Production Of Antibodies Against Clenbuterol, Penicillin And Gentamycin And Primary Research On Detection Of Clenbuterol Residue By Indirect Competitive ELISA

Clenbuterol is a sympathomimetic agent with principally p-adrenergic activity and a selective action on p-receptors. It is used in veterinary medicine as bronchodilatator and tocolytic. It has also been illegally appled in animal feed additives as a repartitioning agent which would lead to resiudes in the animal products with the potential of food-borne poisoning. Gentamycin is active against many gram-positive and gram-negative bacteria. It is administered by intramuscularly or intravenously for general infections or orally for gastro-intestinal infection. It is also used intramammarily for bovine mastitis and topically in ocular and auricular infections. Improper use of the drug may result in residual problems because of its slow elimination. Penicillin G is inactivated by penicillinases and is unstable in gastric acid. It is often used for the treatment of infections by susceptible bacteria through parenteral application or intramammary infusion for bovine mastitis. Its reside in milk is of great concern to the consumers for its potential of allergic reactions as well as to the dairy industry for its inhibitory effect on the starter cultures. Therefore,development of fast methods for detection of residues of these drugs in the foods of animal origins is of great public health significance and socio-economic importance.The above three drugs are haptens. Their immunogenicity can be imporved by coupling them to suitable carrier protein molecules. N-ethyl-N'-[3-(dimethylamino)propyl] carbodiimide hydrochloride (EDC) is a bifunctional coupling reagent. It is efficient for one-step coupling to form a wide variety of hapten-carrier protein immunogens. Conjugation may occur at either the C- or N-terminal of the hapten or at any carboxyl- or amine-containing side chains. Imject Immunogen EDC Conjugation Kits from Pierce are designed to simplify the production of clenbuterol-BSA,clenbuterol-OVA,penicillm-BSA,penicillin-OVA and gentamycin-BSA,gentamycin-KLH. After Dialysis for 3 days at 4"C for desalting,the BSA conjugates were immunized to rabbits after mixing with the aluminum adjuvant. Blood samples were taken 7 days after the third vaccination. Serum samples were prepared and stored at -20 C.Antibody titers were measured by indirect Enzyme-Linked Immunosorbant Assay (ELISA). The antigen (hapten-OVA or -KLH conjugates) was immobilized overnight at 4 C to the polystyrene microtiter plate. Unbound sites were blocked by 1% casein for 1 hr at 37C. Serial dilutions of anti-serum samples were added to the wells and allowed to bind for 2 hrs at 37C,followed by three times of washing. Goat anti-rabbit HRP-IgG conjugate (1:500) was added and incubated for 2 hrs at 37 C. After another cycles of washing,100 uL of the substrate o-phenylenediamine solution was added for color development at 37 C for 20 minutes. The reaction was stoped by addition of 50 uL of 2 M H2SO4. Absorbance was measured at 490 nm on a microplate-based ELISA reader. Antibody titers were approximated by plotting dilutions of the anti-sera against OD490nm.The antibody titers of penicillin (Scheme 1) and clenbuterol (Scheme 2) are 1600 X and 400 X respectively while the titers of penicillin,clenbuterol and gentamycin in Scheme 3 are 400 X,400 X and 200 X respectively. We also compared the blocking effects of 1% ovoalbumin,1-3% gelatin,1% bovine serum albumin and 1% casein. Casein at 1% wasfound the most satisfactory blocking solution.Indirect competitive ELISA was examined for detection of clenbuterol resides in rabbit urine. Clebuterol-OVA conjugate was immobilized onto the microplate wells and unbound sites blocked by 1% casein. Affinity-purified antibody (10 x dilution) was added together with different concentrations of clenbuterol. The remaining procedures were same as above. Good linear relationship was found between the concentration of added clenbuterol and OD490nm values. This approach could detect as low as 10 ng/ml of clenbuterol residue in rabbit urine. Further efforts are needed to optimize the detecting system.