A double-antibody sandwich enzyme-linked immunosorbent assay for the detection of Pseudomonas syringae Pseudomonas syringae

A pseudomonas, double antibody technology, applied in the field of immunoassay, to achieve the effect of high sensitivity, high uniformity and low cost

Active Publication Date: 2016-03-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Reports at home and abroad mainly focus on the pathogenic mechanism and the typing of isolated strains. There is no report on the immunological detection of Pseudomonas syringae pv.

Method used

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  • A double-antibody sandwich enzyme-linked immunosorbent assay for the detection of Pseudomonas syringae Pseudomonas syringae

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Experimental program
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Effect test

Embodiment Construction

[0026] specific implementation plan

[0027] The present invention is further illustrated by the following examples.

[0028] 1. Instrument:

[0029] TGL-40B desktop low-speed centrifuge, Shanghai Anting Scientific Instrument Factory;

[0030] KFLOW pure water machine, KFLOW company;

[0031] ZD-9556 horizontal shaker, Taicang Science and Education Equipment Factory;

[0032] 96-well 8×12 detachable microplate, Xiamen Yijiamei Laboratory Equipment Co., Ltd.;

[0033] MuLtiskaMks microplate reader, ThermoLabsystems;

[0034] Adjustable pipette, ThermoLabsystems;

[0035] Vortex mixer, Shanghai Huxi Instrument Analysis Factory.

[0036] 2. Reagents:

[0037] Tetramethylbenzidine (TMB), Shanghai Jingchun Industrial Co., Ltd.;

[0038] All other reagents were of analytical grade.

[0039] 3. Steps

[0040] 1. Preparation of monoclonal antibodies

[0041] a. Experimental animals: 5 8-week-old BALB / c mice were selected for immunization;

[0042] b. Antigen preparation: ...

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Abstract

The invention relates to a double-antibody enzyme-linked immunosorbent assay for detecting clove pseudomonas spot pathogenic and variant bacteria, which belongs to the field of the immunoassay. Inactivated clove pseudomonas spot pathogenic and variant inactivated bacteria are used for immunizing an 8-month-old BALB / c mouse, and six hybridoma cell strains for producing specific monoclonal antibodies can be obtained by virtue of immunization, cell fusion and screening. The six antibodies are respectively marked by HRP, and every two of the six antibodies are paired by adopting the clove pseudomonas spot pathogenic and variant bacteria as a target. The antibody 1D3 (CGMCCNo.9312 monoclonal cell strain Q) and the antibody 6C6 (CGMCCNo.9313 monoclonal cell strain R) are respectively used as a coating antibody and an ELISA antibody to establish the double-antibody enzyme-linked ELISA method of the clove pseudomonas spot pathogenic and variant bacteria, and LOD is 1.5*10<5>cfu / mL. By adopting the ELISA method to detect the double-antibody enzyme-linked immunosorbent assay for detecting clove pseudomonas spot pathogenic and variant bacteria, the specificity is good, the sensitivity is high, the cost is low, and the high-flux determination of the double-antibody enzyme-linked immunosorbent assay for detecting clove pseudomonas spot pathogenic and variant bacteria can be realized.

Description

technical field [0001] The invention relates to a double-antibody sandwich method for quantitatively detecting Pseudomonas syringae spotted pathogenic variant, which belongs to the field of immune analysis. Background technique [0002] Pseudomonas syringa epv. maculicola is an important pathogen of cruciferous vegetables in the world, mainly causing cruciferous bacterial black spot. The pathogen belongs to the Prokaryotic Kingdom Bacteria, Proteobacteria, and Pseudomonas in classification. The optimum culture temperature of the bacteria is 25-27°C, and the main hosts are cruciferous vegetables such as cabbage, radish, and cauliflower. [0003] Strengthening quarantine work and preventing the spread of infected seeds in non-infested areas are important measures to control the pathogen. Reports at home and abroad mainly focus on the pathogenic mechanism and the typing of isolated strains. At present, there is no report on the immunological detection of Pseudomonas syringae ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577
CPCG01N33/532G01N33/56911G01N33/577G01N2333/21
Inventor 匡华王文彬胥传来徐丽广刘丽强宋珊珊吴晓玲
Owner JIANGNAN UNIV
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