Development Of Immunoassay Determination Of Ractopamine Residues In Animal Tissue

The hapten RAC was coupled to BSA for use as the immunogen RAC-BSA,and coupled to HRP for use as the enzyme tracer,the coupling agent,butane-1,4-diol diglycidyl ether,was used for coupling RAC to proteins.The hapten RAC was coupled to the protein OVA with method of amber acid anhydride-mixed acid anhydride to be used as coating conjugate for indirect competitive ELISA by which the antiserum titre was detected.The antiserum obtained from Japan big rabbit immunised by RAC-BSA showed high affinity to RAC.A rapid direct competitive enzyme-linked immunosorbent assay(ELISA) procedure employing a polyclonal antibody purified by immunity affinity chromatography from the antiserum was established.The antibody showed high sensitivity and specificity in phosphate buffer,with an IC₅₀ of 0.9 ng/mL,and the limit of detection was 0.1 ng/mL, lowed than other congeneric reports.Besides,the antibody showed no cross-reactivity with clenbuterol,salbutamol,(-)-isoproterenol,terbutaline,dobutamine and isoxsuprine except dobutamine which showed the cross-reactivity for 7.5%.The matrix effect of the four animal samples was easily eliminated by one step of extraction with PBS,without any organic solution or clean-up procedure,and the recoveries of RAC was more than 60%at three different level.The detection limit of the method in blank samples was 0.5μg/kg.It is a basic of RAC-ELISA kit development.In addition,the stored condition of RAC-ELISA kit was studied to validate the stabiblity of antibody and enzyme tracer.The results showed that it could be stored at 4℃over half a year without any decline in detect capability.It benefits for the RAC-ELISA kit's application and important to control the use of ractopamine.In the end,the RAC chemiluminescent immunoassay was studied primarily on the basic of the RAC-ELISA development.It's important for the development of more sensitive determination method of RAC.