Development Of Polyclonal Antibody And Immunoassay For Phenobarbital

Phenobarbital(PB),which has the effects of sedation,hypnosis,anticonvulsion,antiepileptic and so on,is often used to treat symptoms of insomnia in people.But PB has many toxic effects on nerve and bone marrow,teratogenicity,carcinogenesis and so on.Long-term taking will accumulate PB in human body and endanger human health.The Food Safety Law of China stipulates that chemical substances other than food additives and substances that may endanger human health are not allowed in food production.In the list of substances that may be illegally added in health foods released by the Food and Drug Administration on March 6,2012,contains phenobarbital.Therefore,it is very important to strengthen the monitoring and control of PB in health food.Immunoassay has the characteristics of sensitivity,rapidity and high throughput,it can be used as a complementary method for instrument detection,and meet the safety and rapid screening needs of actual samples,It has become a research hotspot in the field of food safety in recent years.In this study,a haptens were desigbed and synthesized to the preparation of high-affinity polyclonal antibodies against PB.The indirect competitive enzyme-linked immunosorbent assay(ELISA)and the indirect competitive chemiluminescence enzyme-linked immunosorbent assay(CLEIA)for PB were established.The main contents and results are showed as follow:(1)A PB hapten PB-1 and artificial antigen was designed and synthesized.From its molecular structure,retaining the benzene ring of its characteristic structure,we extended the carboxyl structure at the position of the carbon atom of the pyrimidine ring.The artfical antigens,PB-1-BSA,PB-1-KLH and PB-1-OVA was obtained by using active ester method to conjugate with carrier protein.The titer of antiserum PB-1 was 1:32,000,and the inhibition rate of 1?g/m L PB was 70%.Based on this,a highly specific polyclonal antibody against PB was prepared.(2)An indirect competitive enzyme-linked immunoassay(ic ELISA)for the rapid determination of PB was developed.The optimized conditions are as follows.:the concentration of the coating is 0.1?g/m L,antibody dilution factor is 32,000,the optimal reaction buffer system is 0.02 M p H=6.4 PBST buffer solution,and the Tween-20 content is0.20%,primary antibody reaction for 30 min,secondary antibody reaction for 30 min.The50%inhibitory concentration(IC₅₀)was 0.55?g/m L.The limit of detection was 0.02?g/m L with a linear range between 0.08?g/m L and 3.637?g/m L.The cross reaction rate of this method with phenobarbital sodium was 96%,and with the other five phenobarbital structural analogues(barbital?pentobarbital?thiobarbital?okasepine?carbamazepine)was lower than 0.1%.The coefficient of variation among and between batches was lower than15%.The recovery rates of oral liquid,tablet and capsule samples were between 71.1%and96.3%.This proposed ic ELISA provided a valid method for a rapid detection for PB in real samples.(3)An indirect competitive chemiluminescene enzyme immunoassay(ic CLEIA)for the rapid determination of PB was developed.The optimized conditions are as follows:the concentration of the coating is 0.0625?g/m L,antibody dilution factor is 16,000,the optimal reaction buffer system is 0.02 M p H=6.4 PBST buffer solution,and the Tween-20content is 0.20%,primary antibody reaction for 40 min,secondary antibody reaction for 40min.The 50%inhibitory concentration(IC₅₀)was 0.24?g/m L.The limit of detection was0.01?g/m L with a linear range between 0.03?g/m L and 1.83?g/m L.The cross reaction rate of this method with phenobarbital sodium was 104%,and with other five phenobarbital structural analogues(barbital?pentobarbital?thiobarbital?okasepine?carbamazepine)was lower than 0.1%.The coefficient of variation among and between batches was lower than15%.The recovery rates of oral liquid,tablet and capsule samples were between 80.3%and99.3%.The results show that the method is suitable for the detection of actual samples.