title: Establishment of human heterohybridoma and lymphoblastoid cell lines specific for the Rh D and C antigens.
abstract: 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 microg mL-1 and haemagglutination titre of 1/20 480. This antibody was not able to agglutinate a sample of native weak D RBCs (Du); however, agglutination was achieved with papain-treated Du 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.
authors.affiliation: National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: R P K
authors.initials: RP
authors.lastname: Pasha
authors.forename: A
authors.initials: A
authors.lastname: Roohi
authors.forename: F
authors.initials: F
authors.lastname: Shokri
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D000918:Antibody Specificity; D001798:Blood Proteins / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line; D002472:Cell Transformation, Viral; D005260:Female; D006384:Hemagglutination; D004854:Herpesvirus 4, Human; D006801:Humans; D006825:Hybridomas / Q000166:cytology* / Q000276:immunology; D008214:Lymphocytes / Q000166:cytology* / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology; D051379:Mice; D011233:Precipitin Tests; D012204:Rh-Hr Blood-Group System / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D008565:Membrane Proteins; C066468:RHCE protein, human; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen