Monoclonal antibody specific to the Dia blood group antigen generated by phage display technology

Abstract

Background: Alloanti-Di^a can be implicated in mild to severe blood transfusion reactions. Given the concomitance of a high prevalence of the Di^a antigen and antibody circulating in some populations, an anti-Di^a typing reagent is required in order to enable safe blood transfusions. Limitations of hybridoma technology to produce such a reagent led to the use of phage display technology to generate an anti-Di^a monoclonal antibody.

Materials and methods: A library of phages displaying murine single-chain variable fragment antibody (scFv-phages) was consecutively adsorbed with different panels of Di(a-b+) red cells to eliminate scFc-phages that potentially bind irrelevant blood group antigens. Thereafter, the subtractive library was specifically selected for the scFv-phages that bound Di^a antigen by sequentially biopanning the library with Di(a+b+) cell ghosts and Di(a+b-) intact red cells. A specific interaction between the selected scFv-phages and Di^a epitope was validated with the Di^a peptide by a competitive haemagglutination inhibition assay and confirmed with the red cells by flow cytometry.

Results: An scFv-phage clone specifically bound the Di^a epitope, as shown by its binding competition with the human anti-Di^a to the Di^a peptide in a haemagglutination inhibition test. Moreover, it was highly reactive to Di(a+b+) red cells but not to Di(a-b+) red cells, as determined by flow cytometry.

Discussion: In this study, a Di^a-specific scFv-phage antibody was successfully produced. The selection protocol might be a prototypic platform for producing monoclonal antibodies to relevant blood group antigens. The scFv-phage produced in this way warrants further development for use as a reagent for Di^a red cell typing.