Development Of The Monoclonal Antibodies Against GM And Application For The Determination Of GM Residues

Aminoglycosides antibiotics are frequently used antibiotics with broad spectrum of antibacterial activity, mostly including Gentamicin (GM), Streptomycin(SM) and Kanamycin (KM) etc. They are very useful to treat cow mastitis and endometritis, and are frequently added into feeds for the prevention or treatments of animal diseases. However, illegal administration will lead to residues in edible tissues, especially in the milk of galactophorous animals, and finally do latent harm to human health, resulting in hearing toxicity and kidney toxicity. Many countries and international organizations have established the maximum residue limit of aminoglycosides antibiotics in animal tissues for human consumption. And the tolerance limit of Gentamicin in milk is 0.2mg/L in China.Many chemical and physics methods for detecting Gentamicin residued in edible tissues of animals have been established. Though some of these methods are sensitive and accurate, they are not suitable for screening a large number of samples because these methods are time-consumption, costly and highly skilled. Enzyme-linked immunosorbent assay (ELISA) as a rapid, special and sensitive biological method, is gradually applied in drug residues assay. This research synthesized the antigen and devoloped the antibodies, and established the ELISA method for detecting GM. In the simulative experiment, the residues of GM in milk were detected.To prepare monoclonal antibodies (mAbs) against GM, it was bound to BSA or HSA using EDC as the coupling reagent, and identified the conjugation by SDS-PAGE. The amidocyanogen of GM was linked to carboxyl of carrier protein terminal via the coupling reagent, which are specific for individual GM were readily exposed to the immune system and this resulted in the production of antibodies that were highly specific for the hapten.Mice (BALB/c) were immunized subcutaneously (sc) with GM-BSA conjugates. After three booster injections, the mice were primed with a final booster injection (intraperitoneally). .Three days later, spleen cells were isolated. The fresh spleen cell was used for fusion with myeloma cell. iELISA and CiELISA were employed to screen the positive cell. The positive cells were cloned by limiting dilutions (three times). Thisprocedure resulted in four stable clones: 1A10, 1B11, 5D11 and 6E10.The hybridized myeloma cell were expanded cultured and freezed into liquid nitrogen.Mice (BALB/c) were injected intraperitoneally with lAlO cell to produce ascites. Determination the suitable concentration of the coated antigen and ascites titre by chessboard test, and the ascites titre reached l:80000.Indirect competitive ELISA (CiELISA) was established with this ascites. Calibration graphs were prepared. The limit of detection of this assay was established to be 10.66ng /ml, and the limit of determination was 13.39 ng/ml, standard deviation was less than 3%.The antibody can be used in practical detection.To test the speciality of antibody, eight kinds of drug were used in the competitive ELISA as the competitive antigen. The result proved that the ascites shows no cross-reaction to other drug.A CiELISA method which was used to detect GM added to milk was preliminary established. The milk samples were dealed with 5 methods, the standard curve and rate of recovery were compared with eath other in order to select the best method.The skimmed milk diluted into 5 times can be used to detection. Limit of detection in milk was established to be 3.85ng /ml, and the limit of determination was 6.597ng/ml, standard deviation was less than 5%.