Phase Separation Fluoroimmunoassay of Methyltestosterone
GAO Jun 1, LI Yuan zong 1, GUO Zhen quan 2, CHANG Wen bao 1* , CI Yun xiang 1 (1. Department of Chemistry, 2. Department of Biology, Peking University, Beijing, 100871)
A novel immunoassay of methyltestosterone (MT) in serum was developed. The antigen of MT (Ag MT ) was synthesized by covalently bonding MT to BSA. The antibody of Ag MT (Ab MT ) was raised from rabbit. Ag MT was conjugated to poly N isopropylacrylamide (PNIP) to form Ag MT bound thermally reversible hydrogel PNIP Ag MT . A competitive immunoassay method based on the competition of PNIP Ag MT and free MT with limited amount of fluorescein isothiocyanate (FITC) labeled Ab MT was established. The separation PNIP in the assay process was achieved by precipitation of the immunocomplex above its critical solution temperature. The amount of MT was determined by measuring the fluorescence intensity of Ab MT FITC. The linear range for MT is 27.5 ng/mL～1.1 μg/mL and the detection limit is 27.5 ng/mL. The recoveries of MT from human serum are satisfactory.