is stored at room temperature for standby. The amino acid of the Fc fragment is shown in SEQ ID NO 1.
(4) And (3) sequentially sticking the processed materials such as the sample pad 2, the gold-labeled binding pad 3, the hemofiltration membrane 4, the nitrocellulose membrane 7 with the detection line 5 and the quality control line 6, the absorption pad 8 and the like on a bottom plate 1, wherein the bottom plate 1 is preferably a PVC bottom plate, and cutting, assembling and sealing to obtain the novel coronavirus antibody detection test strip for the cats, and storing the test strip at room temperature for later use. The assembled test strip is shown in figure 1.
Example 2: novel coronavirus antibody detection test strip using method
(1) Taking the test strip out of the plastic package, and placing the test strip on a dry desktop, wherein a quality control area (C) and a test area (T) are displayed on the test strip;
(2) adding the obtained blood sample into the sample adding hole;
(3) unscrewing a cover of the diluent bottle, and dripping 2 drops of diluent into a sample adding hole into which a blood sample is dripped; results were read within 15min, not after 20 min.
(4) And (4) interpretation of results:
positive: a purple red strip appears in the test area (T) and a purple red strip appears in the quality control area (C)
Negative: only one purple red strip appears in the quality control area (C), and no strip appears in the test area (T);
invalid result: only one purple red strip appears in the test area (T), and no purple red strip appears in the quality control area (C); no purple red band appears in the test area (T) and the quality control area (C).
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Sequence listing
<110> university of science and technology in Huazhong
Wuhan Nadakang Biotechnology Ltd
<120> novel coronavirus antibody detection test strip and application thereof
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Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser
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Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu
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Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr
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Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser
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Claims (7)
1. A novel coronavirus antibody detection test strip is characterized by comprising a bottom plate (1), a sample pad (2), a gold-labeled binding pad (3), a hemofiltration membrane (4), a detection line (5), a quality control line (6), a nitrocellulose membrane (7) and an absorption pad (8); the sample pad (2), the gold-labeled combination pad (3), the blood filtering membrane (4), the nitrocellulose membrane (7) and the absorption pad (8) are sequentially pasted on the bottom plate (1) from left to right, the sample pad (2) is connected with the gold-labeled combination pad (3), the gold-labeled combination pad (3) is connected with the blood filtering membrane (4), the blood filtering membrane (4) is connected with the nitrocellulose membrane (7), and the nitrocellulose membrane (7) is connected with the absorption pad (8);
the nitrocellulose membrane (7) is sequentially provided with a detection line (5) and a quality control line (6) from left to right;
the gold-labeled binding pad (3) is coated with a conjugate marker of a novel coronavirus recombinant antigen containing an Fc label and colloidal gold, and the amino acid of the Fc label is shown as SEQ ID NO. 1;
the detection line (5) on the nitrocellulose membrane (7) is coated with a novel coronavirus recombinant antigen; the quality control line (6) on the nitrocellulose membrane (7) is coated with an anti-mouse IgG-Fc fragment antibody secondary antibody, wherein the amino acid of the Fc fragment is shown as SEQ ID NO. 1;
the novel coronavirus recombinant antigen coated by the gold-labeled binding pad (3) and coupled with the colloidal gold and the novel coronavirus recombinant antigen coated by the detection line (5) contain the same antigenic determinant;
the conjugate of the novel coronavirus recombinant antigen containing the Fc label and coated by the gold-labeled binding pad (3) and the colloidal gold is used for being specifically bound with a novel coronavirus antibody in a sample to be detected, and the novel coronavirus recombinant antigen coated by the detection line (5) is used for being specifically bound with the novel coronavirus antibody in the sample to be detected;
the conjugate of the novel coronavirus recombinant antigen containing the Fc tag and the colloidal gold coated by the gold-labeled binding pad (3) is used for specifically binding with an anti-mouse IgG-Fc fragment antibody secondary antibody coated by a quality control line (6).
2. The novel coronavirus antibody detection test strip of claim 1, wherein the anti-mouse IgG-Fc fragment antibody secondary antibody is a rabbit anti-mouse IgG-Fc fragment antibody secondary antibody.
3. The novel coronavirus antibody detection test strip of claim 1, wherein the novel coronavirus recombinant antigen is a novel coronavirus recombinant S protein.
4. Use of a test strip according to any one of claims 1 to 3 for the detection of novel coronavirus antibodies.
5. The use according to claim 4, comprising the steps of:
s1: dropwise adding a sample to be detected on the sample pad (2), gradually carrying out chromatography on the sample to be detected to a gold-labeled binding pad (3), a blood filtering membrane (4), a nitrocellulose membrane (7) and an absorption pad (8) after 5-15 min, and judging the condition of the new coronavirus antibody in the sample to be detected according to a color development result;
s2: if purple red strips appear in the detection line (5) and the quality control line (6), the new coronavirus antibody in the sample to be detected is positive;
if a mauve strip appears only in the quality control line (6) and no mauve strip exists in the detection line (5), the new coronavirus antibody in the sample to be detected is negative;
if a mauve strip appears in the detection line (5) and no mauve strip exists in the quality control line (6), the detection card is invalid; and if no mauve strip exists in the detection line (5) and the quality control line (6), the detection card is invalid.
6. Use according to claim 4, wherein the sample to be tested is diluted with phosphate buffer and applied to the sample pad (2).
7. The use of claim 4, wherein the pH of the sample to be tested is 6.5-8.0.
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| CN202010866730.5A CN114113586A (en) | 2020-08-26 | 2020-08-26 | Novel coronavirus antibody detection test strip and application thereof |
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| CN2629040Y (en) * | 2003-05-26 | 2004-07-28 | 漳州市格林沃生物技术有限公司 | Improved simple and fast epidemic diseases diagnostic test paper strap |
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