Preparation of a novel monoclonal antibody againstα-galactosidase from Bacteroides fragilis for detection of minimal residual enzyme in universal red blood cells / 军事医学

ABSTRACT

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.