CN114002427B - Method and kit for detecting novel coronavirus antigen - Google Patents

Method and kit for detecting novel coronavirus antigen Download PDF

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CN114002427B
CN114002427B CN202111644663.3A CN202111644663A CN114002427B CN 114002427 B CN114002427 B CN 114002427B CN 202111644663 A CN202111644663 A CN 202111644663A CN 114002427 B CN114002427 B CN 114002427B
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variable region
chain variable
seq
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antibody
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CN114002427A (en
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陈亚萍
尹午山
王美娜
张磊
贾晨晨
石云敬
赵雷
张丛
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Beijing Genesee Biological Technology Co ltd
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    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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    • G01—MEASURING; TESTING
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Abstract

The invention provides a novel coronavirus antigen detection method and a kit. The kit comprises a reagent strip, wherein the reagent strip comprises a bottom plate, and a sample pad, a colloidal gold pad, a nitrocellulose membrane and absorbent paper which are sequentially connected along the direction of sample chromatography and are positioned on the bottom plate; the colloidal gold pad is attached with a quality control marker marked by the colloidal gold and a second novel coronavirus nucleocapsid protein antibody; the nitrocellulose membrane is provided with a T line and a C line, the T line contains a first novel coronavirus nucleocapsid protein antibody, and the C line contains a ligand which is specifically combined with a quality control marker marked by colloidal gold; the first novel coronavirus nucleocapsid protein antibody has a first heavy chain variable region comprising the CDR sequences shown in SEQ ID NO 1, 2 and 3 and a first light chain variable region comprising the CDR sequences shown in SEQ ID NO 4, 5 and 6. The provided kit is used for in vitro detection of the novel coronavirus, and has extremely high sensitivity and specificity.

Description

Method and kit for detecting novel coronavirus antigen
Technical Field
The invention relates to the field of medicine detection, in particular to a novel coronavirus antigen detection method and a kit.
Background
The new coronavirus (SARS-COV-2) can probably cause the new coronavirus pneumonia (COVID-19) after infecting human body, has extremely strong infectivity and pathogenicity, is recognized as a serious epidemic disease by the world health organization, and has great influence on human society.
After the emergence of the new coronavirus epidemic situation, in order to realize the diagnosis of diseases and block the virus transmission chain, a plurality of new coronavirus in-vitro diagnosis technologies come out, and the main methods comprise new coronavirus nucleic acid detection, new coronavirus antibody detection and new coronavirus antigen detection. Antibody detection is a main detection means at the initial stage of epidemic situations, but the antibody detection is indirect detection, has the defects of incapability of directly judging whether the virus is carried or not, long window period and the like, and is gradually eliminated in the follow-up process. Nucleic acid detection has been used as a gold standard because of its high sensitivity, but nucleic acid detection requires specialized equipment and operators, and is slow and cannot be widely used in areas lacking these resources. The rapid detection of the antigen becomes the most important means for detecting the novel coronavirus in the world due to the characteristics of high detection speed, simple and convenient operation, high result accuracy and the like.
The detection principle is that a trace amount of novel coronavirus antigens contained in a sample are specifically recognized by a novel coronavirus antibody, and a detection signal is amplified through a beacon combined with the antibody so as to realize the detection purpose. The key raw material of the technology is the antibody of the novel coronavirus, which determines the detection accuracy.
However, there are still a series of problems in the detection of new coronaviruses. The main performance is as follows: 1) the binding affinity of the antibody and the antigen is not high enough, so that the detection sensitivity is low, and the detection omission easily occurs in the practical application. 2) The antibody is easily interfered by other substances in the sample, so that the detection specificity is poor, and false positive results are easy to occur in practical application. Further improvements are needed in the detection of new coronaviruses.
Disclosure of Invention
The invention aims to provide a novel coronavirus antigen detection method and a kit, which are applied to the detection of novel coronaviruses and have extremely high sensitivity and specificity.
