The Fluorescence Labeling Technique Of Phycoerythrin And Application In Tobacco Mosaic Virus Detection

Phycoerythrin is a new type of labeling probe with remarkable fluorescent characteristics, which was widely used in fluorescence immunoassay. The fluorescence labeling technique of phycoerythrin and application for the immunoassay of Tobacco mosaic Virus were studied in this research using it as a labeling probe.The phycoerythrin was isolated and purified from the algae. The TMV antiserum was purifed with n-octanoic acid precipitation. The main materials suitable for fluorescence labeling were obtained through the measurement of the purity, concentration and antibody titer.The effects of different pH values, ionic strength and additives on PE fluorescence intensity was measured in this study. The results showed that the stability of fluorescence intensity of PE was better maintained in a range of pH from 5 to 9 and ionic strength within 150~200mmol/L PBS (or 100~200mmol/L NaCl solution added), but pH3 and pH12 had an obvious effect on the fluorescence intensity of this protein. The additive of 0.1% Tween20 and lmmol/L EDTA was also favourable to the phycoerythrin's stability.Several heterobifunctional reagent combinations were screened, the labeling protocol was established, and Experiment conditions for crosslinking reaction were optimized. SPDP was used to activated the phycoerythrin, 2-IT was used to modified multiclonal antibodies, labeling probe was prepared by the two-step conjugation. The better protocol was as follows: the proper molar ratio of SPDP to PE was 100: 1, and the corresponding reaction time was 0.5-1h;, the proper molar ratio of 2-IT to multiclonal antibodies was 200: 1, and the corresponding reaction time was 2h.Phycoerythrin fluorescence immunoassay protocol was established by the use...