Thermally Reversible Hydrogel Based Fluoroimmunoassay of Methyltestosterone

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