基于纳米金标记和银增强放大的血吸虫抗体的免疫分析

Metalloimmunoassay of schistosoma japonium antibody based on silver-enhancement and colloidal gold-label

该文提出了一种新的、高灵敏的基于银增强放大和免疫胶体金技术的血吸虫抗体的免疫分析方法。免疫反应在聚苯乙烯微孔板中以间接分析模式(血吸虫抗原-兔抗血吸虫抗体-纳米金标记的羊抗兔IgG 二抗)进行,通过反应上去的纳米金催化银离子的还原,使大量的银沉积在纳米金的周围,用酸将银溶解后,通过阳极溶出伏安法(ASV)对银离子进行检测,溶出峰电量的大小与待分析物血吸虫抗体(一抗)的浓度成正比。对免疫分析的一些实验条件如抗原与待测抗体的反应时间、纳米金标记二抗与待测抗体的反应时间、纳米金标记抗体稀释度以及银增强时间进行了优化。阳极溶出峰电量与日本血吸虫抗体的浓度在6.4μg/L～100mg/L 范围内呈良好的线性关系,检测下限为3.0μg/L。将该方法应用于兔血清中日本血吸虫抗体的测定,取得了满意的结果。

A silver-enhanced colloidal gold metalloimmunoassay has been proposed for the determination of Schistosoma japonium antibody (SjAb) in rabbit serum.The adult worm antigen of Schistosoma japonium (SjAg) was adsorbed passively on the walls of a polystyrene microwell and then reacted with the desired SjAb.The colloidal gold-labeled goat anti-rabbit IgG secondary antibody was adsorbed on the walls of the polystyrene microwells through the reaction with the SjAb,followed by the silver enhancement process and the dissolution of silver metal atoms in an acidic solution,and the determination of dissolved silver ions by the anodic stripping vohammetry (ASV) at a glassy-carbon electrode.Assay conditions were optimized, including the reaction time of SjAg with SjAb,the time of SjAb with the colloidal gold-labeled secondary antibody,the dilution ratio of the colloidal gold-labeled secondary antibody and the silver enhancement time.The integration of the anodic stripping peak current depended linearly on the SjAb logarithmic concentration over the range of 6.4 μg/L and 100 mg/L.A detection limit as low as 3.0 mg/L SjAb was achieved,which was better than the piezoelectric body acoustic wave sensor (detection limit of 7.2 mg/L) and the renewable amperometric immunosensor(detection limit of 0.36 mg/L).Rabbit serum samples with varying infection degrees were analyzed,and the results demonstrated that the proposed method meets the requirement of clinical analysis.