异硫氰酸荧光素发光纳米微球标记双抗夹心固体基质室温燐光免疫分析法测定人IgG

基于Sol-gel技术合成了粒径为20nm、包含了异硫氰酸荧光素（FITC）的SiO2纳米微球（简写作FITC-SiO2）.该发光纳米微球在聚酰胺膜（ACM）基质上可发射强而稳定的室温燐光.研究发现,由该发光微球标记的羊抗人IgG抗体（简记为FITC-SiO2–Ab1）,不仅能在ACM固体基质上与人IgG发生定量的特异性免疫反应,并能在免疫反应后保持优良的燐光特性,且室温燐光信号进一步增强.据此建立了固体基质室温燐光免疫分析（SS-RTP-IA）测定人IgG的一种新方法.本方法线性范围为0.08~20.0pg人IgG/斑（样品体积0.4μL/斑,对应浓度0.20~50.0 ng/mL）,工作曲线的回归方程△Ip=19.97＋15.92mIgG（pg/斑）、相关系数r=0.9997,按3Sb/k计算本方法的灵敏度,其检出限为0.015 pg/斑.与用FITC标记羊抗人IgG的方法相比,灵敏度有较大提高.分别对0.20和50.0ng/mL样品重复测定11次,RSD为3.45%和4.1%,表明本方法的重现性好.

Determination of Human IgG by Solid Substrate-room Temperature Phosphorescence Immunoassay

Silicon dioxide nanoparticle containing fluorescein isothiocyanate（FITC-SiO2） with the particle size of 20nm was synthesized by Sol-Gel method.It can emit strong and stable solid substrate-room temperature phosphorescence（SS-RTP） signal on the substrate of polyamide membrane.It was found in the research that the specific reaction between goat-anti-human IgG antibody labeled with FITC-SiO2 and human IgG（immunoglobulin） can be carried on the polyamide membrane quantitatively,the antigen-antibody conjugate remained good phosphorescence properties and the signal of phosphorescence was enhanced after the immunoassay reaction.Based on the facts above,a new method of solid substrate-room temperature phosphorescence immunoassay（SS-RTP-IA） to determine human IgG was established.The linear range of this method is 0.080-20.0pg human IgG spot–1（sample volume 0.40μl spot–1,corresponding concentration range of human IgG is 0.20-50.00ng ml–1）.The regression equation of working curve is ΔIp = 19.97 ＋15.92m IgG（pg spot–1）,n = 6,correlation coefficient is 0.9997.Detection limit of this method calculated by 3Sb/k is 0.015 pg spot–1.Compared with the immunoassay using FITC to label goat-anti-human IgG,the sensitivity was improved obviously.For the sample containing 0.20（ng ml–1） and 50.0（ng ml–1） human IgG,with a sample volume of 0.40μl,the relative standard deviation（RSD） is 3.4% and 4.1 %（n = 11）,respectively.This indicated very good repeatability.