Solid substrate room temperature phosphorescence immunoassay based on an antibody labeled with tetramethylrhodamine B isothiocyanate Original Research Article

Solid substrate room temperature phosphorescence immunoassay (SS-RTP-IA) based on an antibody labeled with tetramethylrhodamine B isothiocyanate (TRITC) was described. The SS-RTP properties of rabbit anti-goat antibody labeled with TRITC (RAGAb-TRITC) and its immune complex with goat-immunoglobulin G (G-IgG) were studied on a polyamide membrane (PM). The results showed that RAGAb-TRITC can react specifically with G-IgG on a PM, and retain the excellent SS-RTP property of TRITC, λexmax/λemmax=558 nm/700 nm. The dependence of the phosphorescence intensity on the amount of antigen G-IgG (in a sample volume of 0.4 μl) was linear. Compared with enzyme-linked immunosorbent assay (ELISA), this assay showed a lower detection limit (0.37 pg per spot), a better linear relationship and a wider dynamic range (0.62–30 pg). The method was applied directly to determination of G-IgG in goat serum and the results agreed well with those obtained by ELISA. Therefore, this study shows the high sensitivity in SS-RTP and specificity of the immunological reaction. Moreover, it is a simple but powerful procedure.