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A kind of imazalil hapten ym-a, artificial antigen, heavy chain antibody and its preparation method and application

A technology of YM-A and artificial antigen, which is applied in the biological field to achieve the effect of simple preparation method, strong specificity and high sensitivity

Active Publication Date: 2021-12-24
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is to overcome the defects and deficiencies of the existing imazalil detection method, and provide a kind of imazalil hapten YM-A, artificial antigen, heavy chain antibody and its preparation method and application

Method used

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  • A kind of imazalil hapten ym-a, artificial antigen, heavy chain antibody and its preparation method and application
  • A kind of imazalil hapten ym-a, artificial antigen, heavy chain antibody and its preparation method and application
  • A kind of imazalil hapten ym-a, artificial antigen, heavy chain antibody and its preparation method and application

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Effect test

Embodiment 1

[0048] The preparation of embodiment 1 imazalil hapten YM-A

[0049] A preparation method of imazalil hapten YM-A, comprising the following steps:

[0050] Dissolve 1mol imazalil in 50mL dioxane solution, add 0.5mol KMnO 4and NaIO4 under magnetic stirring at 20°C for oxidation reaction, after reflux for 4h, the reactants were layered, the organic layer was distilled under reduced pressure to remove the solvent, and the concentrate was purified by silica gel column chromatography to obtain the reaction product A; 100mg of the reaction product A Add 75 mg of carboxymethylhydroxylamine hemihydrochloride and carry out oximation condensation derivatization reaction under magnetic stirring in an oil bath at 60°C. After reflux overnight (24h), the reaction product is extracted with ethyl acetate and saturated NaCl, and the liquid-liquid layer is separated. After the filtrate was distilled under reduced pressure to remove the solvent, the organic phase was dried and concentrated, and...

Embodiment 2

[0052] The preparation of embodiment 2 imazalil artificial antigen

[0053] 1. Experimental method

[0054] A preparation method of imazalil artificial antigen, comprising the following steps:

[0055] The imazalil hapten YM-A prepared in Example 1 was coupled to ovalbumin OVA (albumin) and lactoferrin LF (lactoferrin) by the active ester method, and the imazalil hapten YM-A was dissolved in DMF Add 1.5eq NHS and EDC to the solution and react overnight at 4°C, which is recorded as solution A; add solution A dropwise to the PBS buffer solution of the carrier protein, and continue to react at 4°C for 8 hours; Dialyze at ℃ for 3 days, change the dialysate twice a day, dialyze 6 times in total, collect the solution (immunogen solution) in the dialysis bag, and obtain the imazalil artificial antigen (imazalil complete antigen YM-A-LF and inhibitory imazalil complete antigen YM-A-OVA), and utilize the ultraviolet spectrophotometer to the imazalil hapten YM-A that embodiment 1 prep...

Embodiment 3

[0059] The identification of embodiment 3 imazalil artificial antigen

[0060] 1. Experimental method

[0061] Healthy alpacas were used as experimental animals, and the imazalil complete antigen YM-A-LF prepared in Example 2 was used as the immunogen, and subcutaneously injected into the back and neck of the alpaca, and each immunization dose was 0.5 mL (which contained 0.5 mg immunogen). For the first immunization, 0.5 mL of complete Freund's adjuvant was emulsified with antigen for immunization, and 4 weeks later, 0.5 mL of incomplete Freund's adjuvant was used for booster immunization after emulsification with antigen, and then booster immunization was performed every 2 weeks. Before immunization, 10 mL of blood-separated serum was used as a negative control. From the second immunization, 10mL of blood was taken one week after each immunization for serum titer and competition reaction detection to determine whether the imazalil complete antigen YM-A-LF is immunogenic.

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Abstract

The invention discloses an imazalil hapten YM‑A, an artificial antigen, a heavy chain antibody, a preparation method and an application thereof. The present invention firstly provides a kind of imazalil hapten YM-A, and its structural formula is as shown in formula (I): the skeleton structure overlap degree of this imazalil hapten YM-A and test substance imazalil is high, effective The immunogenicity of the imazalil hapten YM‑A was greatly improved; the artificial antigen obtained by coupling the imazalil hapten YM‑A with lactoferrin was further used as an immunogen to immunize alpacas, and the sheep with good immune effect Camel serum was separated using Protein A and Protein G affinity chromatography columns alternately, and the heavy chain antibody of imazalil was successfully prepared; and an analytical method based on the heavy chain antibody was established for the detection of imazalil by using the heavy chain antibody. The lowest detection limit of imazalil was 12.39ng / mL, IC 50 The concentration is 122.18ng / mL, and the linear range is 29.41-507.63ng / mL; and the preparation method of the imazalil hapten YM‑A, artificial antigen and heavy chain antibody is simple, easy, low-cost, and has broad application prospects.

Description

technical field [0001] The invention belongs to the technical field of biotechnology. More specifically, it relates to an imazalil hapten YM-A, an artificial antigen, a heavy chain antibody and a preparation method and application thereof. Background technique [0002] Imazalil, also known as imendazim, is a systemic fungicide, which has a good control effect on common penicillium and green mold in fruits and vegetables, and is widely used in various fruits and vegetables after harvest. Preservation and preservation of vegetables. Studies have found that if the human body takes imazalil for a long time, it will cause dysfunction of the endocrine system, and the nervous system and liver will also be affected. Due to the abuse of imazalil, my country's requirements for imazalil residues in food are becoming more and more stringent. Compared with the national standard GB 2763-2019, the latest national standard GB 2763-2019 adds 8 new varieties to be tested. [0003] The dete...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D233/60C07K14/77C07K14/79C07K16/44C07K16/06G01N33/543
CPCC07D233/60C07K14/77C07K14/79C07K16/44G01N33/543C07K2317/10G01N2033/184
Inventor 沈玉栋张咏仪徐振林王弘杨金易孙远明肖治理雷红涛
Owner SOUTH CHINA AGRI UNIV