CN114544960B - SARS-CoV-2 antigen detecting test paper strip - Google Patents

SARS-CoV-2 antigen detecting test paper strip Download PDF

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CN114544960B
CN114544960B CN202210424086.5A CN202210424086A CN114544960B CN 114544960 B CN114544960 B CN 114544960B CN 202210424086 A CN202210424086 A CN 202210424086A CN 114544960 B CN114544960 B CN 114544960B
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monoclonal antibody
variable region
cdr
chain variable
heavy chain
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CN114544960A (en
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张海江
张尧
李亚坤
沈迩萃
刘芸
杨增敏
郑明卉
伍树明
陈晓
王建英
刘永江
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Beijing Kangleweishi Biological Technology Co ltd
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Abstract

The invention discloses a test strip for detecting novel coronavirus infection. In particular to a colloidal gold test strip prepared by the group of antibodies for detecting the infection of the new coronavirus. The method comprises the steps of preparing and purifying a monoclonal antibody by cloning and expressing a purified prototype strain RBD-SD1 recombinant protein, marking a 4C4# monoclonal antibody by using colloidal gold, preparing a gold-labeled pad, assembling a test strip, and coating a 7H1# monoclonal antibody on a cellulose acetate membrane as a detection line and a rabbit anti-mouse antibody as a quality detection line. The prepared colloidal gold test strip for detecting the infection of the new coronavirus has the characteristics of convenience and quickness in operation, no need of special instruments and the like, has clear detection results, is suitable for quick diagnosis in clinical sites and large-scale epidemiological investigation of epidemic areas and the like, and is used as an auxiliary of a nucleic acid detection means.

Description

SARS-CoV-2 antigen detecting test paper strip
Technical Field
The invention relates to the field of medicines and disease detection, in particular to a detection antibody and a detection test strip for a novel coronavirus prototype strain S protein.
Background
The novel coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) is a novel beta coronavirus-novel coronavirus (SARS-CoV-2 Virus) which causes novel coronavirus pneumonia and is called new coronavirus pneumonia for short, and the new coronavirus pneumonia refers to pneumonia caused by 2019 novel coronavirus (COVID-19, SARS-CoV-2) infection.
The prevention and treatment of the new coronavirus is the first major thing in the world at present. Although inactivated vaccines exist for the new coronavirus and recombinant protein vaccines are marketed as preventive measures, the most effective method is isolation and monitoring. However, due to differences in sampling positions and manipulation techniques, missed detection often occurs, such as pharyngeal swabs, nasal swabs or anal swabs, which bring physiological discomfort to the person to be detected to a certain extent and are not user-friendly. There is a great clinical need for other effective detection methods as an auxiliary detection method.
In addition, the development of nucleic acid detection methods is limited by the accumulation of people in a certain region of the population, the accumulation of people during large-scale screening, the overloading of equipment, etc. The colloidal gold method is suitable for large-scale screening because of its simplicity, rapidness. The novel coronavirus (SARS-CoV-2) has four major structural proteins: spike protein (S protein), Nucleocapsid protein (N protein), Membrane protein (M protein), Envelope protein (E protein). Several antigen detection kits currently on the market mostly detect N protein, but not much detect S protein, and especially detect RBD region of S protein. Therefore, the screening of the antibody suitable for S protein detection is very important, and the colloidal gold test strip based on the screened antibody also has very strong market development prospect.
The experimental principle of the colloidal gold test strip is similar to that of the double-antibody sandwich method. Under the traction of the water absorbing material, the antigen to be detected moves upwards on the test strip and is firstly combined with the gold-labeled antibody to form an antigen-antibody complex, when the antigen-antibody complex moves upwards to the detection line, the other combination site of the detected antigen is combined with the monoclonal antibody (T line antibody) coated on the antigen-antibody complex to form a gold-labeled complex with two antibodies combined with one antigen (double antibody sandwich), and the detection line is red due to the deposition of gold particles. When the gold-labeled antibody not bound to the antigen ascends to the control line, it binds to the "anti-gold-labeled antibody", and therefore the control line also appears red. The sample to be tested has no tested antigen, and only the gold-labeled antibody on the test strip goes up and is combined with the antibody (C line antibody) at the control line, so that the gold particles are deposited at the position to be red.
