A Biotin-Streptavidin Amplified Enzyme-Linked Immunosorbent Assay with Improved Sensitivity for Rapid Detection of Ractopamine in muscular tissue

An effective biotin-streptavidin amplified enzyme-linked immunosorbent assay (BA-ELISA) was optimized and characterized for the rapid detection of Ractopamine (RAC) residue in muscular tissue. Purification of the RAC antiserum by protein A-Sepharose 4B followed with bovine serum albumin (BSA)-Sepharose 4B affinity chromatography enhanced the sensitivity and reduce the background adsorption. Blocking with 0.5% skimmed milk power and diluting streptavidin-HRP conjugates with 0.5% BSA/phosphate-buffered saline (PBS) effectively remove the nonspecific adsorption in biotin-streptavidin amplified ELISA system. The established method allowed RAC determination with an IC50 value of 0.3 +/- 0.02 ng ml(-1) and a limit of detection of 0.02 +/- 0.003 ng ml(-1), more sensitive than the other reported methods. The variation coefficients of intra-assay and inter-assay were all below 7%. RAC residue in pig muscular tissue could be quantified without matrix effects after a 5-fold extraction and 2-fold dilution with PBS. Recoveries of RAC in pig muscular tissue ranged from 75% to 82.75%. The results were also compared with those from HPLC and a good correlation was obtained (r (2) = 0.9822). The characters show that the established biotin-streptavidin amplified ELISA could be potentially useful in rapid detection of RAC in animal-derived foods.