Thermally Reversible Hydrogel Based Fluoroimmunoassay of Methyltestosterone

ABSTRACT

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-N-isopropylacrylamide (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 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%.

Keywords::

• methyltestosterone
• immunoassay
• poly-N-isopropyl acrylamide
• thermally reversible hydrogel.