Disclosure of Invention
The invention aims to disclose a group of antibodies for detecting the infection of the new coronavirus and a detection test strip prepared from the group of antibodies, and provides an auxiliary means for detecting the antigen of the new coronavirus by using a colloidal gold method besides nucleic acid detection.
The invention firstly researches and obtains 9 monoclonal antibodies aiming at novel virus RBD region combined with SD1 sequence: 1G1, 4C4, 5B9, 6D11, 7C4, 7H1, 7H9, 8B5, 8D 1. The purpose of the invention is to obtain a neutralizing antibody, but in the subsequent further research, the paired combination of the two monoclonal antibodies 4C4 and 7H1 is particularly suitable for preparing colloidal gold test paper, has very good effect and extremely high application value, and thus the invention is completed.
Therefore, the invention provides a test paper strip for detecting new coronavirus pneumonia, which is characterized by comprising 4C4# monoclonal antibody and 7H1# monoclonal antibody, wherein the CDR 1: SKSVSTSGYS (SEQ ID NO:1), CDR 2: YRA (SEQ ID NO:2), CDR 3: CMQSMEELT (SEQ ID NO: 3); CDR1 in the heavy chain variable region: SGFSLTSY (SEQ ID NO:4), CDR 2: VIWAGGS (SEQ ID NO:5), CDR 3: CARDYYGPFDY (SEQ ID NO: 6);
the light chain variable region of the 7H1# monoclonal antibody comprises CDR 1: SQSVDYDGDS (SEQ ID NO:7), CDR 2: YAA (SEQ ID NO:8), CDR 3: CQQSVEDPPT (SEQ ID NO: 9); CDR1 in the heavy chain variable region: SGFSLSTSGM (SEQ ID NO:10), CDR 2: HIWWDDD (SEQ ID NO:11), CDR 3: CARRMSPYYALDY (SEQ ID NO: 12).
Further, the amino acid sequence of the variable region of the 4C4# monoclonal antibody light chain: DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYRASNLESGVPARFSGSGSESDFTLTIDPVEEDDAAMYYCMQSMEELTFGAGTKLELK (SEQ ID NO: 13); heavy chain variable region amino acid sequence: SQVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARDYYGPFDYWGQGTTLTVSS (SEQ ID NO: 14);
the amino acid sequence of the variable region of the light chain of the 7H1# monoclonal antibody: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSVEDPPTFGGGTKLQIK (SEQ ID NO: 15);
heavy chain variable region amino acid sequence: SQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSHLTISKDTSRNQVFLKITSVDTADTATYYCARRMSPYYALDYWGQGTSVTVSS (SEQ ID NO: 16).
Preferably, the test strip takes the 4C4# monoclonal antibody as a capture antibody to mark colloidal gold, a gold-labeled pad is manufactured, a 7H1# monoclonal antibody coated nitrocellulose membrane (NC membrane) is taken as a detection line, and a rabbit anti-mouse antibody is taken as a quality control line.
Specifically, the reagent strip is formed by sequentially pasting a nitrocellulose membrane, a sample pad, a gold label pad and absorbent paper from bottom to top. Preferably, the nitrocellulose membrane is 20mm, the sample pad is 21mm, the gold mark pad is 9mm, and the absorbent paper is 30 mm; and the sample pad is pressed by 1.5mm of the gold-labeled pad, the gold-labeled pad is pressed by 1.5mm of the nitrocellulose membrane, and the absorbent paper is pressed by 1.5mm of the nitrocellulose membrane. Further preferably, the assembled test paper board is cut into test strips by a paper cutter, preferably with a reagent strip width of 3 mm.
The invention also provides a novel coronavirus monoclonal antibody which is a 4C4# monoclonal antibody, wherein a CDR 1: SKSVSTSGYS, CDR 2: YRA, CDR 3: CMQSMEELT, respectively; CDR1 in the heavy chain variable region: SGFSLTSY, CDR 2: VIWAGGS, CDR 3: CARDYYGPFDY, respectively; or 7H1# monoclonal antibody, CDR 1: SQSVDYDGDS, CDR 2: YAA, CDR 3: CQQSVEDPPT, respectively; CDR1 in the heavy chain variable region: SGFSLSTSGM, CDR 2: HIWWDDD, CDR 3: CARRMSPYYALDY is added.
