Preparation Of Monoclonal Antibodies Against Phenylethanolamine A And Development Of An ELISA Method

Phenylethanolamine A(PA)is a new ?-adrenergic agonist and it has been used as a growth promoter in livestock production to improve feed utilization and the lean meat percentage of carcasses of livestock.However,it has toxic response such as nervousness,cardiac palpitation and vomitness after being taken by humans.PA has been prohibited from being used in feeds and animal drinking water in the bulletin no.1519 issued by the Ministry of Agriculture of China.Several methods are known to detect PA such as the liquid chromatography(LC),liquid chromatography-mass spectrometry(LC-MS),gas chromatography-mass spectrometry(GC-MS),high performance liquid chromatography(HPLC).But all the methods mentioned above have some shortcomings,such as the requirement of expensive instruments,the pretreating of the sample and the long detecting time as well as the impossibility of large amount of detection.Therefore establishing a rapid,simple,and sensitive detection method for PA has important practical significance.This study used monoclonal antibody preparations and enzyme labeled immunosorbent assay(ELISA)to detect the PA on the basis of synthetic,which simple and has a high degree of selectivity and sensitivity which are suitable for rapid detection of large quantities of samples.1.Synthesis and identification of PA artificial antigens.PA does not have the ability of completing the antigenicity because of it'-s light molecular weight.According to the characteristics of the molecular structure of PA,we transformed the molecular structure of PA through the reduction reaction and introduced a derivative of an amino group.The derivative of PA was conjugated with BSA and OVA proteins by the Diazotization method,and then we got complete antigen.We can use the UV scanning,SDS-PAGE and immunological method to judge whether the synthesis of PA with artificial completely antigens succeed or not.The protein concentrations of PA-BSA and PA-OVA were4.82 mg/ml and 5.61mg/ml respectively by the measurement using the BCA protein assay kit.2.Preparations of the anti-PA monoclonal antibodies.PA-BSA was used to immunize BALB/c mice by routine immunization.The titer of blood serum collected after the fifth immunization reached 1:25600.With the help of hybridoma technique to fuse spleen cells and myeloma cells and enzyme labeled immunosorbent assay(ELISA)technology to screen the fused cells.We got one strains hybridoma cells secreting anti-PA monoclonal antibodies which named D6H8.We prepared 13mL ascites by using D6H8 hybridoma cell line.The protein concentration of ascites was 55.38mg/mL and the titer of ascites protein was 1:12800.3.The establishment of ELISA detection method.Through the establishment of indirect competition ELISA method(CiELISA),the best dilution concentration of PA-OVA was 1:400 and the best working concentration of ascites was 1:4000.When the linear range was 5?1000 ng/mL,the standard curve of PA to antibody-antigen reaction was prepared with the linear equation y=0.3861x-0.1845(R2=0.9902),ICso=58.88ng/mL,LOD=3.833ng/mL.The cross experiment showed that the monoclonal antibody had 100%inhibition rate for PA,it had no cross reaction with Butanol amine,clenbuterol hydrochloride,isoproterenol hydrochloride,and phenylephrine hydrochloride.4.The addition and reclamation test of PA in the pork*When the extracting solution was diluted for 16 times,the intervention disappeared.The standard of working linear equation was established.When the range was from 10 to 1000 ng/mL,the linear equation was y=0.3785x-0.2588(R2=0.9898),IC50=93.97ng/mL,LOD=5.92ng/mL.When adding 50,400,800 ng/mL PA to the pork,the recovery ratio was 85.96%?104.32%.