Abstract

Background - Alloanti-Dia can be implicated in mild to severe blood transfusion reactions. Given the concomitance of a high prevalence of the Dia antigen and antibody circulating in some populations, an anti-Dia 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-Dia 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 Dia 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 Dia epitope was validated with the Dia peptide by a competitive haemagglutination inhibition assay and confirmed with the red cells by flow cytometry.
Results - An scFv-phage clone specifically bound the Dia epitope, as shown by its binding competition with the human anti-Dia to the Dia 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 Dia-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 Dia red cell typing.

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Authors

Phatchira Thattanon - Graduate Programme in Biomedical Sciences

Jeeraphong Thanongsaksrikul - Graduate Programme in Biomedical Sciences

Sawang Petvises - Department of Medical Technology; Faculty of Allied Health Sciences, Thammasat University, Rangsit Campus, Thailand

Oytip Nathalang - Graduate Programme in Biomedical Sciences

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