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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.
Using 4-methoxy-N- (2-N ’, N’-dimethylaminoethyl-N’-allyl) naphthalimide ammonium chloride (DMNAA) as the fluorescent monomer, A pH-sensitive fluorescent macromolecular poly N-isopropylacrylamide-4-methoxy-N- (2-N ’, N’- dimethylaminoethyl-N’-allyl) N, N-dimethylaminopropylmethacrylamide [P (NIP-DMAPM-DMNAA)]. The S. japonicum antigen (SjAg) was immobilized on P (NIP-DMAPM (NIP-DMAPM-DMNAA) -SjAg conjugate was prepared and immunized with Schistosoma japonicum antibody (test, SjAb). The pH value was adjusted so that the fluorescent polymer phase-change isolated macromolecule-immunohistochemistry Finally, SjAb was quantified by measuring the fluorescence signal of the polymer itself using the affinity capture of P (NIP-DMAPM-DMNAA) -SjAg-SjAb by protein A. The novel polymer has good fluorescence characteristics , Rapid response to pH and pH 7.2 at 37 ℃, which results in low damage when the immune complexes are separated.Compared with the traditional phase separation immunoassay, the new method through the double phase separation of polymer phase transition and protein A capture, Elimination of non-specific components and unreacted specificity The use of macromolecules own fluorescence signal detection, without additional markers, greatly improving the simplicity of the immunoassay.With the Schistosoma japonicum antibody as the analysis object, measured linear range of 1 ~ 1500ng / mL, the antibody The limit of detection was 1.3 ng / mL and the relative standard deviation was 3.6% (n = 10) with satisfactory results.