Preferably, the amino acid sequence of the variable region of the 4C4# monoclonal antibody light chain: DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYRASNLESGVPARFSGSGSESDFTLTIDPVEEDDAAMYYCMQSMEELTFGAGTKLELK, respectively; heavy chain variable region amino acid sequence: SQVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARDYYGPFDYWGQGTTLTVSS, respectively;
the amino acid sequence of the light chain variable region of the 7H1# monoclonal antibody is as follows: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSVEDPPTFGGGTKLQIK
Heavy chain variable region amino acid sequence: SQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSHLTISKDTSRNQVFLKITSVDTADTATYYCARRMSPYYALDYWGQGTSVTVSS are provided.
More preferably, the 4C4# monoclonal antibody is secreted by a CoV S-RBD 4C4 monoclonal antibody cell strain; the 7H1# monoclonal antibody is secreted by a CoV S-RBD 7H1 monoclonal antibody cell strain.
The classification of CoV S-RBD 4C4 is named as monoclonal antibody cell strain, which is preserved in China general microbiological culture Collection center (CGMCC) at 25.02.2022, and the preservation address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, with the deposit number: CGMCC No. 45104.
The classification of CoV S-RBD 7H1 is named as monoclonal antibody cell strain, which is preserved in China general microbiological culture Collection center (CGMCC) at 25.02.2022, and the preservation address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, with the deposit number: CGMCC number 45103.
The invention also provides a nucleic acid molecule for encoding the novel coronavirus monoclonal antibody. Preferably, the DNA sequence of the light chain variable region of 4C4# monoclonal antibody is: GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGAGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGAGTCAGACTTCACTCTCACCATCGATCCTGTGGAGGAAGATGATGCTGCAATGTATTACTGTATGCAAAGTATGGAAGAGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 17); the heavy chain variable region DNA sequence is: TCCCAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCAGCTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAGCACAAATTATAATTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGAGATTACTATGGCCCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO:18);
the DNA sequence of the light chain variable region of the 7H1# monoclonal antibody is as follows: GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTGTTGAGGATCCTCCGACGTTCGGTGGAGGCACCAAGCTACAAATCAAA (SEQ ID NO:19), the heavy chain variable region DNA sequence is: TCCCAGGTTACTCTAAAAGAGTCTGGCCCTGGGATATTGAAGCCCTCACAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGATAAGTACTATAACCCATCCCTGAAGAGCCATCTCACAATCTCCAAGGATACCTCCAGAAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACTTACTACTGTGCTCGAAGAATGAGCCCCTACTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 20).
According to the invention, the 4C4# monoclonal antibody and the 7H1# monoclonal antibody are obtained by screening, and the test strip is prepared by a double-antibody sandwich principle, so that the novel coronavirus can be specifically detected. The colloidal gold test strip prepared by two monoclonal antibodies retains the characteristic of strong specificity and enhances the sensitivity and stability of the test strip. The test strip has good stability, simple operation and easy judgment and storage.
Drawings
FIG. 1 shows antigen detection of gold-labeled antibody.
FIG. 2 is a photograph of a monoclonal antibody pair cross-validated chromogenic test strip (T-line antibody 1G 1).
FIG. 3 is a photograph of a monoclonal antibody pair cross-validated chromogenic test strip (T-line antibody 4C 4).
FIG. 4 is a photograph of a monoclonal antibody pair cross-validated chromogenic test strip (T-line antibody 5B 9).
FIG. 5 is a photograph of a monoclonal antibody paired cross-validated chromogenic strip (T-line antibody 6D 11).
FIG. 6 is a photograph of a monoclonal antibody pair cross-validated chromogenic test strip (T-line antibody 7C 4).
FIG. 7 is a photograph of a monoclonal antibody pair cross-validated chromogenic test strip (T-line antibody 7H 1).
FIG. 8 is a photograph of a monoclonal antibody pair cross-validated chromogenic test strip (T-line antibody 7H 9).
FIG. 9 is a photograph of a monoclonal antibody pair cross-validated chromogenic test strip (T-line antibody 8B 5).
