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Establishment of human heterohybridoma and lymphoblastoid cell lines specific for the Rh D and C antigens
Author(s) -
Pasha R. P. K.,
Roohi A.,
Shokri F.
Publication year - 2003
Publication title -
transfusion medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.471
H-Index - 59
eISSN - 1365-3148
pISSN - 0958-7578
DOI - 10.1046/j.1365-3148.2003.00424.x
Subject(s) - antibody , antigen , microbiology and biotechnology , hemagglutination , virology , rh blood group system , biology , monoclonal antibody , titer , agglutination (biology) , immunology
summary . Human monoclonal antibodies specific for the D antigen of the Rh system are valuable tools for blood group typing and prevention of erythroblastosis. In this study, peripheral blood lymphocytes obtained from an Rh‐negative woman immunized with Rh‐positive fetuses were immortalized with Epstein–Barr virus (EBV), and transformed lymphoblastoid cell lines (LCLs) secreting antibodies to Rh antigens were generated. The presence of specific antibody was assessed by direct haemagglutination using Rh‐positive, papain‐treated red blood cells (RBCs), and the production of human antibody was assayed by enzyme‐linked immunosorbent assay (ELISA). Specificities of the antibodies were determined by a panel of RBCs of known Rh phenotypes. Five LCLs produced antibody specific for the D antigen, and one LCL showed specificity towards the C antigen of the Rh blood group system. High‐titre anti‐Rh antibody‐producing LCLs were subsequently selected and fused with a human x mouse heteromyeloma cell line. A hybridoma line producing human antibody of the immunoglobulin M (IgM) isotype, which strongly reacted with the D antigen, was established. The hybridoma was cloned, and the monoclone has been stable for growth and antibody production during 8 months of continuous culture, with a mean antibody concentration of 11·5 µg mL −1 and haemagglutination titre of 1/20 480. This antibody was not able to agglutinate a sample of native weak D RBCs (D u ); however, agglutination was achieved with papain‐treated D u RBCs. Immunoprecipitation of the D antigen by this antibody, followed by Western blot analysis, did not reveal any immobilized D‐specific polypeptide. As this human antibody readily agglutinates D + RBCs in saline, it has the potential to be used as an efficient reagent in routine blood group typing.