Preparation Of Monoclonal Antibodies Against Clenbuterol And The Establishment Of An Indirect Competition ELISA Method

Clenbuterol(CL) is a kind of β2-adrenrgic receptor agonist which first is used to treat bronchial asthma and chronic bronchitis as bronchial-dilating agents in clinical treatment, because of its toxicity to stop using.CL can assign the animal’nutrition, again, reduce the fat to deposit and synthesis, promote protein synthesis speed. However, it can keep higher residue amount in the body when animal are feed CL as an adjunct and it also bring serious danger to human when they eat the animal food produce. Therefore, the countries all over the world have been forbidden strictly using it as the additive of feed.In China, it is banned, but there are still illegal people widely using in the animal food production for permits.Many physical and chemical methods to detect residue of CL have been established at home and abroad. Such as HPLC, GS, GC-MS, immunogical analysis including the ELISA, CGIA.Although chromatography technology are precise and efficient to detect the residue of CL, it is not suitable for large batch sample screening. Because of their complication, long time consuming, big cost and high technical request. ELISA can solve these shortcomings, it is simple,accurate,efficient,cheap and unique test methods, suitable for rapid screening of large samples.In this research, we synthesized the antigen and prepared anti-clenbuterol monoclonal antibody by using monoclonal antibody and ELISA technique.Finally,we established the CiELISA method to detect residue of Clenbuterol.1. Synthesis and Identification of CL artificial antigens. CL-conjugated antigens were synthesis by coupling CL with BSA and OVA.The synthesized antigens were demonstrated to be successful by UV-scanning and immunological methods. The coupling ratio respectively is12:land10:1.2. The preparation of the Monoclonal Antibodies against CL.After comparing experiments, we selected CL-BSA as immunogen and CL-OVA as coating antigen. Mice (BALB/c) were immunized by CL-BSA, in the way of routine method.The titer of blood serum collected after the fifth immunization reached1:16,000.Then a hybridoma cell line named8D2which could ecrete anti-clenbuterol monoclonal antibody steadily by hybridoma’s technique was received and then ascetic fluid was prepared by inside body induced ascites method.The protein concentration of ascetic fluid was21.8mg/mL, titer of the McAb was1:120,000.3. The establishment of ELISA method.The CiELISA’s method was established base on the McAb, The optimum conditions were established through the matrix text. The coating antigen was attenuated byl:10,000,the monoclonal antibody in the abdominal dropsy was attenuated by 1:60,000,Under the conditions,The standard curve of CL to antibody-antigen reaction was prepared with the linear equation Y=-O.5O15x+0.7438(R2=0.9919), IC50=2.15ng/mL,LOD under0.5ng/mL,LOQ=0.97ng/mL.The cross reaction experiment showed that the McAb expressed high specificity to CL and there were almost no cross reaction with other pVadrenergic receptor agonists.4.The addition and reclamation test of CL.The CiELISA’s method was initially applied in the addition and reclamation test of the liver tissue and muscle tissue of swine.The sample was determined after pre-processing.The intervention disappeared when the extraction of liver and muscle sample was diluted4times.The standard curve equation was:Y=-0.4114x+0.7063(R2=0.9886) ranging from0-50ng/mL, IC50was4.39ng/g, LOD was0.5ng/g. When adding5,10,50ng/mL CL to the liver tissue of swine, the recovery ratio was between85%and100%.The intraassay coefficient of variation was between4.12%and6.71%,and the interassay coefficient of variation was between5.98%and7.13%.