Hybridoma cell line 3g4 1D6, anti-GII.4 type norovirus P protein monoclonal antibody and application

A hybridoma cell line, 3G41D6 technology, applied in the biological field, can solve the problems of unsatisfactory human norovirus, lack of cross-reactivity, missed detection, etc., and achieve good matching effect, clear results and good cross-reactivity

Active Publication Date: 2022-07-29
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 line 3g4 1D6, anti-GII.4 type norovirus P protein monoclonal antibody and application
  • Hybridoma cell line 3g4 1D6, anti-GII.4 type norovirus P protein monoclonal antibody and application
  • Hybridoma cell line 3g4 1D6, anti-GII.4 type norovirus P protein monoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of hybridoma cell lines and acquisition of monoclonal antibodies

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

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

Embodiment 2

[0041] Example 2 Establishment of 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) conjugated labeling kit, and the sensitivity of the labeled antibody was determined by direct ELISA, and the detection antibody was determined. Using the double-antibody sandwich ELISA method, 1 μg / mL GII.4P particles were used as the detection antigen, and the determined 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 used as capture antibodies to be paired with them, respectively, to determine the optimal paired antibody and the optimal working concentration. The results showed that the HRP-labeled 3G7 1B10 antibody (HRP-3G7 1B10) had the best sensitivity, so HRP-3G7 1B10 ...

Embodiment 3

[0046] Performance testing of the method of Example 3

[0047] The double-antibody sandwich ELISA method established in Example 2 is tested for performance, as follows:

[0048] 1. Specificity test

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Abstract

The invention discloses hybridoma cell line 3G41D6, anti-GII.4 type norovirus P protein monoclonal antibody and application. The deposit number of the hybridoma cell line 3G41D6 is: GDMCC No: 61138. The hybridoma cell line 3G41D6 can secrete anti-GII.4 type norovirus P protein monoclonal antibody. The monoclonal antibody secreted by the hybridoma cell line 3G41D6 was used as the capture antibody, and the monoclonal antibody secreted by the hybridoma cell line 3G71B10, which was labeled with horseradish peroxidase under the deposit number of GDMCC No: 61139, was used as the detection antibody. Double antibody sandwich ELISA detection method. The invention has the advantages of simple and quick operation, clear results and the like, provides scientific basis for the research on the in vitro diagnostic method of norovirus, and has application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a hybridoma cell line 3G4 1D6, an anti-GII.4 type norovirus P protein monoclonal antibody and applications. Background technique [0002] Norovirus (NoV) has become the leading cause of acute nonbacterial gastroenteritis worldwide and can infect people of all ages, especially children under 5 years of age. NoV can infect at low doses of about 100 viral particles per milliliter and is easily spread through food, water, and the vomit and feces of infected people. To meet public health demands, fast 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-stranded RNA genome of about 7.5-7.7 kb, belonging to the family Caliciviridae, the genus Norovirus, and its genome is highly variable. The genome of NoV consists of three open reading frames (ORF1, ORF2, ORF3), of which ORF2 encod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/543G01N33/569G01N33/577
CPCC07K16/10G01N33/577G01N33/543G01N33/56983G01N2333/08
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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