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Antibody or antigen-binding fragment thereof against novel coronavirus nucleocapsid protein and application thereof

A technology of nucleocapsid protein and coronavirus, applied in the direction of antiviral immunoglobulin, virus/bacteriophage, virus, etc., can solve the problem of long detection time of new coronavirus, inability to apply early preliminary screening, and specificity that cannot meet the needs, etc. problems, to achieve the effect of standardization and automation of operating procedures, high sensitivity and specificity, and easy operation

Active Publication Date: 2021-07-09
AMPSOURCE BIOPHARMA (SHANGHAI) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the current antibody detection has a high false positive rate, and the specificity cannot meet the needs
[0004] Nucleic acid detection has high specificity and sensitivity, but this detection method has high technical requirements, is prone to false negatives, requires special handling of samples, and requires professional equipment such as PCR amplifiers and gel electrophoresis. It takes a long time for the test, and professional technicians are required to operate and judge the test results, which cannot be applied to the early preliminary screening of communities, grassroots hospitals, airports, customs and even families.

Method used

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  • Antibody or antigen-binding fragment thereof against novel coronavirus nucleocapsid protein and application thereof
  • Antibody or antigen-binding fragment thereof against novel coronavirus nucleocapsid protein and application thereof
  • Antibody or antigen-binding fragment thereof against novel coronavirus nucleocapsid protein and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1 The acquisition of hybridoma cell lines and the preparation of monoclonal antibodies

[0106] This embodiment is used to obtain hybridoma cell lines and prepare monoclonal antibodies. The specific steps are as follows:

[0107] 1.1 Animal immunity

[0108] After fully emulsifying the SARS-CoV-2 N protein antigen (Sino Biological, 40588-V08B) with complete Freund's adjuvant, immunize male Balb / C mice (Shanghai Slack Experimental Animal Co., Ltd.) by multi-point immunization, 50 μg / monkey, the immunization cycle is once every three weeks;

[0109] On the 10th day after the third immunization, blood was collected from the eye socket, and the serum antibody titer was tested according to the indirect ELISA method to monitor the degree of immune response of the mice.

[0110] 1.2 Determination of immune serum titer

[0111] Establish an indirect ELISA method to measure the titer of immune serum: Coat polystyrene micro-96-well plates with 0.1 μg / mL recombinant SAR...

Embodiment 2

[0134] Example 2 Double Antibody Sandwich ELISA Screening for the Best Matching Monoclonal Antibody

[0135] The antibody produced by monoclonal hybridoma cells only targets one epitope of the antigen, and the "double-antibody sandwich method" is used to screen the best paired solid-phase antibody and labeled antibody.

[0136]A 6×6 matrix was used for pairwise screening of antibodies, and the above-mentioned 6 monoclonal antibodies were coated as capture antibodies, which were combined with 6 biotin-labeled monoclonal antibodies Bio-mAb6, Bio-mAb7, Bio-mAb8, Bio-mAb9, Bio-mAb10, and Bio-mAb11 were paired for rapid screening of captured and labeled mAb pairs in a sandwich ELISA.

[0137] By comparing the detection sensitivity, it was shown that when the monoclonal antibody mAb7 or mAb11 was used as the capture antibody and paired with Bio-mAb6, Bio-mAb8, Bio-mAb9 and Bio-mAb10, respectively, they could generate stronger signals.

Embodiment 3

[0138] Example 3 Functional Identification of Anti-N Protein Monoclonal Antibody

[0139] 3.1 Determination of monoclonal antibody affinity constant

[0140] The binding affinity constants of the purified mouse monoclonal antibodies mAb6, mAb7, mAb8, mAb9 and the N protein of the novel coronavirus were determined by biofilm interferometry (BLI). The ForteBio Octet RED&QK platform of PALL Company was used for determination, and the method was referred to the instruction manual of the platform.

[0141] First, the biotinylated SARS-CoV-2 N protein was immobilized on the surface of the SA sensor, and the above-mentioned monoclonal antibody against the SARS-CoV-2 N protein was used as the analyte. Process the data, and use the analysis software to fit the model of 1:1 binding. The fitted data basically overlaps with the experimental data, and the association and dissociation rate constants are obtained. K a and K d ,use K d remove K a Get the equilibrium dissociation cons...

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Abstract

The present invention provides an antibody or an antigen-binding fragment thereof against a novel coronavirus nucleocapsid protein and applications thereof, wherein the antibody is selected from any one of mAb6 antibody, mAb7 antibody, mAb8 antibody and mAb9 antibody; and the The antibody is secreted by the hybridoma cells with the deposit number of CCTCC NO: C2020236, CCTCC NO: C2020237, CCTCC NO: C2020238 or CCTCC NO: C2020239. The antibody can be used to detect the presence of novel coronavirus or its antigen in environmental samples and / or biological samples. In addition, the present invention also provides a novel coronavirus detection kit prepared by using the antibody, which can detect nucleocapsid protein in the early stage of virus infection, and provides a fast and accurate means for clinical detection of novel coronavirus.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to an antibody against a novel coronavirus, in particular to an antibody against a nucleocapsid protein of a novel coronavirus or an antigen-binding fragment thereof and applications thereof. Background technique [0002] The novel coronavirus (SARS-CoV-2) belongs to the genus Betacoronavirus, which is known to be the MERS virus that can cause Middle East Respiratory Syndrome and the SARS virus that is known to cause Severe Acute Respiratory Syndrome. The acute respiratory infectious disease caused by the new coronavirus is the novel coronavirus pneumonia (COVID-19). Its clinical manifestations are mainly fever, fatigue, and dry cough. Upper respiratory symptoms such as nasal congestion and runny nose are rare, and hypoxia and hypoxia may occur. . [0003] At present, the existing clinical novel coronavirus detection reagents include the enzyme-linked immunosorbent m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N5/20G01N33/577G01N33/569G01N33/552G01N33/546G01N33/543G01N33/535C12R1/91
CPCC12N5/163C12N2710/00G01N33/535G01N33/543G01N33/546G01N33/569G01N33/552G01N33/577C07K16/1003
Inventor 李强武翠翁仕强张晓峰周宇
Owner AMPSOURCE BIOPHARMA (SHANGHAI) INC
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