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Antibody or antigen binding fragment thereof for novel coronavirus nucleocapsid protein and application of antibody or antigen binding fragment thereof

A technology of nucleocapsid protein and coronavirus, which is applied in the direction of antiviral immunoglobulin, virus/bacteriophage, virus, etc., and can solve the problems of high technical requirements, prone to false negatives, high false positive rate, etc.

Active Publication Date: 2021-04-20
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 for novel coronavirus nucleocapsid protein and application of antibody or antigen binding fragment thereof
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  • Antibody or antigen binding fragment thereof for novel coronavirus nucleocapsid protein and application of antibody or antigen binding fragment thereof

Examples

Experimental program
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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 invention provides an antibody or an antigen binding fragment thereof for novel coronavirus nucleocapsid protein and application of the antibody or the antigen binding fragment thereof. The antibody is selected from any one of an mAb6 antibody, an mAb7 antibody, an mAb8 antibody and an mAb9 antibody; and the antibody is secreted by a hybridoma cell with preservation number of CCTCC NO: C2020236, CCTCC NO: C2020237, CCTCC NO: C2020238 or CCTCC NO: C2020239. The antibody can be used for detecting the existence of novel coronavirus or an antigen thereof in an environmental sample and / or a biological sample. In addition, the invention further provides a novel coronavirus detection kit prepared from the antibody. According to the detection kit, the nucleocapsid protein can be detected in the early stage of virus infection, and a rapid and accurate means is provided for clinical detection of novel coronaviruses.

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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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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