Study Of Enzyme Immunoassay For Ractopamine

In recent years,food poisoning events is not uncommon.The global food safety problem has become the the primary problem that media and the public generally concerned about.The reason for “the clenbuterol event” in the late 80 s of last century was that people appeared acute poisoning symptoms for eating foods containing clenbuterol.This once caused the panic of the people in the world.So the rapid detection is very necessary on market of meat products containing clenbuterol.Firstly,Mixed acid anhydride method was used to translated RAC-BSA.It was found that the maximum peak of the synthetic shifted compared with RAC and BSA by UV-VIS spectrophotometer.The coupling ratio was 3.9375.It was proved that RAC-BSA was synthesized successfully.Secondly,three indirect competitive enzyme immunoassays were established.Then The quality of each method was evaluated.The results of ic-ELISA indicated that,the optimum dilution multiple of artificial antigen was 2000-fold,the anti-body was 3000-fold,the secondary antibody was 1000-fold.Blocking buffer was dried skim milk at the concentration of 1.5%.The competitive reaction time was 30 min.The half-inhibitory concentration(IC50)value was 0.956 ng/mL.The linearity range was 0.102~9.074 ng/mL.There was no cross reaction with other analogues and derivatives.The recovery rate was between 85.6%~93.2%.The intra-assay coefficient of variation was between 3.58%~5.19%,and the inter-assay coefficient of variation was between 3.61%~5.78%.The results of ic-BA-ELISA indicated that,the optimum dilution multiple of artificial antigen of was 4000-fold,the anti-body was 12000-fold,the B-Ab2 was 2000-fold,the SA-HRP was 6000-fold.Blocking buffer was dried skim milk at the concentration of 2%.The competitive reaction time was 45 min,and the chromogenic reaction time was 20 min.The half-inhibitory concentration(IC50)value was 0.726 ng/mL.The linearity range was 0.094~5.596 ng/mL.There was no cross reaction with other analogues and derivatives.The recovery rate was between 87.8%~93.7%.The intra-assay coefficient of variation was between 2.09%~4.74%,and the inter-assay coefficient of variation was between 1.48%~4.74%.The results of ic-CLEIA indicated that,the ideal chemiluminescence fluid's formula: Liquid A was 4 mM of iodine phenol solution and 6 mM luminol solution mixed by the samevolume;Liquid B was 0.5 ?L/mL 30% H2O2 solution.The optimum dilution multiple of artificial antigen was 8000-fold,the anti-body was 6000-fold,the secondary antibody was 5000-fold.Blocking buffer was dried skim milk at the concentration of 2%.The competitive reaction time was 30 min.The half-inhibitory concentration(IC50)value was 0.475 ng/mL.The linearity range was 0.021~18.62 ng/mL.There was no cross reaction with other analogues and derivatives.The recovery rate was between 94.3%~102.1%.The intra-assay coefficient of variation was between 1.49%~2.30%,and inter-assay coefficient of variation was between 1.18%~5.78%.The results showed that the sensitivity,accuracy and precision of the three indirect competitive enzyme immunoassay methods met the requirement of related detection.And the sensitivity of ic-CLEIA was superior to other two methods.The three methods with demonstrated excellent sensitivity,accuracy and precision can provide the basis for rapid detection of RAC content in animal-derived food for supervision department.