African Journal of Food Science Vol. 1 (2), 024-029, October 2007          
ISSN 1996-0794 © 2007 Academic Journals

Full Length Research Paper

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

Wentao Xu¹, Kunlun Huang¹, Aike Deng¹, Baiqiang Zhai¹, Heng Zhao^1,2, Yingcong Li¹, Zhihong Liang¹ and Yunbo Luo¹*

¹College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.

²Current address: Cell Biology and Immunology Group, Wageningen University, P.O. Box 338, 6700 AH, The Netherlands.

*Corresponding author. E-mail: lyb@cau.edu.cn.

Accepted 17 September, 2007

Abstract

Ractopamine has been developed to be the main β-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 β-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).

Key words: Purification, ractopamine, swine, immunoaffinity column, enzyme linked immunosorbent assay.