Hybridoma cell strain 3G7 1B10, anti-GII.4 type norovirus P protein monoclonal antibody and application

A technology of 3G71B10 and hybridoma cell lines, applied in the direction of antiviral immunoglobulin, immunoglobulin, chemical instruments and methods, etc., can solve the problems of unsatisfactory human norovirus, missed detection, lack of cross-reactivity, etc. Achieve good pairing effect, good cross-reactivity and clear results

Active Publication Date: 2021-01-01
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with molecular biology detection methods such as RT-PCR, norovirus immunoassay methods lack cross-reactivity for different genotypes, and often miss detection, which cannot meet the clinical needs of multiple genotype detection of human norovirus

Method used

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  • Hybridoma cell strain 3G7 1B10, anti-GII.4 type norovirus P protein monoclonal antibody and application
  • Hybridoma cell strain 3G7 1B10, anti-GII.4 type norovirus P protein monoclonal antibody and application
  • Hybridoma cell strain 3G7 1B10, anti-GII.4 type norovirus P protein monoclonal antibody and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The preparation of embodiment 1 hybridoma cell line and the acquisition of monoclonal antibody

[0025] 1. Preparation of GII.4 type norovirus P protein immunogen

[0026] Amplification primers were designed according to the GII.4-2012 type GZ2014-L307 strain (GenBank KT202798) P region, the upstream primer P1 and the downstream primer P2 respectively introduced BamHI and EcoRI restriction sites (underlined part), and the downstream primer 5' Adding the CDCRGDCFC amino acid sequence (italics) at the end can promote the formation of Norovirus P particles, as shown in Table 1. The GII.4-2012 type GZ2014-L307 strain cDNA was used as a template for PCR amplification, and the amplified product and the pGEX-4T-1 vector (GE General Healthcare, USA) were subjected to BamHI and EcoRI double enzyme digestion, respectively. The amplified product was connected to the pGEX-4T-1 vector, transformed into Escherichia coli BL21 competent cells, and verified by colony PCR and sequencing...

Embodiment 2

[0041] The establishment of embodiment 2 double antibody sandwich ELISA method

[0042] 1. Screening of paired antibodies and determination of optimal working concentration

[0043] First, the two monoclonal antibodies were labeled according to the instructions of the horseradish peroxidase (HRP) coupling labeling kit, and the sensitivity of the labeled antibody was measured by direct ELISA, and the detection antibody was determined. Using the method of double-antibody sandwich ELISA, 1 μg / mL GII.4 P particles were used as the detection antigen, and the identified HRP-labeled monoclonal antibodies (HRP-3G7 1B10, HRP-3G4 1D6) were used as the detection antibodies, and the other two monoclonal antibodies ( 3G7 1B10, 3G4 1D6) were paired with the capture antibody respectively, and the best paired antibody and the optimal working concentration were determined. The results showed that HRP-labeled 3G7 1B10 antibody (HRP-3G7 1B10) had the best sensitivity, so HRP-3G7 1B10 was select...

Embodiment 3

[0046] The performance test of embodiment 3 method

[0047] The performance test of the double-antibody sandwich ELISA method established in Example 2 is as follows:

[0048] 1. Specificity test

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Abstract

The invention discloses a hybridoma cell strain 3G7 1B10, an anti-GII.4 type norovirus P protein monoclonal antibody and an application. A preservation number of the hybridoma cell strain 3G7 1B10 isGDMCC No:61139. The hybridoma cell strain 3G7 1B10 can secrete the anti-GII.4 type norovirus P protein monoclonal antibody. A double-antibody sandwich ELISA detection method is established by taking ahorseradish peroxidase labeled monoclonal antibody secreted by the hybridoma cell strain 3G7 1B10 as a detection antibody and taking a monoclonal antibody secreted by a hybridoma cell strain 3G4 1D6with a preservation number of GDMCC No:61138 as a capture antibody. The hybridoma cell strain 3G7 1B10 has the advantages of simplicity, convenience and quickness in operation, clear result and the like, provides a scientific basis for research of a norovirus in-vitro diagnosis method, and has an application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a hybridoma cell line 3G7 1B10, an anti-GII.4 type norovirus P protein monoclonal antibody and applications. Background technique [0002] Norovirus (Norovirus, NoV) has become the main cause of acute non-bacterial gastroenteritis in the world, and it can infect people of all ages, especially children under 5 years old. NoV can infect at low doses of about 100 virus particles per milliliter and is easily transmitted through food, water, and the vomit and faeces of infected persons. To meet public health demands, rapid and sensitive biosensing systems are required to detect and prevent the spread of viruses. [0003] NoV is a non-enveloped virus with a single-stranded positive-sense RNA genome of about 7.5-7.7kb, belonging to the family Caliciviridae and the genus Norovirus, and its genome is highly variable. The genome of NoV consists of three open reading frames (ORF1,...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569G01N33/535C12R1/91
CPCC07K16/10G01N33/577G01N33/56983G01N33/535C07K2317/33G01N2333/08
Inventor 薛亮高珺珊吴清平张菊梅蔡伟程左月婷蔡淑珍
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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