Development of monoclonal antibody for Diego blood group typing

Antibodies against the Diego (DI) blood group antigens, anti-Dia and anti-Dib are implicated in mild to severe hemolytic disease of the fetus and newborn and hemolytic transfusion reactions. Owing to the high prevalence of the Dia antigen in Asian populations, including this antigen in the reagent red cells is relevant. To date, polyclonal human anti-Dia is routinely used in blood bank laboratories. Because of its high cost and short supply, the production of monoclonal antibodies is suggested. This study aimed to produce monoclonal antibodies for Diego blood group antigen typing using antibody phage display technology. A library of phages displaying mouse single-chain variable fragment antibody (scFv-phages) was incubated with panels of Di(a−b+) red blood cells (RBCs) containing other clinical important blood group antigens to eliminate unwanted binders. Thereafter, the scFv-phages specific to the Dia antigen were selectively fished out from the subtractive library by Di(a+b+) RBC ghosts and Di(a+b−) RBCs. Specific binding of the selected scFv-phages was validated with the Dia peptide by hemagglutination inhibition (HI) assay and confirmed testing with Di(a+b+) and Di(a−b+) RBCs by flow cytometry. The M13 wild-type phage served as a negative control. The selected scFv-phages were checked and analyzed to determine phage peptide sequence and immunoglobulin domains were predicted using DNA sequencing and IMGT visualization, respectively. Following the screening process, five scFv-phage clones were primarily chosen to test binding activity with the Dia epitope using reverse competitive inhibition test. Three scFv-phage clones showed positive binding activity; however only an scFv-phage clone no. 4 had complete VH and VL domains contributing antigen-binding ability. Predicting their immunoglobulin domains was closely related to the Mus musculus immunoglobulin sequence. Concerning flow cytometry analysis, the percentages of scFv-phage clone no. 4 reacting with Di(a+b+) RBCs were significantly higher than with Di(a−b+) RBCs. On the contrary, the M13 wild-type phage significantly differed with scFv-phage clone no. 4 testing with Di(a+b+) RBCs (P < 0.05) and no significant difference was observed when testing with Di(a−b+) RBCs. In conclusion, phage-derived monoclonal mouse antibody specific to the Dia blood group antigen was successfully produced. It warrants further development for use as a diagnostic reagent.