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Ivermectin derivative, monoclonal antibody for resisting abamectin type drug and application thereof

A monoclonal antibody and ivermectin technology, applied in the direction of sugar derivatives, chemical instruments and methods, instruments, etc., can solve the problems of poor broad spectrum of antibodies and single types of detection samples, and achieve low detection cost and high accuracy , The effect of simple detection method

Active Publication Date: 2019-01-11
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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  • Ivermectin derivative, monoclonal antibody for resisting abamectin type drug and application thereof
  • Ivermectin derivative, monoclonal antibody for resisting abamectin type drug and application thereof
  • Ivermectin derivative, monoclonal antibody for resisting abamectin type drug and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of immunogen and coating

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

[0034] (1) 5′-O-t-BuMe 2 Synthesis of Si-IVM: Put 4.0 g of IVM in a 50 mL round bottom flask, add 20 mL of tetrahydrofuran to dissolve, then add 1.8 g of imidazole and mix. Under mechanical stirring, dissolve 2.0gt-BuMe in 10mL of tetrahydrofuran 2 SiCl was added dropwise and reacted at 30°C for 4 hours. The reaction was monitored by TLC. After the reaction was completed, 100 mL of ethyl acetate was added to the reaction mixture and mixed, the mixture was washed with water three times to separate the ethyl acetate layer, and the organic layer was collected. Anhydrous MgSO 4 Dry and concentrate under reduced pressure to obtain a slightly yellow 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 group was collected, concentrated...

Embodiment 2

[0044] Example 2 Preparation of monoclonal antibodies

[0045] 2.1 Animal immunity

[0046] The immunogen 4"-CMO-IVM-KLH prepared by the inventor’s National Reference Laboratory for Veterinary Drug Residues was used to immunize female Balb / C mice (purchased from the Experimental Animal Center of Hubei Provincial Center for Disease Control and Prevention). The immunization procedure is to obtain the immunogen 4"-CMO-IVM-KLH is mixed with the same amount of protein content of 50μg and Freund's adjuvant and injected into mice to produce specific serum.

[0047] The immunization procedure is as follows: the basic immunization emulsifies the immunogen with an equal volume of Freund's complete adjuvant, and then injects multiple points under the skin of the back of the mouse. After that, boosts the immunization every 2 weeks and switches to Freund's incomplete adjuvant emulsification. Finally, three days before the fusion (preferably one month after the end of the immunization) intraperit...

Embodiment 3I

[0051] Example 3 Establishment of IVM indirect competitive ELISA detection method

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

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

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

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

[0057] Coating liquid: take Na 2 CO 3 1.59g, NaHCO 3 2.93g, add three distilled water to 1000mL, adjust the pH to 9.6;

[0058...

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Abstract

The invention discloses a novel ivermectin hapten. The novel ivermectin hapten is obtained through reacting with carboxymethoxylamine hemihydrochloride at the C4-OH position of ivermectin and substituting hydroxyl on C4 with carboxymethyl hydroxyamino; the derivative is coupled with carrier protein to be used as an immunogen, and can be used for preparing a monoclonal antibody for resisting an abamectin type drug. The invention further discloses one broad-spectrum monoclonal antibody for resisting the abamectin type drug; the monoclonal antibody can be used for simultaneously and specificallyidentifying abamectin, ivermectin, eprinomectin and emamectin; and an enzyme immunoassay method and a kit, which are established by utilizing the monoclonal antibody are suitable for detecting residues of the abamectin type drug in animal tissues and have the advantages of sensitivity in detection, high accuracy, good precision and the like.

Description

Technical field [0001] The present invention relates to a new ivermectin derivative and its application in preparing a monoclonal antibody against avermectin drugs. The present invention also relates to a new ivermectin derivative and its application in the preparation of monoclonal antibodies against avermectin drugs. Its application. Background technique [0002] Abamectin drugs include avermectin (AVM), ivermectin (IVM), eprinomectin (EPR), selamectin (SEL), moxiectin (MOX), and more Lamectin (DOR) and Emamectin (EMA), etc., belong to macrolide 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 avermectin drugs are neurotoxic, hepatotoxic, immunotoxic, and may be ecotoxic. In view of the harm of such drugs to human health and the ecological...

Claims

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Application Information

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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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