Specifically, the invention provides the following technical scheme:
in a first aspect of the present invention, there is provided a kit comprising a test strip, the test strip comprising:
the device comprises a bottom plate, and a sample pad, a colloidal gold pad, a nitrocellulose membrane and absorbent paper which are positioned on the bottom plate;
along the direction of sample chromatography, the sample pad, the colloidal gold pad, the nitrocellulose membrane and the absorbent paper are sequentially connected;
the colloidal gold pad is attached with a quality control marker marked by the colloidal gold and a second novel coronavirus nucleocapsid protein antibody or antigen binding fragment,
a T line (detection line) and a C line (quality control line) are arranged on the nitrocellulose membrane, the T line contains a first novel coronavirus nucleocapsid protein antibody or antigen binding fragment, and the C line contains a ligand which is specifically bound with the colloidal gold-labeled quality control marker;
the first novel coronavirus nucleocapsid protein antibody or antigen-binding fragment has a first heavy chain variable region and a first light chain variable region,
the first heavy chain variable region comprises CDR sequences shown in SEQ ID NO. 1, 2 and 3, or sequences with at least one conservative amino acid substitution with the sequences shown in SEQ ID NO. 1, 2 and 3;
the first light chain variable region comprises CDR sequences shown in SEQ ID NO. 4, 5 and 6, or sequences with at least one conservative amino acid substitution with the sequences shown in SEQ ID NO. 4, 5 and 6.
In a second aspect, the present invention provides a novel coronavirus antigen detection method, comprising: the kit is used for detecting a sample to be detected. The sample to be tested is at least one selected from saliva, nasal swab, nasopharyngeal swab, pharyngeal swab, blood or urine.
The beneficial effects obtained by the invention are as follows: the kit provided by the invention contains the novel coronavirus nucleocapsid protein antibody or the antigen binding fragment for in-vitro diagnosis of the novel coronavirus, and has extremely high sensitivity and specificity.
Drawings
Fig. 1 is a schematic structural diagram of a test strip provided in an embodiment of the present invention, in which reference numeral 1 is a sample pad, 2 is a colloidal gold pad, 3 is a nitrocellulose membrane, 4 is absorbent paper, 5 is a bottom plate, 6 is a detection line, and 7 is a quality control line.
Detailed Description
The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. In describing the present invention, reference will now be made to terms used herein for explanation and illustration, which are for the purpose of facilitating an understanding of the concepts and are not to be construed as limitations on the scope of the invention.
Antibodies
Herein, the term "antibody" is an immunoglobulin molecule capable of binding to a specific antigen. Comprises two light chains with lighter molecular weight and two heavy chains with heavier molecular weight, wherein the heavy chains (H chains) and the light chains (L chains) are connected by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies widely and is called variable region (V region), and the carboxy-terminal (C-terminal) is relatively stable and varies little and is called constant region (C region). The V regions of the L chain and H chain are referred to as VL and VH, respectively.
Certain regions in the variable region, which have a higher degree of variation in amino acid composition and arrangement order, are called Hypervariable regions (HVRs), which are the sites where antigens and antibodies bind and are therefore also called complementarity-determining regions (CDRs). The heavy chain variable region and the light chain variable region both have three CDR regions.
The invention provides a kit, which comprises a test strip, wherein the test strip comprises: the device comprises a bottom plate, and a sample pad, a colloidal gold pad, a nitrocellulose membrane and absorbent paper which are positioned on the bottom plate; along the direction of sample chromatography, the sample pad, the colloidal gold pad, the nitrocellulose membrane and the absorbent paper are sequentially connected; the colloidal gold pad is attached with a quality control marker marked by colloidal gold and a second novel coronavirus nucleocapsid protein antibody or antigen binding fragment; a T line (detection line) and a C line (quality control line) are arranged on the nitrocellulose membrane, the T line contains a first novel coronavirus nucleocapsid protein antibody or antigen binding fragment, and the C line contains a ligand which is specifically bound with the colloidal gold-labeled quality control marker; the first novel coronavirus nucleocapsid protein antibody or antigen-binding fragment has a first heavy chain variable region comprising the CDR sequences shown in SEQ ID NOs 1, 2 and 3 or a sequence having at least one conservative amino acid substitution with the sequences shown in SEQ ID NOs 1, 2 and 3; the first light chain variable region comprises CDR sequences shown in SEQ ID NO. 4, 5 and 6, or sequences with at least one conservative amino acid substitution with the sequences shown in SEQ ID NO. 4, 5 and 6. "antigen-binding fragment" refers to an antibody fragment having the ability to specifically bind to a novel coronavirus nucleocapsid protein antigen. "conservative amino acid substitution" refers to the replacement of an amino acid by another amino acid residue that is biologically, chemically, or structurally similar. Biologically similar means that the substitution does not destroy the biological activity of the novel coronavirus nucleocapsid protein antibody or the novel coronavirus nucleocapsid protein antigen. Structurally similar refers to amino acids having side chains of similar length, such as alanine, glycine, or serine, or side chains of similar size. Chemical similarity refers to amino acids that are identically charged or are both hydrophilic or hydrophobic. For example, the hydrophobic residues isoleucine, valine, leucine or methionine. Or substitution of polar amino acids such as lysine with arginine, aspartic acid with glutamic acid, asparagine with glutamine, threonine with serine, and the like. The conservative amino acid substitutions mentioned may be one, two or three amino acid substitutions.
