以萘酰亚胺衍生物基荧光高分子为载体和指示剂的相分离免疫分析方法

A Phase-separation Immunoassay Method Using Naphthalimide Derivative Based Fluorescent Polymer as Phase Transition Carrier and Indicator

以4-甲氧基-N-(2-N',N'-二甲基氨基乙基-N'-烯丙基)萘二甲酰亚胺氯化铵(DMNAA)为荧光单体,合成了一种pH敏感荧光高分子聚N-异丙基丙烯酰胺-4-甲氧基-N-(2-N',N'-二甲基氨基乙基-N'-烯丙基)萘二甲酰亚胺氯化铵-N,N-二甲基氨丙基甲基丙烯酰胺[P(NIP-DMAPM-DMNAA)].采用共聚法将日本血吸虫抗原(SjAg)固定在P(NIP-DMAPM-DMNAA)上,制备P(NIP-DMAPM-DMNAA)-SjAg连接物,与日本血吸虫抗体(待测,SjAb)发生免疫反应后,调节pH值,使荧光高分子相变分离高分子-免疫组分连接物,最后,利用蛋白A对抗体的亲和性捕获P(NIP-DMAPM-DMNAA)-SjAg-SjAb,通过测定高分子自身的荧光信号来定量SjAb.该新型高分子具有良好的荧光特性,对pH响应快速,37℃下相转变pH值为7.2,分离免疫复合物时造成的损害低.与传统相分离免疫分析比较,新方法通过高分子相变分离和蛋白A捕获双重分离作用,消除了非特异性组分和未反应的特异性免疫成分等的干扰;利用高分子自身的荧光信号检测,无须另外的标记物,大大提高了免疫分析的简便性.以日本血吸虫抗体为分析对象,测得线性范围为1～1500ng/mL,抗体检出限为1.3ng/mL,相对标准偏差为3.6%(n=10),结果令人满意.

A phase-separation immunoassay method for Schistosomia japonicum antibody （SjAb） was developed using a pH-sensitive fluorescent polymer prepared by polymerization of N-isopropylacrylamide （NIP）, N-（3-dimethylaminopropyl）methacrylamide （DMAPM） and 4-methoxy-N-（2-N＇,N＇-dimethylaminoethyl-N＇-allyl）naphthalimide chloride quaternary ammonium salt （DMNAA） as a carrier and indicator. The polymer P（NIP-DMAPM-DMNAA） possesses relatively strong fluorescence and can be precipitated out of water above a critical pH 7.2 and re-dissolved below pH 7.2. The characteristic of this ＂smart＂ polymer made it possible to carry out the immunochemical steps of an immunoassay in a true solution and then to quickly separate the resulting immunoreaction complexes from the reaction mixture. After the polymerization of acryloyl-Schistosomia japonicum antigen （acryloyl-SjAg） with aforementioned monomers,P（NIP-DMAPM-DMNAA）-SjAg conjugates were gotten. In a fluorescence immunoassay procedure, analyte SjAb was reacted with the polymer-SjAg forming P（NIP-DMAPM-DMNAA）-SjAg-SjAb complexes, then the pH of solution was adjusted above the pHt of polymer to precipitate the polymer-immune complex, and the polymer-immune complex precipitate was separated and re-dissolved by the adjustment of pH, finally the antibody binding to the polymer-immune complex was absorbed by protein A entrapped in sol-gel matrixes and quantified by fluorescence measurement. The calibration graph for S jAb was linear over the range of 1 - 1500 ng/mL with a detection limit of 1.3 ng/mL. Owing to neutral pH and fluorescence response signal of P（NIP-DMAPM-DMNAA） itself, the damage to antigen-antibody immune complex was greatly decreased in the course of separation and the additional labeling was not needed for fluorescence measurement for immunoassay. The proposed method has been successfully used for SjAb analysis of the cow serum samples with satisfactory results.