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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 for the detection of α-galactosidase from Bacteroides fragilis origin for the determination of residual enzymes on enzymatically converted universal red blood cells. Methods BALB / c mice were immunized with gene recombinant α-galactosidase (more than 90% purity) of Bacteroides fragilis to prepare monoclonal antibodies. The monoclonal antibodies against ascites were prepared by hybridoma cell lines secreting antibodies, Purified rP rotein A column to obtain high purity antibody; indirect ELISA assay monoclonal antibody titer, Western blot evaluation of monoclonal antibody specificity. The amount of residual enzyme in the universal erythrocytes and washings prepared by enzymolysis was detected by indirect ELISA. Results The McAb with high titer and high purity was obtained. The result of Western blotting showed that the antibody could specifically bind to α-galactosidase. The limit of detection of α-galactosidase by indirect ELISA was 1 ng / ml. Red blood cells by 1: 4 volume ratio after 4 times the washing, the residual enzyme <10 ng / ml. Conclusion The established method for the determination of trace residual α-galactosidase during blood group transformation can be used to evaluate the safety of universal erythrocytes prepared by enzymolysis.