The sequences shown are shown below, and it is to be noted that these CDR sequences are derived from the IMGT database (http:// www.imgt.org). It will be appreciated by those skilled in the art that the CDR sequences from the heavy chain variable region and the light chain variable region will be slightly altered when the database of the selection assay is altered and such alterations are intended to be included within the scope of the present invention.
Figure 308285DEST_PATH_IMAGE001
In at least some embodiments, the first heavy chain variable region is selected from: (1) the sequence shown in SEQ ID NO. 7, or (2) a sequence having more than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the sequence shown in SEQ ID NO. 7.
In at least some embodiments, the first light chain variable region is selected from: (1) the sequence shown in SEQ ID NO. 8, or (2) a sequence having more than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the sequence shown in SEQ ID NO. 8.
In at least some embodiments, the first heavy chain variable region is encoded by the sequence set forth in SEQ ID NO 7.
QSVEESGGRLVTPGTPLTLTCTASGFSPSSYGMSWVRQAPGKGLEWIGYINIEDYAYYAPWAKGRFTISKTSSTTVDLKVTSPTTEDTATYFCGRGGYAADIWGPGTLVTVSS（SEQ ID NO:7）
In at least some embodiments, the first light chain variable region is encoded by the sequence set forth in SEQ ID No. 8.
DVVMTQTASPVSAAVGGTVTISCQSSESVYTNNRLSWFQQKPGQRPKLLIYRASKLASGVPSRFSGSGSGTQFTLIISDVQCDDAATYYCAGVGSGGTDIAFGGGTKVEIK（SEQ ID NO:8）
The provided antibodies or antigen-binding fragments may further comprise a heavy chain constant region and a light chain constant region in addition to the first heavy chain variable region and the first light chain variable region mentioned above. The heavy and light chain constant region sequences may be derived from sequences of human origin.
In at least some embodiments, the nucleotide sequence encoding the first heavy chain variable region is selected from the group consisting of: (1) a sequence shown as SEQ ID NO. 9; or (2) a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the sequence shown in SEQ ID NO. 9. In at least some embodiments, the nucleotide sequence encoding the first light chain variable region is selected from the group consisting of: (1) 10, SEQ ID NO; or (2) a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the sequence shown in SEQ ID NO. 10.
In at least some embodiments, the nucleotide sequence encoding the first heavy chain variable region is set forth in SEQ ID NO 9.
CAATCTGTAGAGGAGTCAGGTGGTAGACTTGTGACCCCCGGTACCCCACTTACTCTGACTTGTACGGCTAGTGGCTTCTCCCCGTCATCCTACGGGATGAGTTGGGTCCGACAGGCTCCCGGCAAGGGATTGGAATGGATAGGGTACATTAATATTGAAGACTACGCCTACTATGCCCCCTGGGCCAAGGGTCGATTCACCATCTCTAAAACTTCCAGCACGACTGTGGACTTGAAGGTAACATCACCAACAACTGAAGATACGGCAACATACTTCTGCGGACGCGGCGGATATGCGGCTGATATTTGGGGACCAGGAACCTTGGTTACCGTTTCTTCA（SEQ ID NO:9）
In at least some embodiments, the nucleotide sequence encoding the first light chain variable region is set forth in SEQ ID NO 10.
GATGTCGTTATGACACAGACCGCAAGTCCTGTGAGCGCAGCAGTAGGGGGCACGGTCACCATCAGTTGCCAAAGCTCTGAAAGCGTGTATACCAACAATAGGCTTTCTTGGTTTCAGCAGAAACCAGGGCAACGCCCTAAACTTCTTATCTACCGAGCGTCAAAGCTGGCTAGTGGCGTACCTAGCCGGTTCTCCGGTAGCGGAAGTGGGACCCAATTCACGCTCATTATCTCTGACGTACAATGCGACGACGCTGCCACATACTACTGTGCCGGGGTCGGGAGCGGTGGCACAGACATAGCTTTCGGTGGAGGCACGAAGGTAGAGATTAAA（SEQ ID NO:10）
In at least some embodiments, the second novel coronavirus nucleocapsid protein antibody or antigen-binding fragment has a second heavy chain variable region selected from the CDR sequences set forth in SEQ ID NOs 11, 12, and 13, or a sequence having at least one conservative amino acid substitution with the sequences set forth in SEQ ID NOs 11, 12, and 13; the second light chain variable region comprises CDR sequences shown in SEQ ID NO. 14, 15 and 16, or sequences with at least one conservative amino acid substitution with the sequences shown in SEQ ID NO. 14, 15 and 16.
