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.

Monoclonal and Polyclonal Antibodies Specific to Human Fibromodulin

“…Cell lines containing I83-E95, 232-B4 and Chinese hamster ovary (CHO) (National cell bank of Iran, Tehran, Iran) as well as lymphoblastoid cell line (LCL) (14) were grown in RPMI 1640 (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS) (Gibco) at 37 C in a humidified incubator with 5% CO 2 atmosphere.…”

Frequency analysis of B lymphocytes specific for Rh antigens in naturally immunized Rh‐negative women

“…Rh D‐negative women, who are naturally immunized to D antigen in previous pregnancies, may serve as donors of specific B lymphocytes and have reportedly been recruited in studies aiming to establish human monoclonal anti‐D immunoglobulin [7–9]. These individuals vary greatly with respect to the level of anti‐D immunoglobulins they produce.…”