Immunoaffinity column as cleanup tool for an enzyme linked immunosorbent assay of ractopamine detection in various tissues of swine

Abstract

Ractopamine has been developed to be the main -agonist substance used illegally in meat producing animals. A
simple and efficient extraction and purification procedure for ractopamine was developed by means of the
immunoaffinity column (IAC) as a cleanup tool. Purified polyclonal antibodies against RCT were produced and
coupled covalently to CNBr-activated Sepharose 4B. Both the binding conditions and elution protocols were
optimized and the capacity, reusability, precision and accuracy of IAC were determined. The IAC was successfully
employed to isolate and purify the RCT from the various tissues of swine. Subsequently, enzyme linked
immunosorbent assay (ELISA) procedures were established further on to measure RCT. The antibodies showed
negligible cross-reactivity with other -agonists. IAC-ELISA allowed 0.2 ng/mL of RCT to be detected in urine and 0.5
ng/mL to be detected in other various tissues of swine, which makes this method an acceptable screening tool to
access RCT. IAC-ELISA for the detection of RCT was validated by LC-MS and the correlations between the results
from LC-MS and those from IAC-ELISA were all high (above 0.89).