Phatchira Thattanon1, Jeeraphong Thanongsaksrikul1, Sawang Petvises2, Oytip Nathalang1. 1. Graduate Programme in Biomedical Sciences, Faculty of Allied Health Sciences, Thammasat University, Rangsit Campus, Thailand. 2. Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University, Rangsit Campus, Thailand.
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.
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.
Authors: J D Marks; W H Ouwehand; J M Bye; R Finnern; B D Gorick; D Voak; S J Thorpe; N C Hughes-Jones; G Winter Journal: Biotechnology (N Y) Date: 1993-10