The isolation and characterisation of human monoclonal HLA-A2 antibodies from an immune V gene phage display library.
Molecular cloning techniques and V gene phage display have revolutionised the production of human monoclonal antibodies. Antibodies of a defined specificity can be obtained by selecting phage display libraries on antigen in a process known as panning. We have applied these techniques to the isolation of three HLA-A2-specific single chain variable domain fragments (scFv) from a patient alloimmunised by blood transfusion. Analysis of specificity with cells of HLA genotyped donors revealed the following: i) in addition to the major reactivity with HLA-A2, cross-reactivity with the HLA-A28 epitope; and ii) inhibition of scFv binding to the antigen by the patients' antibodies. The heavy chain variable genes of all three were derived from the germline gene Cos-3, carry the hallmarks of somatic hypermutation, and are most likely derived from clonally related B cells. The light chain variable domains were encoded by DPK1 and DPK8 from the VkappaI family. These data show that phage display can be used to clone HLA-specific alloantibodies that recognise the native antigen from alloimmunised patients.
Watkins
N A
NA
University of Cambridge, Department of Haematology, United Kingdom.
Brown
C
C
Hurd
C
C
Navarrete
C
C
Ouwehand
W H
WH
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D018076:DNA, Complementary; D005260:Female; D015789:HLA-A2 Antigen / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D008875:Middle Aged; D008969:Molecular Sequence Data; D019151:Peptide Library
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D015789:HLA-A2 Antigen; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library