控温相变免疫分析测定雌酮

Determination of Estron by Temperature Controlled Phase Separation Immunoassay

通过热引发聚合与氧化还原引发聚合 ,合成水凝胶 ,作为雌酮全抗原 (E1 BSA)载体 ;以异硫氰基荧光素标记雌酮的单克隆抗体 (McAb )作为示踪剂 ,建立了控温相变竞争免疫分析测定雌酮 (E1 )的方法并应用于分析血清样品。实验表明 :以热引发聚合合成的水凝胶为载体的荧光免疫分析方法具有更高的灵敏度 ,它的线性范围为 1 0～ 70 0 μg L ,检出限为 1 μg L(n=1 0 ,3倍空白 )。经水凝胶性质的比较 (包括LCST、非特异性吸附、抗原与抗体反应时间及固定化抗原活性 ) 。

Thermally sensitive hydrogel-antigen conjugates were synthesized with two different methods. Both kinds of conjugates can be used for the determination of free estrone (E-1) in serum in a temperature controlled phase-separation immunoassay. Antigen (bovine serum albumin (BSA)-E-1) was activated. by acrylic acid N-hydroxysuccinimide ester (NAS), and then copolymerized with N-isopropylacrylamide (NIPA) in the presence of ammonium persulphate (as an initiator) and N,N,N',N'-tetramethylene diamine(TEMED, as an accelerator). The copolymer prepared was called A. Another copolymer was prepared by copolymerization of NIPA and NAS with azoisobutyronitrile (AIBN) as an initiator. This copolymer reacted with BSA-E-1 to form copolymer-BSA-E-1 conjugate named B-Some properties of A and B were investigated and compared. A (or B) immunologically reacted with fluoresceinated anti-E-1 monoclonal antibody (McAb*) competing with free E-1. Thus a competitive temperature controlled phase-separation inummoassay was established for the determination of E-1. The calibration range (for B) was 10similar to700 mug/L. The detection limit was 1 mug/L (n=3). The results demonstrated that the immunoassay of E-1 based on B was more sensitive and efficient than that based on A.