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HiTrap rProtein 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.fragilisα-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 spe-cificity were detected by ELISA and Western blotting, respectively.Purified monoclonal antibody and rabbit polyclonal an-tibody 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.fragilisα-galactosidase.Moreover, indirect ELISA was sensitive enough to detect the minimal amount of residualα-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 min-imal amount of residual α-galactosidase in blood conversion is established for safety evaluation of universal RBCs prepara-tion by enzymatic treatment.
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