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ELIS method and kit for panoramic identification of intra-genus broad spectrum of human norovirus antibody

A technology for identifying people and kits, which is applied in ELISA methods and kits to comprehensively identify the broad-spectrum of human Norovirus antibodies, and can solve the problems of measuring the broad-spectrum of NoV serum antibodies

Pending Publication Date: 2021-12-03
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

However, few methods can directly measure the broad spectrum of NoV serum antibodies

Method used

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  • ELIS method and kit for panoramic identification of intra-genus broad spectrum of human norovirus antibody
  • ELIS method and kit for panoramic identification of intra-genus broad spectrum of human norovirus antibody
  • ELIS method and kit for panoramic identification of intra-genus broad spectrum of human norovirus antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Preparation of a broad-spectrum kit for panoptic identification of human norovirus antibodies

[0036] 1. Dilute the NoV capsid protein of 28 genotypes (9 GI types and 19 GII types, each genotype is shown in Table 1) to 2 μg / μl, take 100 μl and coat it in a 96-well microtiter plate, Each protein was coated in triplicate wells. Glutathione S-transferase (GST)-tagged protein was used as a positive control. Place overnight at 4°C.

[0037] 2. Take out the 96-well plate and wash it 3 times with PBST. Add 5% skimmed milk powder solution and incubate at 37°C for 2h.

[0038] 3. After the incubation, take out the 96-well plate, wash it with PBST for 3 times, control the dry water and place it at 4°C for later use, and obtain a broad-spectrum kit for the panorama identification of human norovirus antibody ( figure 2 ).

[0039] Table 1

[0040]

[0041]

Embodiment 2

[0042] Example 2 Determination of Norovirus Antibody Titer

[0043] Take 100 μl of 2 μg / μl GII.4 and GII.8 capsid proteins and coat them in a 96-well microtiter plate, overnight at 4°C. The next day, the 96-well plate was washed with PBST three times, and then 200 μl of 5% skimmed milk powder solution was added to each well to block at 37° C. for 2 hours. After blocking, wash the plate 4 times with PBST, dilute the GII.4 monoclonal antibody and GII.4+GII.8 antiserum to be tested from 1:2000 and 1:500 respectively, and add 100 μl to each well at 37°C Incubate for 1 hour. After incubation, wash the plate 4 times with PBST, add 100 μl of HRP-labeled goat anti-mouse secondary antibody serum diluted 1:3000, and incubate at 37°C for 0.5h. After that, the plate was washed 5 times with PBST, 100 μl of TMB single-component chromogenic solution was added, and incubated at 37° C. in the dark for 5 minutes. Immediately add 50 μl 2M H at the end 2 SO 4 The reaction was terminated, and...

Embodiment 3

[0045] Embodiment 3 Norovirus antibody is broad-spectrum determination in the genus

[0046] Take out the sealed 96-well plate ( figure 2 ), GII.4 monoclonal antibody and GII.4+GII.8 antiserum were diluted 1:5000 and 1:500 respectively, and 100 μl was added to each well and incubated at 37°C for 1 hour, and GST protein antibody for positive control was added at the same time. Wash the plate 4 times with PBST, add 100 μl of HRP-labeled goat anti-mouse secondary antibody at a dilution ratio of 1:3000, and incubate at 37°C for 0.5h. After that, the plate was washed 5 times with PBST, 100 μl of TMB single-component chromogenic solution was added, and incubated at 37° C. in the dark for 5 minutes. Finally join 2M H immediately 2 SO 4 The reaction was terminated, and the OD 450nm was measured with a microplate reader. An OD value below 0.2 was defined as no binding; an OD value between 0.2-0.6 was defined as moderately strong binding; an OD value greater than 0.6 was defined as...

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Abstract

The invention discloses an ELISA method and a kit for panoramic identification of intra-genus broad spectrum of a human norovirus antibody. The kit contains 28 Nov capsid proteins of human norovirus genotypes, wherein the Nov capsid proteins are respectively coated in an elisa plate. The intragenus broad-spectrum ELISA method for identifying the human norovirus antibody has the advantages of simplicity in operation, short period, high flux, good stability and the like, and can determine the broad-spectrum range of the norovirus antibody among different types in one step. The kit can be widely applied to scientific research, inspection and quarantine and other institutions and viral vaccine or antibody research and development companies.

Description

Technical field: [0001] The invention belongs to the technical field of microbial safety detection, and in particular relates to a broad-spectrum ELISA method and kit for panoramic identification of human norovirus antibody genus. Background technique: [0002] Norovirus (NoV) is the most common cause of epidemic gastroenteritis, and about 18% (95% CI: 17%–20%) of acute gastroenteritis cases worldwide are associated with NoV infection. NoV causes direct medical costs of US$ 4.2 billion and social costs of US$ 60.3 billion annually worldwide. NoV can infect people of all ages, especially children under the age of 5 and adults over the age of 65. It has been reported that 212,000 deaths worldwide are caused by NoV infection every year. my country is also one of the main areas where NoV is prevalent. [0003] NoV belongs to the Caliciviridae family and is a non-enveloped single-stranded positive-sense RNA virus with a diameter of 27–37 nm. The full length of the genome is a...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543G01N33/58
CPCG01N33/56983G01N33/54306G01N33/581G01N2333/08G01N2469/20
Inventor 薛亮高珺珊吴清平张菊梅蔡伟程蔡淑珍
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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