The sequences shown are shown below, and it is to be noted that these CDR sequences are derived from the IMGT database (http:// www.imgt.org /). It will be appreciated by those skilled in the art that the CDR sequences from the heavy chain variable region and the light chain variable region will be slightly altered when the database of the selection assay is altered and such alterations are intended to be included within the scope of the present invention.
Figure 304054DEST_PATH_IMAGE002
In at least some embodiments, the second heavy chain variable region is selected from: (1) 17 or (2) a sequence having more than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the sequence shown in SEQ ID NO. 17.
In at least some embodiments, the second heavy chain variable region is the sequence set forth in SEQ ID NO 17.
EVQLVESGGGLVKPGGSLKLSCAASGITFSDYYMYWVRQTPEKRLEWVATISDGGSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAMYYCVRDKSMGFGAWFAYWGQGTLVTVSA（SEQ ID NO:17）
In at least some embodiments, the second light chain variable region is selected from: (1) 18 or (2) a sequence having more than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the sequence shown in SEQ ID NO. 18.
In at least some embodiments, the second light chain variable region is the sequence set forth in SEQ ID NO 18.
EIVLTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQRPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGHSIPYTFGGGTKLEIK（SEQ ID NO:18）
The provided antibodies or antigen-binding fragments may further comprise a second heavy chain constant region and a second light chain constant region in addition to the above-mentioned second heavy chain variable region and second light chain variable region. The second heavy chain constant region and the second light chain constant region sequences may be derived from sequences of human origin.
In at least some embodiments, the nucleotide sequence encoding the second heavy chain variable region is selected from the group consisting of: (1) 19 in SEQ ID NO; or (2) a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the sequence shown in SEQ ID NO. 19. In at least some embodiments, the nucleotide sequence encoding the second light chain variable region is selected from the group consisting of: (1) 20, SEQ ID NO; or (2) a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the sequence shown in SEQ ID NO: 20.
In at least some embodiments, the nucleotide sequence encoding the second heavy chain variable region is set forth in SEQ ID NO 19.
GAAGTCCAACTCGTTGAAAGTGGAGGCGGGTTGGTGAAACCAGGAGGGTCTCTCAAACTGAGCTGCGCTGCGAGCGGTATCACTTTCTCCGACTACTATATGTATTGGGTTAGACAAACTCCCGAGAAACGGCTGGAGTGGGTCGCAACTATCTCTGACGGAGGGAGTTACACGTACTATCCAGACTCCGTAAAGGGCAGGTTTACCATTAGCAGGGATAATGCCAAAAACAATCTTTATCTCCAAATGTCTTCCTTGAAGTCTGAGGATACTGCAATGTACTATTGTGTGCGCGACAAGTCAATGGGATTCGGGGCGTGGTTTGCTTATTGGGGACAGGGAACCCTTGTAACTGTCTCAGCC（SEQ ID NO:19）
In at least some embodiments, the nucleotide sequence encoding the second light chain variable region is set forth in SEQ ID NO: 20.
GAAATTGTCTTGACTCAGAGTCCCACCACAATGGCCGCTTCACCCGGCGAAAAGATAACTATTACGTGTTCTGCGTCCTCAAGTATCAGTAGCAATTATTTGCATTGGTATCAACAACGACCCGGTTTTTCCCCGAAACTTCTGATTTATCGAACCTCAAATCTCGCGTCAGGTGTTCCTGCGCGATTTAGCGGCTCTGGGAGTGGTACAAGCTACTCCCTTACAATTGGCACTATGGAGGCAGAGGACGTTGCCACCTATTATTGCCAACAAGGCCACTCAATCCCCTATACTTTCGGAGGTGGTACAAAGCTCGAAATAAAA（SEQ ID NO:20）
According to a specific embodiment, the quality control marker is at least one selected from the group consisting of goat anti-chicken IgY antibody, goat anti-rabbit IgG antibody, avidin, biotin-BSA, rabbit anti-mouse IgG, mouse IgG antibody, DNP antibody, and DNP-BSA. According to a specific embodiment, the mentioned ligand specifically binding to the quality control marker labeled with the colloidal gold is selected from at least one of chicken IgY antibody, goat anti-chicken IgY antibody, rabbit IgG antibody, goat anti-rabbit IgG antibody, biotin-BSA, avidin, mouse IgG antibody, rabbit anti-mouse IgG, DNP-BSA, and DNP antibody.