FIG. 10 is a photograph of a monoclonal antibody pair cross-validated chromogenic test strip (T-line antibody 8D 1).
Wherein, 1 to 10 in fig. 2 to 10 represent gold-labeled antibodies 1G1, 4C4, 5B9, 6D11, 7C4, 7H1, 7H9, 8B5 and 8D1, respectively.
Detailed Description
The invention is further illustrated by the following specific examples in order to provide a better understanding of the invention, which are not to be construed as limiting the invention.
EXAMPLE I screening of monoclonal antibodies
The screening method of the monoclonal antibody is specifically disclosed in the patent ZL202111165770.8 issued by I. And carrying out ELISA detection on the screened antibodies.
TABLE 1 ELISA method for detecting 4 batches of protein (Note: reading in the Table is OD)450）
Name of coating protein RBD-SD1 RBD-SD1 RBD-SD1 RBD-SD1
Batch number 20201210 20201022 20201020 20201017
7H1 1.639 1.348 1.626 2.255
5B9 1.628 1.197 1.719 2.938
6D11 1.245 1.077 1.307 2.332
4C4 1.188 0.756 1.203 2.287
7H9 1.317 1.016 1.187 2.231
8D1 1.109 0.742 1.291 2.195
8B5 1.122 0.791 1.124 2.231
1G1 0.841 0.55 0.725 1.734
7C4 0.555 0.361 0.708 1.71
EXAMPLE two screening of paired detection antibodies
(1) Firing of colloidal gold
A sodium citrate reduction method is adopted, 90mL of double distilled water is measured and added into a silicified conical flask, a magnetic stirring rotor is placed, a magnetic heating stirrer is adjusted, the rotor rotates at a constant speed, and a constant high temperature is kept. When bubbles begin to form at the bottom of the bottle, 10mL of 1% chloroauric acid solution is added, the stirring knob is adjusted to a proper rotating speed, the speed of the stirring rotor is reduced to the boiling liquid level without swirling, 3mL of 1% trisodium citrate is rapidly added in the middle part which avoids the vortex easily, and then the light yellow chloroauric acid aqueous solution is observed to quickly turn into gray, then turn into black and then gradually stabilize into red. The whole process is about 6min, boiling is continued for 15min, and the volume is made up to 100mL by deionized water after cooling. Thus preparing 30nm colloidal gold particles.
(2) Determination of optimal pH for gold-labeled antibody
With 0.4 mol/LK2C03And (3) regulating the pH of the colloidal gold solution to 4.0-9.0 in sequence by combining with precise pH test paper, and forming a detection gradient every 1 pH value. Add 3mg/mL of 5 uL of mAb to each tube, mix well on a shaker, and allow to stand at room temperature for 30 minutes. The color change of the colloidal gold is observed, the lowest pH value which is consistent with the color of the blank control is recorded, namely the optimal marking pH value is obtained, and the result shows that the optimal pH value is 7.0.
(3) Preparation of gold-labeled antibody
Will most suitable K2C03Adding colloidal gold solution, marking 1G1, 4C4, 5B9, 6D11, 7C4, 7H1, 7H9, 8B5 and 8D1# monoclonal antibodies, dissolving the marked gold-labeled antibodies in the redissolution, uniformly mixing to obtain the gold-labeled antibodies, and dropwise adding the gold-labeled antibodies on the gold-labeled pad. The effect of detecting the gold-labeled antibodies is shown in FIG. 1, and each gold-labeled antibody can be combined with the antigen eggWhite bound, appearing as a band of coloration visible to the naked eye.
(4) Determination of T-line (detection line) monoclonal antibody coating concentration on nitrocellulose membrane
Diluting the T-line 1G1, 4C4, 5B9, 6D11, 7C4, 7H1, 7H9, 8B5 and 8D1# monoclonal antibodies into 1, 0.5, 0.25 and 0.125mg/mL, coating each test strip with 2uL, operating according to the conventional conditions, observing the color development condition of the T-line, and determining the optimal coating concentration of the T-line monoclonal antibodies, wherein the optimal concentration is 0.125 mg/mL.
