制备脆弱拟杆菌来源的新型α-半乳糖苷酶单抗用于检测通用型红细胞中的微量残留酶

Preparation of a novel monoclonal antibody against α-galactosidase from Bacteroides fragilis for detection of minimal residual enzyme in universal red blood cells

目的建立一种检测微量脆弱拟杆菌(Bacteroides fragilis)来源的α-半乳糖苷酶的方法用于测定酶解转变的通用型红细胞上的残留酶。方法用脆弱拟杆菌来源的基因重组α-半乳糖苷酶(纯度大于90%)免疫BALB/c小鼠,制备单抗;用稳定分泌抗体的杂交瘤细胞株制备腹水单抗,再经HiT rap rP rotein A柱纯化获得高纯度的抗体;间接ELISA法测定单抗效价,Western印迹法评价单抗特异性。联合纯化后的单抗和兔多抗采用间接ELISA法检测酶解法制备的通用型红细胞和洗涤液中的残留酶量。结果获得了高效价高纯度的单抗,Western印迹实验显示该抗体可特异性地与新型α-半乳糖苷酶结合;间接ELISA法检测微量α-半乳糖苷酶的下限为1 ng/ml,红细胞按1∶4的体积比经4次洗涤后,其残留酶量<10 ng/ml。结论所建立的在血型转变过程中检测微量残留α-半乳糖苷酶的方法,可用于酶解法制备的通用型红细胞的安全性评价。

Objective To establish a method of quantiying trace α-galactosidase from Bacteroides fragilis in enzymatic conversion of blood group B to O red blood cells （ B-ECO RBCs）. Methods BALB/c mice were immunized with purified recombinant B.fragilis α-galactosidase （the purity 〉 90% ） to prepare monoclonal antibodies. The ascites were prepared using hybridoma cell lines stably secreting antibody and purified by HiTrap rProtein A column. The antibody titer and specificity were detected by ELISA and Western blotting, respectively. Purified monoclonal antibody and rabbit polyclonal antibody were applied to detect residual enzyme in B-ECO RBCs and the washing solution was analyzed by indirect ELISA. Results A high titer and purity antibody was obtained. Western blotting showed that the antibody specifically reacted with B. fragilis α-galactosidase. Moreover, indirect ELISA was sensitive enough to detect the minimal amount of residual ct-gal- actosidase at the concentration of 1 ng/ml. After four repeat washing cycles with 1：4 （v/v） phosphate-buffered saline, the amount of residual enzyme in B-ECO RBCs was less than 10 ng/ml, Conclusion An effective method of detecting the minimal amount of residual α-galactosidase in blood conversion is established for safety evaluation of universal RBCs preparation by enzymatic treatment.