植酸酶鼠源多克隆抗血清的制备

Preparation of Phytase Mouse Polyclonal Antiserum

【目的】制备灵敏性高、特异性强的植酸酶多克隆抗体。【方法】将初步纯化的麸皮植酸酶蛋白经凝胶电泳鉴定纯度后,按50μg蛋白/只.次免疫Balb/C小鼠,共免疫4次,每次间隔4周,最后一次免疫60 d后,摘眼球取血,制备抗血清。利用间接ELISA方法测定抗血清效价,间接竞争ELISA测定敏感性和特异性。【结果】凝胶电泳的麸皮植酸酶只有一个条带;血清植酸酶抗体效价达1×104;半数抑制浓度达148.87 ng.mL-1。此抗血清对市售的普通微生物植酸酶、市售包被微生物植酸酶及浓缩的微生物植酸酶的半数抑制浓度分别达到165.59、163.80和166.51 ng.mL-1,交叉反应率分别为89.85%、90.88%和89.41%。100℃煮沸5 min后的麸皮、市售普通微生物植酸酶、市售包被微生物植酸酶及浓缩酶半数抑制浓度分别为2 871.34、5 208.85、7 914.12和5 804.24 ng.mL-1,交叉反应率分别为5.18%、2.86%、1.88%及2.56%。【结论】制备出了具有高效价、高灵敏度和特异性的鼠源植酸酶多克隆抗体,为植酸酶单克隆抗体制备及植酸酶ELISA检测试剂盒研制奠定了基础。

【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/time,once every four weeks for 4 times,60 days after the last immunization,the serum was prepared followed blood withdrawn by eyeball taken off.Antiserum titer was determined by indirect ELISA,sensibility and specificity by indirect compete ELISA.【Result】The result showed that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.