Preparation Of Monoclonal Antibodies Against Clenbuterol And Development Of An ELISA And Its Kit For Screening Of Clenbuterol Residue In Animal Tissues

Clenbuterol(CL) is a β -agonist registered as a veterinary bronchospasmolytic drug in some countries. It was well known that at comparatively high doses it can be used as a growth-promoting and repartitioning agent in several livestock species, as it improve lean meat deposition and production efficiency. However, there have been several reported incidences resulted from consumption of CL-tained edible tissues of bovine and swine. Its use as a feed additive has been banned within most countries. It is necessary to develop a sensitive and applicable assay for effective surveillance of the illicit use of CL.A fast immunoassay based on specific monoclonal antibodies has been developed and validated to screen the residue of CL in animal tissues and urine. Hybridomas secreting antibodies against CL were compared, selected and cloned followed by a monoclonal antibody raised. The conditions of competitive indirect ELISA(ciELISA) were optimized before a standard curve was obtained. The urine samples of swine and bovine were adjusted pH>8.0 and CL was extracted with tert-butylmethyl ether (MTBE) before determined by ciELISA. The liver samples of swine and bovine were hydrolysed by hydrochloric acid and CL was extracted with potassium dihydrogen phosphate buffer (pH3.0) overnight followed by clean-up by C18 catridge. The CL content in prepared solutions was determined by ciELISA. This immunoassay was confirmed by the GC-MS, compared with Biopharm kit and used to detect the samples of swine fed 2mg/kg CL.Two stable monoclonal hybridomas, C611 23/2 46B3 and C611 23/2 46 A5 were obtained. A McAb from C611 23/2 46 B3 was raised against CL-HAS immunogen which had been prepared via a diazotization. It was highly specific for CL with a titer of 1:64000in ascitic fluid and low cross-reactivities less than 10% to other β -agonists. The optimal conditions of ciELISA were as follows: the concentration of the coated CL-OVA was 0.5 μ g/mL, the working concentrations of the McAb against CL and goat anti-mice IgG were 32000 and 5000 times dilution of the stock, respectively. The incubation time for competition was 1h at 37℃. Under the optimal conditions, a sensitive and specific standard curve was obtained with a linear range from 0.1 ng/mL to 27ng/mL(r=-0.9907). The mean IC50 of repetition was 1.8ng/mL. The limit of detection (LOD) was 0. lng/mL.The extraction recoveries of CL from urine samples spiked at 0.3, 1 and 2ng/mL were determined by ciELISA and ranged from 63.82+13.93% to 111.94±7.41%. The coefficients of variation were less than 20%. The liver samples of swine were spiked CL at 0.3, 1 and 2ng/g and that of bovine at 0.5, 1 and 2ng/g before measured by ciELISA. The recoveries ranged from 80.45±13.79% to 98.45±16.99%, and the coefficients of variation were not below 20%. The LOD for the analysis of the urine of swine and bovine were 0.3ng/mL, and that of the liver of swine and bovine were 0.3 ng/g and 0.5ng/g, respectively.CiELISA and GC-MS were simultaneous performed for confirmation. The values measured by ciELISA is correlated to corresponding those determined by GC-MS (r=0.99), which indicated that this method was accurate and reliable. This immunoassay has been successfully used to analyze the urine and the liver tissues of swine fed 2mg/kg CL. The result demonstrated that this method could safely be used to analyze the urine and the liver of treated animal. It has a more extensive range of linearity and was more specific for the analysis of urine and liver than the kit bought from Biopharm Company in Germany, and two kits have the same LOD.Conclusion can be drawn that the immunoassay presended here is more accurate and specific than Biopharm kit and can be safely used as a screening assay to monitor the residue of CL in the urine and liver of food-producing animal.