The kit may contain instructions and sample collection tools as required in addition to those mentioned above. One skilled in the art can take samples and perform the detection of the novel coronavirus antigen according to the instructions and the sample collection tool.
The invention also provides a novel coronavirus antigen detection method, which comprises the following steps: the kit is used for detecting a sample to be detected. The sample to be tested includes but is not limited to saliva, nasal swab, nasopharyngeal swab, pharyngeal swab, blood or urine, etc.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
And obtaining the mAb #4 antibody by using a B cell single cell cloning method. The method comprises the following specific steps:
rabbits are immunized with the novel coronavirus nucleocapsid protein as an antigen. Whole blood from rabbits was collected and PBMCs were prepared. Antigen-specific memory B blood cells are then sorted from PBMCs using a flow cytometer. Antibody variable regions were amplified by PCR method and cloned into pCMV _ HC and pCMV _ LC antibody expression vectors. Finally, antibody expression is carried out, and antigen specificity high affinity antibodies are screened. Among them, the antibody numbered mAb #4 had the strongest activity. The heavy chain variable region sequence is shown as SEQ ID NO. 7, and the light chain variable region sequence is shown as SEQ ID NO. 8.
The pCMV _ HC and pCMV _ LC vectors were transfected into CHO cell lines by the liposome method for expression of recombinant antibodies. The antibody is separated and purified by a Protein A affinity column, and the purity reaches more than 95 percent.
mAb #9 antibody was obtained by the hybridoma method. The method comprises the following specific steps:
mice were immunized with the novel coronavirus nucleocapsid protein as an antigen. Mouse spleens were collected and splenocytes prepared. Mouse spleen cells were fused with Myeloma cells and screened for stable hybridoma cells. Then screening by ELISA to produce antigen-specific antibodies.
Sequencing the obtained high specificity antibody. One of the antibodies was mAb #9, whose heavy chain variable region sequence is shown in SEQ ID NO. 17 and light chain variable region sequence is shown in SEQ ID NO. 18.
The coding sequence of the antibody was then introduced into the pCMV _ HC and pCMV _ LC antibody expression vectors. The pCMV _ HC and pCMV _ LC vectors were transfected into CHO cell lines by the liposome method for expression of recombinant antibodies. The antibody is separated and purified by a Protein A affinity column, and the purity reaches more than 95 percent.
Example 2
Example 2 provides a test strip, as shown in fig. 1. In fig. 1, reference numeral 1 denotes a sample pad (which is a glass cellulose film), 2 denotes a colloidal gold pad, 3 denotes a nitrocellulose film, 4 denotes absorbent paper, 5 denotes a base plate, 6 denotes a detection line (also referred to as a T line), and 7 denotes a quality control line (also referred to as a C line). The preparation method of each part in the test strip is as follows:
1. preparation of nitrocellulose membranes
Preparing a coating buffer solution: 0.05M PBS buffer (pH8.5) is used as coating buffer, and the solution is filtered through a 0.22 μ M membrane and kept at 4 ℃ for later use. Wherein the buffer solution formula comprises: NaCl 40g, KCl 1g, Na2HPO4·12H2O 14.5g、KH2PO41g, double distilled deionized water to 1000 mL.
Preparing a nitrocellulose membrane: the novel coronavirus nucleocapsid protein recombinant antibody (mAb #4) obtained in example 1 was diluted to 0.5-2mg/mL with coating buffer, machine adjusted, and scored as a T-line; and (3) marking the goat anti-chicken IgY as a C line, drying for 2-18 hours at 45 ℃, and packaging for later use.
2. Preparation of colloidal gold and colloidal gold labeled monoclonal antibody
(1) The following reagents were prepared:
a. preparing chloroauric acid: dissolving chloroauric acid with double distilled deionized water to prepare 0.04% solution for later use. The formula of the chloroauric acid solution is as follows: 0.4g of chloroauric acid is made up to 1000mL with double distilled deionized water.
b. Preparing trisodium citrate: dissolving sodium citrate with double distilled deionized water to prepare 1% solution for later use. The formula of the trisodium citrate solution is as follows: 10g of trisodium citrate is made up to 1000mL with double distilled deionized water.
c. Preparation of 0.1M potassium carbonate: prepared by double-distilled deionized water for standby. The formula of the 0.1M potassium carbonate solution is as follows: 13.8g of potassium carbonate was made up to 1000mL with double distilled deionized water.
d. Preparation of 5% BSA solution: prepared with 0.02MPBS for use. The formulation of the 5% BSA solution was: BSA 5g was made up to 100mL with 0.02M PBS pH7.4.
