Preparation And Identification Of Anti-idiotypic And Anti-metatypic Antibodies Ageinst Ivermectin

Ivermectin(IVM) is a kind antibiotics that belongs to the family of marocyclic lactone.It has been widely used in livestock due to its broad spectrum of insecticide characteristicsm. The effective doses of IVM are small, but as fat-soluble drugs, IVM in animals'bodies have long residual effect, so they are ranked as highly toxic compound by WHO.So its residue in animal tissue should not be ignored.Now,the main methods used for IVM detection are chromatography technology, such as:high-performace liquid chromatography(HPLC),gas chromatography(GS) etc; immunological analysis,including the ELISA,colloidal gold immunological method (CGIA) etc. Also chromatography technology are precise and sensitive,but not suitable for large batch samples'screening, because of their complication, long time consuming, big cost and high technical request. Immunological analysis can slove these problems, ELISA is an accurate, reliable, rapid and unique test mehtods,suitable for rapidscreeing of lagre samples.Conventional ELISA for IVM drugs are dependent on the competitive immunoassays,Although noncompetitive immunoassay procedures such as two-site immunometric assays offer a much higher sensitivity, these assay principles are not applicable to hapten measurement, because the molecular mass of haptens precludes simultaneous binding of two antibody molecules. During the last decade, various attempts have been made to construct noncompetitive immunoassays for haptens. Among them, anti-idiotype antibodies and anti-metatypic antibodies have been successfully introduced as key antibody reagents facilitating noncompetitive assays.In order to obtain for ivermectin (IVM) anti-idiotype or anti-metatype antibodies, we developed the polyclonal antibody and monoclonal antibody against IVM. These antibodies were purified and named with PAb1 and MAb1.The PAb1 and MAb1 were added to the 20% methanol-PBS that containing 10μg/mL IVM, then the mixture was stirred at 4℃for 24h, two kinds of antigen-antibody complex were prepared. BALB/c were immunized with the two kinds of complex, PAbl-IVM and MAb1-IVM. It was found that the immune effect was better when immunized with PAb1-IVM than with MAb1-IVM. After several fusion and screening experiments,3 hybridoma cells which could steadily secrete anti-idiotype antibody against IVM, were achieved and named with Ab2-1, Ab2-2 and Ab2-3. Characterization of Ab2 was investigated, the results revealed that Ab2 had the high specificity. Ab2 could only react with antibody against IVM(Abl), whereas not react with the other antibodies, and affinity constant of antibody is 5.04×1010L/mol. When IVM-OVA was used as coating antigen, MAb1 as reaction antibody, IC50 of Ab2 was 33.771μg/mL; and when Ab2 was used as coating antigen, MAbl as reaction antibody, IC50 of IVM was 0.4μg/mL, and a correlation curve between Ab2 and IVM was established, it is y=1.9076X-6.4507. From the results above, it is concluded that the Ab2 was internal image of IVM and exhibitedβ-type binding property. Ab2βcan replace some toxic toxins standard(e.g. AFB, TTX and morphine) in competitive ELISA immunoassay system. The preparation of Ab2 may build good foundation for the establishment of the Non-competitive ELISA to examine the small molecular drug. By the way, we could not obtain any practical quantities of anti-metatype antibody.