|
The novel avermectin chemical structure of the antibiotic (AVMs), the unique mechanism of action, strong insecticidal activity and a broad spectrum insecticide, a strong drive to kill nematodes and in vitro arthropods. AVMs has made a significant contribution to crop protection, animal and human health, and is currently the most widely used one of the antiparasitic properties. AVMs abamectin (Avermectin, AVM),, ivermectin (Ivermectin, IVM), doramectin (Doramectin, DOR) drugs. Although AVMs effect dose is small, but as a fat-soluble drugs, AVMs longer residual effect of time in animals, so WHO classified as highly toxic compounds. AVMs residues in animal tissues has been one of the objects of research areas focus on monitoring of veterinary drug residues. At home and abroad have established a variety of physical and chemical detection of Ivica prime residual detection methods, although some methods are more sensitive and accurate, but the method is complicated, time-consuming, costly, and technical requirements of reasons not suitable for screening large numbers of samples. As a sensitive, rapid, and specificity of the method, the enzyme-linked immunosorbent assay is gradually applied to the detection of drug residues. In order to explore a way to quickly and efficiently detecting ivermectin residues, the present study used the monoclonal antibody technology, and enzyme-linked immunosorbent assay techniques, synthetic artificial antigen, prepared anti--ivermectin monoclonal antibody, the establishment of detecting IVM residual ELISA assay. Haptens synthesized by carbodiimide method with the the NHS method and the mixed acid anhydride method, the coupling of the hapten to the carrier protein, was prepared by artificial antigen IVM-BSA and IVM-OVA. The UV scanning preliminary determination coupled successful combination of two complete antigen IVM-BSA, IVM-OVA ratio of 16:1 and 21:1, respectively. EDC synthesis IVM two kinds of artificial antigen as immune antigen which IVM-BSA, a stable hybridoma cell lines secreting anti the IVM specific monoclonal antibody 6A9 hybridoma technique. Prepared titers of 1: 32000, and the protein concentration of 19.9mg/mL. Tylosin, erythromycin, acetyl erythromycin cross reaction rate of 0.5% or less, and ivermectin similar objects avermectin cross reaction rate at 15%. Mouse ascites monoclonal antibody 6A9 hybridoma cell lines established indirect competitive ELISA method for detecting IVM residues. Using coating antigen dilution of 1:4000 ascites antibody concentration of 1:8000, the drug concentration in 5 ~ 2000ng/mL the range of the standard curve linear equations y = 0.0725x - 0.795 (R2 = 0.9874), IC50 is 141.25ng/mL, LOD below 5ng/mL, quantitative limit of 9.25ng/mL. The preliminary add recycling test used in pig liver tissue and muscle tissue samples after pre-treatment for the determination. Liver, muscle sample processing solution for more than 4 times diluted sample matrix interference disappeared and in 5 ~ 5000ng/mL range within, ELISA standard curve linear equation: Y = 0.2019x-2.1081, IC50 141.25ng / g. 200,1000,5000 ng / mL ivermectin standard liquid do add a recovery test to detect muscle, the liver drugs recoveries between 66.8% to 97.4%, the intra difference between 3.82% to 6.74%, approved between the difference between 4.33% to 9.42%.
|