Human Monoclonal Fab Fragments Recovered from a Combinatorial Library Bind Specifically to the Platelet HPA-1a Alloantigen on Glycoprotein IIb-IIIa

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Abstract
Background and objectives: Certain clinical conditions are related to the presence of platelet-specific alloantibodies in the patient's serum. We studied the molecular diversity of HPA-1a antibodies to analyze some peculiarities of this antibody response. Materials and methods: Human antibody Fab fragments that bind to the platelet alloantigen HPA-1a on glycoprotein IIb–IIIa (GPIIbIIIa) were generated by using a recombinant phage display system. We established an immunoglobulin G1, kappa combinatorial library from the peripheral blood lymphocytes of a person undergoing a severe posttransfusion purpura. Results: Characterization of Fab clones selected from the fifth round of antigen-specific panning of this library demonstrates a highly specific reactivity to the HPA-1a alloantigen. The nucleotide sequence analysis of representative HPA-1a-specific clones reveals at least 3 distinct VL and 3VH gene segments that present an extensive degree of mutation as demonstrated by comparison of gene usage and homologies to the nearest germline genes. Conclusions: These human HPA-1a-specific Fab reagents should allow us to better understand the molecular mechanism involved in HPA-1a alloimmunization.
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