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Preparation method of anti-carbamate oxime ester pesticide butanocarb monoclonal antibody

A technology of monoclonal antibody and carbamate oxime, applied in the biological field, can solve the problems that have not yet been reported in the research and the blank of immunological detection technology

Active Publication Date: 2016-01-20
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Butanonecarb immunological detection technology is still blank at home and abroad, and there is no research report on this kind of method.

Method used

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  • Preparation method of anti-carbamate oxime ester pesticide butanocarb monoclonal antibody
  • Preparation method of anti-carbamate oxime ester pesticide butanocarb monoclonal antibody
  • Preparation method of anti-carbamate oxime ester pesticide butanocarb monoclonal antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] The detection of embodiment 1 butanocarb standard substance (Dr.Ehrensorfer, Germany)

[0088] The indirect competitive ELISA method was used to conduct a preliminary exploration of the detection curve. Methods as below:

[0089] Coating: Dilute the coating antigen (H5-OVA) 1000 times with coating buffer (0.05mol / L, pH 9.6) and add to the microtiter plate, 50 μl per well, incubate in a 37°C incubator for 2 hours;

[0090] (2) Plate washing: wash 5 times with washing solution PBST (0.05% Tween 20, 0.01mol / L, pH7.4), and pat dry with absorbent paper;

[0091] (3) Blocking: add 120 μL of 1% gelatin to each well, and incubate at 37° C. for 1.5 h;

[0092] (4) Plate washing: same as (2);

[0093] (5) Add pesticide and antibody mixture: dilute the standard solution of butanonecarb with PBS phosphate buffer 3 times, add 25 μL to each well, then add 25 μL of antibody solution diluted to a certain number of times, incubate at 37 °C for 1 hour, wash 5 times with PBST, And set...

Embodiment 2

[0100] Detect butanonecarb residues in the apple sample of embodiment 2

[0101] H5-OVA-coated ELISA plate 50 μL / well, butanonecarb antibody diluted 8000 times as the working concentration, 1% gelatin as the blocker, PBS phosphate buffer pH value is 7.5, ionic strength is 0.4M, organic solvent is methanol , 50 μL / well in a microtiter plate.

[0102] Sample preparation to be tested:

[0103] The apple sample was purchased from Nanjing Suguo Supermarket. Weighed 5g of the pre-homogenized sample, added the standard substance (butanonecarb) to a final concentration of 60μg / g, mixed well, left at room temperature for 1h, added 10mL of methanol, and oscillated fully. Centrifuge at 4000rpm for 5min, and transfer the supernatant into a 25mL volumetric flask. Add 10mL of methanol to the precipitate, shake fully, centrifuge at 4000rpm for 5min, transfer the supernatant into a 25mL volumetric flask, and dilute to 25mL with the optimal working buffer (without organic solvent). Take par...

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Abstract

The invention relates to a preparation method of a monoclonal antibody with an oxime carbamate pesticide butocarboxim resistant property, and belongs to the biological technical field. The invention discloses a preparation method of a monoclonal antibody which is dedicated for specifically identifying oxime carbamate pesticide butocarboxim and an application of the antibody in the high-sensitive and rapid detection of butocarboxim residual in agricultural products and agricultural production environment. A butocarboxim semi-antigen and a carrier protein are coupled through a lively ester method so as to form a complete antigen, the complete antigen is used to immunize a Balb / C rat, hybridoma cells are prepared from spleen cells and Sp2 / 0 myeloma cells of the immunized rat through a hybridoma technique, so that a monoclonal antibody which can stably secrete an anti-butocarboxim component is obtained. The preparation technique of the antibody is feasible, the whole antigen preparation process does not need special instrument or equipment, and the mass production of the antibody is easy.

Description

(1) Technical field [0001] The invention relates to the preparation of carbamate oxime ester pesticide butanocarb monoclonal antibody, which belongs to the field of biotechnology. It is specially used for the preparation of monoclonal antibodies that specifically recognize the carbamate oxime lipid pesticide butatonocarb, as well as the highly sensitive and rapid detection of butatonocarb residues in agricultural products and agricultural production environments, and is especially suitable for large-scale sample detection and on-site monitoring. (2) Background technology [0002] With the advancement of science and technology, pesticides have become the main means of preventing and controlling pests and diseases, and play a decisive role in stabilizing and increasing agricultural production. Since the 1970s, due to the restriction or prohibition of organochlorine pesticides and the increasing number of insect species resistant to organophosphate insecticides, the amount of c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C12N5/20C07C319/20C12R1/19
Inventor 刘凤权王利民方庆奎华修德
Owner NANJING AGRICULTURAL UNIVERSITY
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