PVY Yunnan isolate TAS-ELISA test kit and its preparing method

A detection kit and the technology of the kit are applied in the field of enzymology or biological devices, and can solve the problems of no report on the large-scale detection of plant viruses, and no TAS-ELISA detection kit.

Inactive Publication Date: 2005-05-04
YUNNAN ACAD OF AGRI SCI +2
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Problems solved by technology

[0007] So far, reports on the study of plant viruses by TAS-ELISA method have increased in recent years, but the large-scale detection of p

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  • PVY Yunnan isolate TAS-ELISA test kit and its preparing method
  • PVY Yunnan isolate TAS-ELISA test kit and its preparing method

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Embodiment Construction

[0060] Samples of diseased plants were collected in the field, and detected by electron microscopy and standard antibodies to contain a large amount of PVY and only the virus. After collection, the samples were stored in a -70°C refrigerator for later use. The extraction buffer was 0.2M PBS (pH 7.4 containing 0.5% mercaptoethanol, 0.01M EDTA).

[0061] Purification of virus purification solution:

[0062] 1) Take 50 g of diseased leaves, add 100 mL of extraction buffer, fully homogenize with an electric mixer, filter with double-layer gauze, add 30% chloroform to the filtrate, stir at 4 ° C for 30 min, centrifuge at 3000 g for 10 min, and collect the supernatant; the extraction buffer is 0.2M PBS (pH7.4, containing 0.5% mercaptoethanol, 0.01M EDTA);

[0063] 2) Add 8% PEG, 0.1M NaCl to the supernatant, dissolve and place at 4°C for 4h or overnight; centrifuge at 10000rpm, 4°C, 20min, take the precipitate, suspend it with 0.05M citric acid buffer (pH7.4), and centrifuge 10000...

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Abstract

This invention relates to a kind of potato virus Y (PVY) yunnan outlier three antibody sandwich enzyme immune adsorbing rule (TAS-ELISA) detection kit and its preparation method. The kit comprises positive and negative contrast, PVY multi-clone antibody, PVY single clone antibody, enzyme standard antibody and other materials and medicines. The PVY multi-clone antibody is got from rabbit immune adsorbing blood serum after separately purifying the PVY yunnan outlier. The PVY single clone antibody is got from mouse immune, cell fusion, clone and purification after separately purifying the PVY yunnan outlier. The detection sensitivity of the TAS-ELISA detection kit in this invention can reach 0.01ng/ml. It coincides with the comparing accuracy rate of electrical glass detection result, and it is 100 times than indirect ELISA and DAS-ELISA detection sensitivity.

Description

Technical field: [0001] The invention relates to a detection kit for triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) of potato virus Y (Potato virus Y, PVY) Yunnan isolate and a preparation method thereof, belonging to the field of enzymology or biological devices. Background technique: [0002] Plant viruses are important pathogens of crop virus diseases. With the development of plant virus-free seedling industrialization and the rise of modern facility agriculture, the detection and prevention and control technology of plant viruses has been paid more and more attention. At present, the detection technology of plant virus has four methods: serological technology, indicator plant determination, electron microscope detection, and molecular detection (including the detection of viral nucleic acid and protein). Various methods confirm each other, and at the same time, there are limitations in detection sensitivity, specialization, equipment and facility ...

Claims

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Application Information

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IPC IPC(8): G01N1/34G01N33/53G01N33/531
Inventor 张仲凯杨录明丁铭周雪平汪继玲李婷婷苏晓霞
Owner YUNNAN ACAD OF AGRI SCI
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