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Monoclonal antibody and kit for cucumber green mottle mosaic viruses (CGMMVs)

A monoclonal antibody, green mottled flower technology, applied in antiviral immunoglobulin, biochemical equipment and methods, instruments, etc., to achieve the effect of strong sensitivity, exquisite design and good specificity

Active Publication Date: 2013-03-27
北京世纪元亨动物防疫技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] But so far, there is no report of a monoclonal antibody against cucumber green mottle mosaic virus CGMMV with good sensitivity, strong specificity and high titer

Method used

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  • Monoclonal antibody and kit for cucumber green mottle mosaic viruses (CGMMVs)
  • Monoclonal antibody and kit for cucumber green mottle mosaic viruses (CGMMVs)
  • Monoclonal antibody and kit for cucumber green mottle mosaic viruses (CGMMVs)

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1, cucumber green mottle mosaic virus monoclonal antibody hybridoma cell, preparation of monoclonal antibody and polyclonal antibody

[0040] 1. Propagation and purification of cucumber green mottle mosaic virus (CGMMV).

[0041]The source of cucumber green mottle mosaic virus was inoculated on gourds, and the diseased leaves were harvested on the 19th day. Add 3.5 times the volume of 0.1mol / L sodium phosphate buffer (containing 0.1% mercaptoethanol, pH7.2) to the diseased leaves, homogenate, filter with double gauze, and centrifuge at low speed (BECKMAN JA14 rotor, 10000r / min, 10min). Take the supernatant, add NaCl to a concentration of 0.1mol / L, add PEG (MV6000) to a concentration of 4% (w / v), TritonX-100 to a concentration of 2.5% (V / V), and stir at 4°C for 4h. Centrifuge (BECKMAN JA14 rotor, 11000r / min, 20min). The precipitate was suspended in 0.1mol / L sodium phosphate buffer and stirred overnight at 4°C. Centrifuge (BECKMANJA17 rotor, 11000r / min, 10mi...

Embodiment 2

[0056] Example 2: Determination of Ascites Titer of Cucumber Green Mottle Mosaic Virus Monoclonal Antibody

[0057] 1. Use the indirect ELISA method to measure the ascites titer of monoclonal antibody to cucumber green mottle mosaic virus.

[0058] The specific method is:

[0059] 1) Coat the microtiter plate with purified cucumber green mottle mosaic virus at a concentration of 2 μg / mL, freeze at 4°C overnight, and wash the microtiter plate.

[0060] 2) Seal the plate with phosphate buffer saline containing 2.5% BSA, wash the microtiter plate at 37°C for 30 minutes.

[0061] 3) Add ascites antibody solution diluted according to a certain ratio, 37°C, 2h. Wash the microtiter plate.

[0062] 4) Add 1:1000 dilution of alkaline phosphatase-labeled horse anti-mouse antibody, 37°C, 2h. Wash the microtiter plate.

[0063] 5) Add the substrate solution, at 37°C, when the color is suitable (5-15min), read the OD value at 450nm with a microplate reader.

[0064] The OD value afte...

Embodiment 3

[0065] Example 3: Ascites Specificity Determination of Cucumber Green Mottle Mosaic Virus Monoclonal Antibody

[0066] 1. Detection of Ascites Specificity of Monoclonal Antibody to Cucumber Green Mottle Mosaic Virus by Indirect ELISA

[0067] To determine the specificity of the monoclonal antibody against cucumber green mottle mosaic virus, it was tested whether there was cross-reaction with CGMMV-genus tobacco mosaic virus, capsicum light mottle virus and tooth blue ringspot virus. For the detection method, see Example 2 (Determination of Ascites Titer of Cucumber Green Mottle Mosaic Virus Monoclonal Antibody). During the detection, the 2ug / ml cucumber green mottle mosaic virus in the first step was replaced with 2ug / ml tobacco mosaic virus, capsicum light mottle virus, tooth leaf blue ringspot virus respectively, and the remaining steps, reagents and reaction conditions were used If it remains unchanged, it is possible to detect whether the 2G5 ascites and the corresponding...

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Abstract

The invention provides a monoclonal antibody and a kit for CGMMVs. Hybridism cell strains secreting the CGMMV monoclonal antibody are obtained, the obtained CGMMV monoclonal antibody has the advantages of good sensitivity, high specificity and titer and the like. The monoclonal antibody can be used for identifying whether viruses to be detected are CGMMVs and CGMMV samples to be detected are affected by CGMMVs. The monoclonal antibody can be used for preparing immunological diagnostic reagents for CGMMVs, for example, enzyme-linked diagnostic reagents, immune colloidal gold test strips, etc.

Description

technical field [0001] The invention belongs to biological detection technology, and relates to a cucumber green mottle mosaic virus monoclonal antibody and a kit. Background technique [0002] Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus in the family Tymoviridae. The virus mainly infects Cucurbitaceae crops, and can cause yellowing deformity of leaves, slow growth, delayed fruiting, yellowing and whitening of most fruits in Cucurbitaceae crops such as cucumbers, watermelons, melons, gourds, winter squash, figs, and leaf melons. Necrotic spots in the form of black-green watery scars are produced, and yield loss can be as high as 15%. Many countries and regions in the world have listed cucumber green mottle mosaic virus as an important quarantine virus of cucurbit crops. In December 2006, my country identified cucumber green mottle mosaic virus as a national agricultural plant quarantine pest, belonging to the third category of national q...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/558C12R1/91
Inventor 陈西钊孙明张丽马永缨迟立超秦亚嫚
Owner 北京世纪元亨动物防疫技术有限公司
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