异硫氰酸荧光素标记抗体的固体基质室温磷光性质研究

Study on the Properties of Solid Substrate Room Temperature Phosphorescence of the Antibody Labeled with Fluorescein Isothiocynate

研究了异硫氰酸荧光素 (FITC)标记的羊抗人抗体 (GAHAb FITC)和兔抗羊抗体 (RAGAb FITC) ,及其以三种免疫方式与人免疫球蛋白 (IgG)反应所得免疫复合物在多种固体薄膜基质上发射室温磷光 (RTP)的适宜条件及其光谱、强度和寿命等性质。研究发现 ,标记抗体及其免疫复合物保留了FITC优良的固体基质室温磷光性质 ,λ(max)ex λ(max)em =5 2 5 6 5 0nm ,其RTP强度与其浓度线性相关 ,并有很高的灵敏度。更为重要的是 ,免疫反应及其RTP检测能结合于同一基质 ,方便地相继完成。在聚酰胺膜 (PM )上 ,以Pb(Ac) 2 为重原子微扰剂 ,稀释比为 1∶10 (φ)的GAHAb FITC ,RAGAb FITC及其与人IgG形成的免疫复合物均能发射较强的RTP信号并有较长的RTP寿命。本结果为建立相应的固体基质室温磷光免疫分析 (SS RTP IA)新方法奠定了相应的实验基础。

The properties of room temperature phosphorescence (RTP) of the labeled goat anti-human antibody.)( with fluoroscein isothiocynate GAHAb-FITC) and labeled rabit anti-goat antibody(RAGAb-FITC) with FITC in different dilution ratios on many kinds of solid substrates were studied in detail. It is found that on the polyamide membrane, only using Pb(Ac)(2) as a heavy atom perturber, both the labeled antibodies and their antigen- anti body conjugated compound with the human immunoglobulin G remained excellent RTP properties of isothiocyanate fluorescein, lambda(ex)((max)) /lambda(em)((max))= 525/650 nm, the relation of RTP intensity to the concentration was linear in a certain range. The conditions for immunological reaction and RTP emission were optimized. The result means that it is possible to develop a new immunoassay named as solid substrate room temperature phosphorescence immunoassay (SS-RTP-1A) which combines both the specificity of immunological reaction and high sensitivity of luminescence analysis.