Abstract: A synthetic derivative of hydrocortisone (HYD) as a hapten was coupled to keyhole limpet hemocyanin (KLH) using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The KLHHYD conjugate as an immunogen was used to immunize mice. A stable hybridoma cell line HYD-C1 secreting monoclonal antibody against HYD was generated to induce large amounts of antibodies in the ascites of mice. Moreover, the HYD hapten and another hapten synthesized from dexamethasone (DEX) were conjugated separately to ovalbumin (OVA) to obtain coating antigens. The final aim of this study was to develop an indirect competitive-enzyme linked immunosorbent assay (ic-ELISA). The results showed that the IC50 of homologous ic-ELISA was 1.63 ng/mL while that of heterologous ic- ELISA was 0.24 ng/mL. Cross inhibition tests revealed that the sensitivity of heterologous ic-ELISA for all glucocorticoids tested was higher than homologous ic-ELISA, which shows the broad-spectrum specificity of heterologous ic-ELISA. The average recoveries of glucocorticoids from spiked liquid milk by heterologous ic-ELISA were in a reasonable range of 77.5% to 111.3%, meeting the requirements for detection. In conclusions, a monoclonal antibody specific for HYD was successfully prepared and used for homologous and heterologous ic-ELISA. Heterologous ic-ELISA was found to be more sensitive and have a broader-spectrum specificity.

Key words: glucocorticoids, homologous strategy, heterologous strategy, broad-spectrum specificity, indirect competitiveenzyme linked immunosorbent assay (ic-ELISA)