(2) Preparing colloidal gold:
boiling 0.04% chloroauric acid in an electric furnace, adding 1% trisodium citrate 6mL per 100mL of 0.04% chloroauric acid, boiling until the liquid is bright red, heating for 15 min, stopping heating, and cooling to room temperature.
(3) Preparing a colloidal gold labeled monoclonal antibody:
adjusting pH of the colloidal gold to 8.5 with 0.1M potassium carbonate, adding the novel coronavirus nucleocapsid antibody (mAb #9) according to 15-40 μ g antibody/mL colloidal gold, mixing for 30min with a magnetic stirrer, adding 5% BSA under stirring to a final concentration of 1%, and continuously stirring for 1 hr. 13000rpm, 4 ℃ centrifugal 30min, abandon the supernatant, the precipitation with one tenth of the initial colloidal gold volume of 5% BSA solution heavy suspension, placed at 4 ℃ for standby.
Adjusting pH of the colloidal gold to 8.5 with 0.1M potassium carbonate, adding IgY into the colloidal gold at a ratio of 10-50 μ g antibody/mL, mixing with magnetic stirrer for 30min, adding 5% BSA under stirring to a final concentration of 1%, and stirring for 1 hr. 13000rpm, 4 ℃ centrifugal 30min, abandon the supernatant, the precipitation with one tenth of the initial colloidal gold volume of 5% BSA solution heavy suspension, placed at 4 ℃ for standby.
3. Preparation of the colloidal gold pad
a. Preparing gold plating solution:
the formula of the gold plating solution is as follows: 20g of casein, 1g of PEG20000, 20mL of Tween-20, and 20g of trehalose, and the volume is adjusted to 1000mL by using 0.02M PBS (pH7.4).
b. Preparing a colloidal gold pad:
the prepared colloidal gold-labeled monoclonal antibody and the prepared colloidal gold-labeled chicken IgY are respectively mixed with the gold-spreading solution uniformly according to the dilution ratio of 1: 5-1: 20, the mixture is uniformly spread on a glass fiber film, each milliliter of the solution is spread by 20 square centimeters, and the mixture is dried for 2-8 hours at 55 ℃ for later use.
c. Assembled test strip
The absorbent paper, the nitrocellulose membrane, the colloidal gold pad and the sample pad were attached to a plastic base plate as shown in fig. 1, and cut longitudinally into small strips of 3.6mm in width to obtain test strips.
Example 3
Example 3 the test strip of example 2 was used to test the detection performance of the novel coronavirus:
435 clinical nasal swab samples confirmed to be negative and positive by PCR were selected, and the results of the tests are shown in Table 1 below. Wherein, in 65 cases of PCR-confirmed positive samples of the novel coronavirus, 60 positive samples and 5 negative samples are detected by adopting the test strip provided by the embodiment 2; 370 PCR-confirmed positive samples of the novel coronavirus were tested positive 0 and negative 370 with the test strip provided in example 2. The specific test results are shown in table 1 below.
TABLE 1 test results
Figure 831594DEST_PATH_IMAGE003
The test sensitivity and specificity of the test strip were calculated as follows, with CI representing the confidence interval:
clinical sensitivity-true positive result amount/positive sample amount 100% = 92.3% (95% CI: 83.0-97.5%)
Clinical specificity-amount of true negative results/amount of negative sample 100% =100% (95% CI: 99.0-100%)
Total coincidence rate of 430/435= 98.9% (95% CI: 97.3-99.6%)
The experimental result shows that the novel coronavirus nucleocapsid protein antibody provided by the invention has extremely high sensitivity and nuclear specificity when being used for in vitro diagnosis.
