Abstract:

Four methods, including saturated ammonium sulfate (SAS), caprylic acid (CA), caprylic acid-ammonium sulfate
(CA-AS) and ammonium sulfate-DEAE-cellulose (SAS-DEAE) were compared for their effectiveness in the purification
of anti-clenbuterol antibodies. Meanwhile, the effect of simple NaIO4 method and its three different modified versions on
antibody labeling with horseradish peroxidase (HRP) was examined. The labeled antibodies were applied to develop a direct
competitive ELISA assay for rapid detection of clenbuterol. Our results indicated that the highest yield of anti-clenbuterol
antibodies was obtained using the CA method, which, however, provided the lowest purity. The SAS-DEAE method
gave rise to the highest purity despite providing a relatively lower yield. The simple NaIO4 method was most effective
in labeling anti-clenbuterol antibodies but resulted in a lower antibody titer. By contrast, the modified NaIO4 methods
provided significantly increased antibody titer. The limit of detection of the ELISA method was 2.7 ng/mL. The recovery
of clenbuterol from spiked urine samples was 94.2%, and the inter-assay and intra-assay coefficient of variation (CV) were
13.8% and 9.1%, respectively. Moreover, no cross-reactivity with clenbuterol analogues was observed. This ELISA method
can provide a simple, sensitive and specific approach to detecting clenbuterol in animal-derived food products.

Key words: clenbuterol, purification, enzyme-labeled antibody, enzyme-linked immunosorbent assay (ELISA)