(5) Determination of coating concentration of C-line (control line) rabbit anti-mouse antibody on nitrocellulose membrane
Diluting the C-line rabbit anti-mouse antibody into 1, 0.5, 0.25 and 0.125mg/mL, coating 2uL of each test strip, operating according to conventional conditions, observing the color development condition of the result C line, and determining the optimal coating concentration of the C-line rabbit anti-mouse antibody. The optimal concentration is 0.125 mg/mL.
(6) Monoclonal antibody pairing cross validation
All gold-labeled antibodies and all T-line antibodies were set at 0.125mg/mL and crossover experiments were performed on paper strips (checkerboard method). The cross-validation results are shown in table 2 below. The two combined color development effects are best: (1) 4C4 gold-labeled antibody + T coil coated with 7C 4; (2) the 4C4 gold-labeled antibody + T coil was coated with 7H 1. 7C4# has been protected in my patent ZL202111165770.8 and 7C4# is a neutralizing antibody, so this patent focuses on the protection of the binding antibodies 4C4# and 7H1# that have not been disclosed.
TABLE 2 monoclonal antibody pairwise Cross validation
Figure 943809DEST_PATH_IMAGE001
NA, no experiment; -, no color band; +/-, a color development strip can be seen by naked eyes, but the picture is not clear; +, the color development band is clearly visible; the color development strip is clear, and the effect is similar to that of the C line; + + + +, the color development band is very clear. For more visual observation, the color test paper is photographed as shown in fig. 2-10, and 7C4# is protected and not described herein. Among them, the 4C4# antibody is the best antibody for gold labeling and is not suitable for T-line antibody. 7H1# is suitable for detection antibody and not suitable for gold labeled antibody. 4C4# and 7H1# are most suitable for development of test strips, and can simultaneously recognize antigens of a prototype strain and a mutant strain.
Example III application of test strip
Sequentially sticking a nitrocellulose membrane of 20mm, a sample pad of 21mm, a gold label pad of 9mm and absorbent paper of 30mm on a PVC bottom plate from bottom to top, wherein the sample pad is pressed by the gold label pad of 1.5mm, the gold label pad is pressed by the nitrocellulose membrane of 1.5mm, and the absorbent paper is pressed by the nitrocellulose membrane of 1.5 mm; after the paper is stuck in sequence, the assembled test paper board is cut into test strips with the width of 4mm by a paper cutter, a T-coil is coated with a 7H1# RBD-SD1 monoclonal antibody, a C-coil is coated with a rabbit anti-mouse antibody, and 4C4 is used as a gold-labeled antibody. The concentrations of the gold-labeled antibody and the T-line antibody were set at 0.25mg/mL, and the concentration of the rabbit anti-mouse antibody was 0.125 mg/mL.
As clinical positive samples are difficult to obtain at present, 196 employees are negative when the novel coronavirus IgG antibody detection test strip provided by the invention is used for detecting the employees of the applicant, and the coincidence rate of the nucleic acid detection results of the employees of the applicant reaches 100%. Therefore, the test strip detection result of the paired antibody combination is consistent with the fluorescence quantitative PCR detection result, and the paired antibody combination can be used as an effective auxiliary means for nucleic acid detection, and the detection result is observed in real time, so that the time consumption of nucleic acid detection is not consumed.
