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A kind of ivermectin derivative and anti-abamectin monoclonal antibody and application thereof

A monoclonal antibody, abamectin technology, applied in the direction of sugar derivatives, chemical instruments and methods, instruments, etc., can solve the problems of poor broad-spectrum antibodies, single type of detection samples, etc., and achieve low detection cost and high accuracy. , the effect of small health hazards

Active Publication Date: 2021-09-17
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Visible, in the prior art, for the immunodetection of abamectin drugs, the design of most haptens is all in the C of AVM or IVM. 4 The -OH position is connected with succinic anhydride or glutaric anhydride to obtain a hapten containing 4 or 5 C indirect arms, and then coupled with a carrier protein as an immunogen. The application of the method is limited

Method used

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  • A kind of ivermectin derivative and anti-abamectin monoclonal antibody and application thereof
  • A kind of ivermectin derivative and anti-abamectin monoclonal antibody and application thereof
  • A kind of ivermectin derivative and anti-abamectin monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of immunogen and coating original

[0033] 1.1 Preparation of hapten 4"-CMO-IVM

[0034] (1) 5′-O-t-BuMe 2 Synthesis of Si-IVM: 4.0 g of IVM was placed in a 50 mL round bottom flask, 20 mL of tetrahydrofuran was added to dissolve it, and 1.8 g of imidazole was added and mixed. Under mechanical stirring, dissolve 2.0gt-BuMe in 10 mL of tetrahydrofuran 2 SiCl was added dropwise and reacted at 30°C for 4h. The reaction was monitored by TLC. After the reaction was completed, 100 mL of ethyl acetate was added to the reaction solution and mixed. The mixed solution was washed three times with water to separate the ethyl acetate layer, and the organic layer was collected. 4 It was dried and concentrated under reduced pressure to give a yellowish viscous substance. The residue was dissolved in an appropriate amount of ethyl acetate, and the product was separated by silica gel column chromatography (eluent ethyl acetate: n-hexane = 1:1, v / v), the second ...

Embodiment 2

[0044] Example 2 Preparation of monoclonal antibodies

[0045] 2.1 Animal immunization

[0046] The immunogen 4"-CMO-IVM-KLH prepared by the inventor's national veterinary drug residue benchmark laboratory was used to immunize female Balb / C mice (purchased from the Laboratory Animal Center of Hubei Provincial Center for Disease Control and Prevention). The immunization procedure was to take the immunogen 4"-CMO-IVM-KLH was mixed with an equal amount of Freund's adjuvant at a protein content of 50 μg and injected into mice to produce specific serum.

[0047] The immunization procedure was as follows: the immunogen was emulsified with an equal volume of Freund's complete adjuvant for the basic immunization, and then subcutaneously injected into the back of the mice at multiple points. After that, the immunization was boosted every 2 weeks, and the incomplete Freund's adjuvant was used for emulsification. Finally, intraperitoneal injection was given three days before fusion (pre...

Embodiment 3I

[0051] Example 3 Establishment of IVM Indirect Competitive ELISA Detection Method

[0052] 3.1 Preparation of reagents (reagents used in this example were prepared by the following methods unless otherwise noted)

[0053] AVM standard stock solution: Weigh a bottle of 5 mg AVM standard product, add 5 mL of methanol to dissolve, and vortex for 2 min, that is, the mother solution of 1 mg / mL.

[0054] Phosphate buffer (pH 7.4): accurately weigh NaCl 8.00g, KH 2 PO 4 0.20g, Na 2 HPO 4 ·12H 2 O2.90g, KCl 0.20g, dissolve in a small amount of deionized water, and dilute to 1000mL.

[0055] Phosphate buffer (pH 8): accurately weigh KH 2 PO 4 0.41g, K 2 HPO 4 5.59g, dissolve in a small amount of deionized water, and dilute to 1000mL.

[0056] Carbonate buffer (pH 9.6): accurately weigh Na 2 CO 3 1.59g, NaHCO 3 2.93g, dissolve in a small amount of deionized water, and dilute to 1000mL.

[0057] Coating solution: take Na 2 CO 3 1.59g, NaHCO 3 2.93g, add three dist...

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Abstract

The invention discloses a novel ivermectin hapten, which is derived from ivermectin C 4 ‑OH position, reacted with carboxymethylhydroxylamine hemihydrochloride, the C 4 The hydroxyl group on the derivative is replaced by carboxymethyl hydroxylamine group, and the derivative is used as an immunogen after being coupled with a carrier protein, and can be used to prepare monoclonal antibodies against avermectins. The invention also discloses a broad-spectrum anti-abamectin monoclonal antibody, which can specifically recognize abamectin, ivermectin, eprinomectin and emamectin at the same time Amectin, the enzyme-linked immunosorbent method and kit established with the monoclonal antibody are suitable for detecting avermectin drug residues in animal tissues, and have the advantages of sensitive detection, high accuracy, and good precision.

Description

technical field [0001] The present invention relates to a new ivermectin derivative and its application in the preparation of monoclonal antibodies against abamectins, and also relates to a monoclonal antibody against abamectins and its application. Background technique [0002] Abamectin drugs include Abamectin (AVM), Ivermectin (IVM), Epilin (EPR), Selamectin (SEL), Moximectin (MOX), Larmectin (DOR) and emamectin (EMA), etc., belong to the macrolide class of drugs, which are widely used in the treatment and prevention of animal parasitic diseases and the control of insects and mites in agricultural products. Abamectin drugs are extremely fat-soluble, and the drugs remaining in the animal body are difficult to degrade. Studies have shown that abamectins are neurotoxic, hepatotoxic, immunotoxic and possibly ecotoxic. In view of the harm of these drugs to human health and ecological environment, countries around the world have strictly stipulated their maximum residue limi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07H17/08C07K16/44G01N33/535G01N33/577
CPCC07H17/08C07K16/44G01N33/535G01N33/577
Inventor 彭大鹏陶燕飞袁宗辉倪腾腾王延新王玉莲潘源虎谢书宇陈冬梅王旭黄玲利郝海红戴梦红程古月瞿伟谢长清
Owner HUAZHONG AGRI UNIV
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