Thermally Reversible Hydrogel Based Fluoroimmunoassay of Methyltestosterone

ABSTRACT A novel immunoassay of methyltestosterone (MT) in serum is developed. We synthesized the antigen of MT (AgMT) by covalently bonding it to BSA and raised its antibody (AbMT) from rabbit. AbMT was conjugated to poly-N-isopropylacrylamide (p-NIPAAm) to form AbMT bound thermally reversible hydrogel (p-NIPAAm-AbMT). A competitive immunoassay method based on the competition of fluorescein isothiocyanate (FITC) labeled MT antigen (FITC-AgMT) and MT with limited amounts of p-NIPAAm-AbMT was established. The separation in the assay process was achieved by precipitation of the immuno-complex above its critical solution temperature. The detection limit of MT is 50 ng/ml. The recoveries of MT in human serum are above 93%.