Finally, it should be noted that: the above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and the embodiments are examples, wherein the details that are not described are all the common general knowledge of those skilled in the art, and the technical solutions described in the foregoing embodiments can be modified or some technical features can be equivalently replaced by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
<110> Beijing Kangle guard Biotechnology Ltd
<120> SARS-CoV-2 antigen detection test paper strip
<160>20
<170>PatentIn Version 3.1
<210>1
<211>10
<212>PRT
<213>4C4# monoclonal antibody light chain CDR1
<400> 1
SKSVSTSGYS 10
<210>2
<211>3
<212>PRT
<213>4C4# monoclonal antibody light chain CDR2
<400>2
YRA 3
<210>3
<211>9
<212>PRT
<213>4C4# monoclonal antibody light chain CDR3
<400>3
CMQSMEELT 9
<210>4
<211>8
<212>PRT
<213>4C4# monoclonal antibody heavy chain CDR1
<400>4
SGFSLTSY 8
<210>5
<211>7
<212>PRT
<213>4C4# monoclonal antibody heavy chain CDR2
<400>5
VIWAGGS 7
<210>6
<211>11
<212>PRT
<213>4C4# monoclonal antibody heavy chain CDR3
<400>6
CARDYYGPFDY 11
<210>7
<211>10
<212>PRT
<213>7H1# monoclonal antibody light chain CDR1
<400>7
SQSVDYDGDS
<210>8
<211>3
<212>PRT
<213>7H1# monoclonal antibody light chain CDR2
<400>8
YAA 3
<210>9
<211>10
<212>PRT
<213>7H1# monoclonal antibody light chain CDR3
<400>9
CQQSVEDPPT 10
<210>10
<211>10
<212>PRT
<213>7H1# monoclonal antibody heavy chain CDR1
<400>10
SGFSLSTSGM 10
<210>11
<211>7
<212>PRT
<213>7H1# monoclonal antibody heavy chain CDR2
<400>11
HIWWDDD 7
<210>12
<211>13
<212>PRT
<213>7H1# monoclonal antibody heavy chain CDR3
<400>12
CARRMSPYYALDY 13
<210>13
<211>110
<212>PRT
<213>4C4# monoclonal antibody light chain variable region
<400>13
DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYRASNLESGVPARFSGSGSESDFTLTIDPVEEDDAAMYYCMQSMEELTFGAGTKLELK 110
<210>14
<211>117
<212>PRT
<213>7H1# Single-antibody heavy chain variable region
<400>14
SQVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARDYYGPFDYWGQGTTLTVSS 117
<210>15
<211>111
<212>PRT
<213>4C4# monoclonal antibody light chain variable region
<400>15 DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSVEDPPTFGGGTKLQIK 111
<210>16
<211>121
<212>PRT
<213>7H1# Single-antibody heavy chain variable region
<400>16 SQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSHLTISKDTSRNQVFLKITSVDTADTATYYCARRMSPYYALDYWGQGTSVTVSS 121
<210>17
<211>330
<212>DNA
<213>4C4# monoclonal antibody light chain variable region DNA
<400>17
GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGAGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGAGTCAGACTTCACTCTCACCATCGATCCTGTGGAGGAAGATGATGCTGCAATGTATTACTGTATGCAAAGTATGGAAGAGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA 330
<210>18
<211>351
<212>DNA
<213>4C4# Single-antibody heavy chain variable region DNA
<400>18
TCCCAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCAGCTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAGCACAAATTATAATTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGAGATTACTATGGCCCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 351
<210>19
<211>333
<212>DNA
<213>7H1# monoclonal antibody light chain variable region DNA
<400>19
GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTGTTGAGGATCCTCCGACGTTCGGTGGAGGCACCAAGCTACAAATCAAA 333
<210>20
<211>363
<212>DNA
<213>7H1# Single-antibody heavy chain variable region DNA
<400>20
TCCCAGGTTACTCTAAAAGAGTCTGGCCCTGGGATATTGAAGCCCTCACAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGATAAGTACTATAACCCATCCCTGAAGAGCCATCTCACAATCTCCAAGGATACCTCCAGAAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACTTACTACTGTGCTCGAAGAATGAGCCCCTACTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA 363

Claims (10)

1. The test strip for detecting the new coronavirus pneumonia is characterized by comprising 4C4# monoclonal antibody and 7H1# monoclonal antibody, wherein the CDR1 in the light chain variable region of the 4C4# monoclonal antibody: SKSVSTSGYS, CDR 2: YRA, CDR 3: CMQSMEELT, respectively; CDR1 in the heavy chain variable region: SGFSLTSY, CDR 2: VIWAGGS, CDR 3: CARDYYGPFDY;
the light chain variable region of the 7H1# monoclonal antibody comprises CDR 1: SQSVDYDGDS, CDR 2: YAA, CDR 3: CQQSVEDPPT, respectively;
CDR1 in the heavy chain variable region: SGFSLSTSGM, CDR 2: HIWWDDD, CDR 3: CARRMSPYYALDY is added.
