Abstract:

【Objective】 The objective of the experiment is to prepare high sensitive and specific polyclonal antiserum against phytase. 【Method】Tentative purified phytase was identified by SDS-PAGE electrophoresis, then immune Balb/C mouse with 50μg phytase protein /time, once every four weeks for 4 times, 60 days after the last immunization, the serum was prepared followed blood withdrawn by eyeball taken off. Antiserum titer was determined by indirect ELISA, sensibility and specificity by indirect compete ELISA.【Result】The result showed that wheat bran phtase SDS-PAGE had only one belt, phytase antiserum titer was 1×104, and IC50 was 148.87ng﹒mL-1. The antiserum IC50 to normal, coated and concentrated microorganism phytase was 165.69 ng﹒mL-1, 163.80 ng﹒mL-1 and 166.51 ng﹒mL-1, and cross-reactivity was 89.85%, 90.88%, and 89.41%, respectively. The antiserum IC50 to inactivated (boiled for 5 min) wheat bran phytase, normal, coated and concentrated microorganism phytase was 2 871.34 ng﹒mL-1, 5 208.85 ng﹒mL-1, 7 914.12 ng﹒mL-1 and 5 804.24 ng﹒mL-1, and the cross-reactivity was 5.18%, 2.86%, 1.88%, and 2.56%, respectively.【Conclusion】In conclusion, phytase polyclonal antiserum with high titer and sensitive and specificity was gotten. This polyclonal antibody may lay a foundation for the further phytase monoclonal antibody preparation and the subsequent ELISA kit development.

Key words: phytase, Balb/C mouse, ELISA, polyclonal antiserum