Mouse Monoclonal Antibody Specific To Human Ena Blood Group Antigen

Abstract

Abstract The aim of this study was to produce mouse monoclonal antibodies (mAbs) specific to human blood group antigens. Two percent solution of normal human red blood cells (RBCs) O, RhD+ in normal saline was freshly prepared, and 100 uL was immunized weekly into BALB/mice, intraperitoneal route for 3 weeks. Prior to each immunization, 100 uL of mouse blood was collected and serum was separated. The rising of anti-RBCs antibody titer was performed by standard hemagglutination compared to pre-immunized serum. Mouse was then sacrificed, and the spleen was collected. The fusion between mouse spleen cells and mouse myeloma (X63Ag8.653) cell lines was performed according to the standard hybridoma technique. Hybrid clones were grown in DMEM high glucose supplemented with 20% FCS at 3 °C, 5% CO2 incubator with 5% humidity. The anti-RBCs in hybrid cell culture supernatant were tested by standard hemagglutination and a monoclonal antibody-producing single clone of the hybrid cell was generated by limiting dilution technique. Antibody screening and identification were performed using screening cells, panel cells, and rare blood type RBCs, followed by enzyme treatment technique. The Isotype of mAb was also identified. The results showed that amongst those obtained, mAb clone 1E10-2B2, was confirmed to be anti-Ena characterized as ficin resistant (anti-EnaFR) and isotype IgG2b, kappa. The anti-Ena mAb is very helpful and can be applied as specific mAb for the phenotyping of Ena which is the high-frequency blood group antigens in all populations. Keywords: Glyphorin, MNS blood group, Ena, alloantibody, HDFN, HTR