The specific clinical data are shown in the attached table:
TABLE 2 attached chart of clinical data
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Figure 384638DEST_PATH_IMAGE013
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Beijing Jiannaixi Biotech Co., Ltd
<120> method and kit for detecting novel coronavirus antigen
<130> PCN1210461
<160> 20
<170> PatentIn version 3.5
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<213> Artificial Sequence
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<223> HCDR1
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Gly Phe Ser Pro Ser Ser Tyr Gly
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<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> HCDR2
<400> 2
Ile Asn Ile Glu Asp Tyr Ala
1 5
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<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> HCDR3
<400> 3
Gly Arg Gly Gly Tyr Ala Ala Asp Ile
1 5
<210> 4
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> LCDR1
<400> 4
Glu Ser Val Tyr Thr Asn Asn Arg
1 5
<210> 5
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> LCDR2
<400> 5
Arg Ala Ser
1
<210> 6
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> LCDR3
<400> 6
Ala Gly Val Gly Ser Gly Gly Thr Asp Ile Ala
1 5 10
<210> 7
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> VH
<400> 7
Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Pro Ser Ser Tyr Gly
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45
Tyr Ile Asn Ile Glu Asp Tyr Ala Tyr Tyr Ala Pro Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys Val
65 70 75 80
Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Gly Arg Gly
85 90 95
Gly Tyr Ala Ala Asp Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser
100 105 110
Ser
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<211> 111
<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> VL
<400> 8
Asp Val Val Met Thr Gln Thr Ala Ser Pro Val Ser Ala Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Ser Cys Gln Ser Ser Glu Ser Val Tyr Thr Asn
20 25 30
Asn Arg Leu Ser Trp Phe Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu
35 40 45
Leu Ile Tyr Arg Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60
Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Ile Ile Ser Asp Val
65 70 75 80
Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Val Gly Ser Gly
85 90 95
Gly Thr Asp Ile Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 9
<211> 339
<212> DNA
<213> Artificial Sequence
<220>
<221>
<222>
<223> nucleotide sequence encoding heavy chain variable region
<400> 9
caatctgtag aggagtcagg tggtagactt gtgacccccg gtaccccact tactctgact 60
tgtacggcta gtggcttctc cccgtcatcc tacgggatga gttgggtccg acaggctccc 120
ggcaagggat tggaatggat agggtacatt aatattgaag actacgccta ctatgccccc 180
tgggccaagg gtcgattcac catctctaaa acttccagca cgactgtgga cttgaaggta 240
acatcaccaa caactgaaga tacggcaaca tacttctgcg gacgcggcgg atatgcggct 300
gatatttggg gaccaggaac cttggttacc gtttcttca 339
<210> 10
<211> 333
<212> DNA
<213> Artificial Sequence
<220>
<221>
<222>
<223> nucleotide sequence encoding light chain variable region
<400> 10
gatgtcgtta tgacacagac cgcaagtcct gtgagcgcag cagtaggggg cacggtcacc 60
atcagttgcc aaagctctga aagcgtgtat accaacaata ggctttcttg gtttcagcag 120
aaaccagggc aacgccctaa acttcttatc taccgagcgt caaagctggc tagtggcgta 180
cctagccggt tctccggtag cggaagtggg acccaattca cgctcattat ctctgacgta 240
caatgcgacg acgctgccac atactactgt gccggggtcg ggagcggtgg cacagacata 300
gctttcggtg gaggcacgaa ggtagagatt aaa 333
<210> 11
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> HCDR1
<400> 11
Gly Ile Thr Phe Ser Asp Tyr Tyr
1 5
<210> 12
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> HCDR2
<400> 12
Ile Ser Asp Gly Gly Ser Tyr Thr
1 5
<210> 13
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> HCDR3
<400> 13
Val Arg Asp Lys Ser Met Gly Phe Gly Ala Trp Phe Ala Tyr
1 5 10
<210> 14
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> LCDR1
<400> 14
Ser Ser Ile Ser Ser Asn Tyr
1 5
<210> 15
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> LCDR2
<400> 15
Arg Thr Ser
1
<210> 16
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> LCDR3
<400> 16
Gln Gln Gly His Ser Ile Pro Tyr Thr
1 5
<210> 17
<211> 121
<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> VH
<400> 17
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Asp Tyr
20 25 30
Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Val Arg Asp Lys Ser Met Gly Phe Gly Ala Trp Phe Ala Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 18
<211> 108
<212> PRT
<213> Artificial Sequence
<220>
<221>
<222>
<223> VL
<400> 18
Glu Ile Val Leu Thr Gln Ser Pro Thr Thr Met Ala Ala Ser Pro Gly
1 5 10 15
Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Ser Asn
