Studies On Solid Substrate Room Temperature Phosphorescence Immunoassay

This thesis proposed to set up SS-RTP-IA (Solid Substrate Room Temperature Phosphorescence Immunoassay), which is the combination of highly sensitive solid substrate room temperature phosphorescence technology and the speciality of immunological reaction. Human IgG was detected with TRITC (tetramethylrhodamine B isothiocyanate) as label through direct mode, human complement3 (C3) was detected with Eosin5-ITC (Eosin5-isothiocyanate) as label through sandwich mode and biotin-avidin sandwich mode in this thesis. Therefore, in methodology the validity of SS-RTP-IA is demonstrated, and the experimental results show high sensitivity and specialty of this assay.1. SS-RTP-IA based on an antibody labeled with TRITC to detect goat IgG through the direct mode was described in this thesis. The SS-RTP properties of rabbit anti-goat antibody labeled with TRITC (RAGAb-TRITC) and its immune complex with goat-immunoglobulin (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, (maxex/(maxem=558nm/700nm. The dependence of the phosphorescence intensity on the amount of antigen G-IgG (in a sample volume of 0.4μl) was linear. Compared with enzyme-linked immunosorbent assay (ELISA), this assay showed a lower detection limit (0.37pg per spot), a better linear relationship and a wider dynamic range (0.62-30pg). The method was applied directly to determination of G-IgG in goat serum and the results agreed well with those obtained by ELISA. 2. Biotin-avidin bridge technology and SS-RTP-IA was combined to form biotin-avidin-SS-RTP-IA (BA-SS-RTP-IA). BA-SS-RTP-IA based on an antibody labeled with Eosin5-ITC to detect human complement3 (C3) was described. The results showed that RAC3-Eosin5-ITC can react specificallywith C3 on a PM, and remain the excellent SS-RTP property of Eosin5-ITC, (maxex/(maxem=535nm/678nm. At the same time, SS-RTP-IA to detect human complement3 through sandwich mode was described. The detection limit was 0.546pg/spot (with a sample volume of 0.4μl). In BA-SS-RTP-IA, the dependence of the phosphorescence intensity on the amount of antigen human complement3 was linear. The detection limit was 1.094pg/spot (with a sample volume of 0.4μl). Compared with ELISA, the concentration of human complement3 in 20 human sera was determined. The results agreed well with those obtained by ELISA.