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Monoclonal antibody, immune fluorescence method and kit for detecting three rusts

A technology of monoclonal antibody and immunofluorescence, applied in the field of agricultural biology, can solve the problems of low degree of instrumentation

Active Publication Date: 2012-09-05
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, this method involves a low degree of instrumentation and is easy to operate, especially the sample pretreatment requirements are simple and easy to promote. Many authoritative organizations in the world have listed it as one of the analytical techniques for priority development.

Method used

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  • Monoclonal antibody, immune fluorescence method and kit for detecting three rusts
  • Monoclonal antibody, immune fluorescence method and kit for detecting three rusts
  • Monoclonal antibody, immune fluorescence method and kit for detecting three rusts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: the propagation of wheat leaf rust

[0039] According to the current prevalence of wheat rust in China, the present invention selects the main popular races PHT and THT of wheat leaf rust as experimental materials, and conducts identification and mass propagation in the high-temperature chamber of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0040] Specific steps are as follows:

[0041] 1. Plant Zhengmai 5389, a susceptible variety of wheat leaf rust, and inoculate with PHT and THT when the second new leaf grows.

[0042] 2. Spray the wheat leaves with a small watering can filled with clean water before inoculation. After the hands of the operator are disinfected with alcohol, dip in clean water and gently wipe off the wax paper layer on the leaves. On the leaves, wipe back and forth several times to evenly spread.

[0043] 3. After wiping the rust, the hands of the experimenter are first rinsed with 70% alcohol, and th...

Embodiment 2

[0048] Embodiment 2: wheat leaf rust fungus immune mice

[0049] 1. Collect freshly multiplied spores of two kinds of wheat leaf rust fungus, count them under a microscope, weigh 0.15 mg each of wheat leaf rust fungus PHT and THT uredospores (5×10 5 spores / mg), add 675 μl normal saline and 675 μl Freund’s complete adjuvant (US Sigma Company, product number F5881), put them into the syringe, connect the two syringes, push back and forth to mix well to obtain the emulsified antigen.

[0050] 2. Take female Balb / C mice aged 4-8 weeks, and take 200 μl of emulsified antigen to immunize by subcutaneous multi-point injection. After 3 weeks, the uredia spore suspension was mixed and emulsified with Freund's incomplete adjuvant, and after 3 weeks, the Freund's complete adjuvant (product number F5506) was used instead of Freund's complete adjuvant and the bacterial solution was mixed and emulsified according to Immunization was carried out with the same dose and method, and subsequent ...

Embodiment 3

[0058] Example 3: Screening of wheat leaf rust hybridoma cell line and preparation of monoclonal antibody

[0059] Take the mouse with higher serum titer in Example 2, boost immunization with 150 μl of the uredospore suspension in Example 2, take the mouse spleen and carry out cell fusion with myeloma cells after 5 days, and screen by "two-step screening method" Positive clones, at the same time, for the cells detected in the positive wells of specific antibodies, they were transplanted and cloned in time. The "limiting dilution method" is used for the cloning of hybridoma cells. The cell suspension is serially diluted to statistically contain only a single cell in each well, and then inoculated into a culture plate, so that a single cell can be propagated to form a homogeneous cell clone. , when there are multiple positive cells, try to take single clone wells for re-cloning, transfer them to 24-well plates, and then transfer them to culture flasks for expanded culture until ...

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Abstract

The invention discloses 'a monoclonal antibody, an immune fluorescence method and a kit for detecting puccinia striiformis f.sp.tritici, puccinia triticina f.sp.tritici and puccicinia graminis f.sp.tritici' and belongs to the field of agricultural biotechnology. The invention mainly provides the monoclonal antibody for detecting puccinia striiformis f.sp.tritici, puccinia triticina f.sp.tritici and puccicinia graminis f.sp.tritici, which is secreted by hybridoma cell line with a collection number CGMCC No.4414 and can identify puccinia triticina f.sp.tritici, puccicinia graminis f.sp.tritici and puccinia striiformis f.sp.tritici at the same time. The invention also provides a detection method based on the combination of the monoclonal antibody and an immune fluorescence technology and a kit. The monoclonal antibody, the detection method and the kit have the advantages of high specificity, high sensitivity, and simplicity and quickness of operation.

Description

technical field [0001] The invention relates to agricultural biotechnology, in particular to a monoclonal antibody, an immunofluorescence method and a kit for detecting three wheat rusts. Background technique [0002] Wheat rust includes pathogenic bacteria including wheat stripe rust (Puccinia striiformis f.sp.tritici), wheat leaf rust (Puccinia triticina f.sp.tritic) and wheat stem rust (Puccicinia graminis f.sp.tritici), is An important disease of a class of worldwide cereal crops. Wheat rust has the characteristics of wide occurrence area, strong outbreak, high epidemic frequency, and heavy damage and loss, which seriously threatens the production safety of wheat in China. In China, wheat stripe rust occurs most widely and is the primary factor affecting the production safety of wheat in China. The disease mainly occurs in relevant provinces (municipalities, autonomous regions) such as the northwest, southwest, middle and lower reaches of the Yangtze River, and North Ch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/16G01N33/577
Inventor 高利陈万权刘太国刘博
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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