2. The test strip of claim 1, wherein the 4C4# mab light chain variable region amino acid sequence: DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYRASNLESGVPARFSGSGSESDFTLTIDPVEEDDAAMYYCMQSMEELTFGAGTKLELK, respectively; heavy chain variable region amino acid sequence: SQVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARDYYGPFDYWGQGTTLTVSS, respectively;
the amino acid sequence of the light chain variable region of the 7H1# monoclonal antibody is as follows: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSVEDPPTFGGGTKLQIK, respectively; heavy chain variable region amino acid sequence: SQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSHLTISKDTSRNQVFLKITSVDTADTATYYCARRMSPYYALDYWGQGTSVTVSS is added.
3. The test strip of claim 1, wherein the 4C4# monoclonal antibody is used as a capture antibody to label colloidal gold to form a gold-labeled pad, the 7H1# monoclonal antibody is used to coat a nitrocellulose membrane as a detection line, and the rabbit anti-mouse antibody is used as a quality control line.
4. The test strip of claim 2, wherein the nitrocellulose membrane, the sample pad, the gold label pad, and the absorbent paper are sequentially pasted from bottom to top.
5. The test strip of claim 3, wherein the nitrocellulose membrane is 20mm, the sample pad is 21mm, the gold label pad is 9mm, and the absorbent paper is 30 mm; the sample pad is pressed by 1.5mm, the gold label pad is pressed by 1.5mm, and the absorbent paper is pressed by 1.5 mm; PVC is used as the bottom plate.
6. The monoclonal antibody of the novel coronavirus is 4C4# monoclonal antibody, and the variable region of the light chain of the monoclonal antibody is shown as CDR 1: SKSVSTSGYS, CDR 2: YRA, CDR 3: CMQSMEELT; CDR1 in the heavy chain variable region: SGFSLTSY, CDR 2: VIWAGGS, CDR 3: CARDYYGPFDY, respectively; or the monoclonal antibody is 7H1# monoclonal antibody, and the light chain variable region of the monoclonal antibody is CDR 1: SQSVDYDGDS, CDR 2: YAA, CDR 3: CQQSVEDPPT; CDR1 in the heavy chain variable region: SGFSLSTSGM, CDR 2: HIWWDDD, CDR 3: CARRMSPYYALDY are provided.
7. The monoclonal antibody against the novel coronavirus of claim 6, wherein the amino acid sequence of the variable region of the light chain of monoclonal antibody No. 4C4 is: DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYRASNLESGVPARFSGSGSESDFTLTIDPVEEDDAAMYYCMQSMEELTFGAGTKLELK, respectively; the heavy chain variable region amino acid sequence is: SQVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARDYYGPFDYWGQGTTLTVSS;
the amino acid sequence of the light chain variable region of the 7H1# monoclonal antibody is as follows: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSVEDPPTFGGGTKLQIK; the heavy chain variable region amino acid sequence is: SQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSHLTISKDTSRNQVFLKITSVDTADTATYYCARRMSPYYALDYWGQGTSVTVSS are provided.
8. The monoclonal antibody against the novel coronavirus of claim 6, wherein the 4C4# monoclonal antibody is secreted by a monoclonal antibody cell strain with the preservation number of CGMCC No. 45104; the 7H1# monoclonal antibody is secreted by a monoclonal antibody cell strain with the preservation number of CGMCC number 45103.
9. A nucleic acid molecule encoding the novel coronavirus monoclonal antibody of claim 6.
10. The nucleic acid molecule of claim 9, wherein the light chain variable region DNA sequence of mab # 4C4 is: GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGAGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGAGTCAGACTTCACTCTCACCATCGATCCTGTGGAGGAAGATGATGCTGCAATGTATTACTGTATGCAAAGTATGGAAGAGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA, respectively; the heavy chain variable region DNA sequence is: TCCCAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCAGCTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAGCACAAATTATAATTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGAGATTACTATGGCCCCTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA;
the DNA sequence of the light chain variable region of the 7H1# monoclonal antibody is as follows: GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTGTTGAGGATCCTCCGACGTTCGGTGGAGGCACCAAGCTACAAATCAAA, respectively; the heavy chain variable region DNA sequence is: TCCCAGGTTACTCTAAAAGAGTCTGGCCCTGGGATATTGAAGCCCTCACAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGATAAGTACTATAACCCATCCCTGAAGAGCCATCTCACAATCTCCAAGGATACCTCCAGAAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACTTACTACTGTGCTCGAAGAATGAGCCCCTACTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA is added.
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