20 25 30
Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Phe Ser Pro Lys Leu Leu
35 40 45
Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu
65 70 75 80
Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly His Ser Ile Pro
85 90 95
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 19
<211> 363
<212> DNA
<213> Artificial Sequence
<220>
<221>
<222>
<223> nucleotide sequence encoding heavy chain variable region
<400> 19
gaagtccaac tcgttgaaag tggaggcggg ttggtgaaac caggagggtc tctcaaactg 60
agctgcgctg cgagcggtat cactttctcc gactactata tgtattgggt tagacaaact 120
cccgagaaac ggctggagtg ggtcgcaact atctctgacg gagggagtta cacgtactat 180
ccagactccg taaagggcag gtttaccatt agcagggata atgccaaaaa caatctttat 240
ctccaaatgt cttccttgaa gtctgaggat actgcaatgt actattgtgt gcgcgacaag 300
tcaatgggat tcggggcgtg gtttgcttat tggggacagg gaacccttgt aactgtctca 360
gcc 363
<210> 20
<211> 324
<212> DNA
<213> Artificial Sequence
<220>
<221>
<222>
<223> nucleotide sequence encoding light chain variable region
<400> 20
gaaattgtct tgactcagag tcccaccaca atggccgctt cacccggcga aaagataact 60
attacgtgtt ctgcgtcctc aagtatcagt agcaattatt tgcattggta tcaacaacga 120
cccggttttt ccccgaaact tctgatttat cgaacctcaa atctcgcgtc aggtgttcct 180
gcgcgattta gcggctctgg gagtggtaca agctactccc ttacaattgg cactatggag 240
gcagaggacg ttgccaccta ttattgccaa caaggccact caatccccta tactttcgga 300
ggtggtacaa agctcgaaat aaaa 324

Claims (11)

1. A kit comprising a test strip, wherein the test strip comprises:
the device comprises a bottom plate, and a sample pad, a colloidal gold pad, a nitrocellulose membrane and absorbent paper which are positioned on the bottom plate;
along the direction of sample chromatography, the sample pad, the colloidal gold pad, the nitrocellulose membrane and the absorbent paper are sequentially connected;
the colloidal gold pad is attached with a quality control marker marked by colloidal gold and a second novel coronavirus nucleocapsid protein antibody or antigen binding fragment;
the nitrocellulose membrane is provided with a T line and a C line, the T line contains a first novel coronavirus nucleocapsid protein antibody or antigen binding fragment, and the C line contains a ligand which is specifically bound with the quality control marker marked by the colloidal gold;
the first novel coronavirus nucleocapsid protein antibody or antigen-binding fragment has a first heavy chain variable region and a first light chain variable region,
the first heavy chain variable region comprises CDR sequences shown in SEQ ID NO 1, 2 and 3;
the first light chain variable region comprises the CDR sequences shown in SEQ ID NO 4, 5 and 6;
the second novel coronavirus nucleocapsid protein antibody or antigen-binding fragment has a second heavy chain variable region and a second light chain variable region,
the second heavy chain variable region is selected from the CDR sequences shown in SEQ ID NO 11, 12 and 13;
the second light chain variable region comprises the CDR sequences shown in SEQ ID NOs 14, 15 and 16.
2. The kit of claim 1, wherein the first heavy chain variable region is: the sequence shown in SEQ ID NO. 7.
3. The kit of claim 1 or 2, wherein the first light chain variable region is: the sequence shown in SEQ ID NO. 8.
4. The kit of claim 1, wherein the nucleotide sequence encoding the first heavy chain variable region is: the sequence shown in SEQ ID NO. 9.
5. The kit of claim 1, wherein the nucleotide sequence encoding the first light chain variable region is: 10, SEQ ID NO.
6. The kit of claim 1, wherein the second heavy chain variable region is: the sequence shown in SEQ ID NO. 17.
7. The kit of claim 1, wherein the second light chain variable region is: 18 in sequence shown in SEQ ID NO.
8. The kit of claim 1, wherein the nucleotide sequence encoding the second heavy chain variable region is: the sequence shown in SEQ ID NO. 19.
9. The kit of claim 1, wherein the nucleotide sequence encoding the second light chain variable region is: 20 in SEQ ID NO.
10. The kit of any one of claims 1 to 8, wherein the quality control marker is at least one selected from the group consisting of goat anti-chicken IgY antibody, goat anti-rabbit IgG antibody, avidin, biotin-BSA, rabbit anti-mouse IgG, mouse IgG antibody, DNP antibody, and DNP-BSA.
11. The kit of claim 10, wherein the ligand specifically binding to the colloidal gold-labeled quality control marker is selected from at least one of chicken IgY antibody, goat anti-chicken IgY antibody, rabbit IgG antibody, goat anti-rabbit IgG antibody, biotin-BSA, avidin, mouse IgG antibody, rabbit anti-mouse IgG, DNP-BSA, and DNP antibody.
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