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Title: Cloning antibody fragments to the α2β1 integrin on platelets by V phage
Author: Hughes, D. L.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2002
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The functional and immunological importance of the α2βl integrin on platelets is well documented. Functionally, α2β1 on platelets acts as one of the collagen receptors with ligand binding mediated by a 200 amino acid N-terminal domain on the α2 subunit designated the I (for 'inserted') domain. The first part of this PhD describes the use of DNA vaccination and V gene phage display to isolate α2 I-domain specific antibody fragments. DNA vaccination represents a versatile alternative to the use of protein for immunisation, particularly for the generation of domain-specific antibodies. In addition, the use of V gene phage display technology to isolate antibodies from immunised animals as recombinant fragments for further engineering may offer advantages over existing hybridoma-fusion techniques. Mice and rabbits immunised with the human α2 I-domain DNA sequence showed antigen-specific antibody responses. Following immunisation, a phage display library was constructed using the heavy and light chain V gene repertoires derived from mouse splenocytes. Selection of this library on recombinant and native antigen resulted in the isolation of two categories of I-domain single chain V(ariable) domain antibody fragments (scFvs). The primary molecular structure of the V domains was determined and the effect of the scFvs on the interaction of the I domain with collagen and collagen-derived peptides was studied in detail. Immunologically, the gene for α2 has several alleles in the Caucasoid population. Allelic variants of α2 can lead to the formation of alloantibodies following transfusion and during pregnancy. A glutamic acid to lysine replacement at residue 505 is the basis of the human platelet antigen (HPA) -5 alloantigen system. Binding of HPA-5 alloantibodies to α2 may lead to reduced platelet survival and thrombocytopenia. The lysine505 form of α2 (HPA-5b) is the second most important platelet-specific alloantigen after the leucine33 form of the β3 integrin (HPA-1a). The second part of this PhD describes transfer experiments of primed human immune lymphocytes to severe combined immune deficiency (SCID) mice with subsequent rescue of the expanded human B cell repertoire by V gene repertoire cloning and phage display.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available