Phage surface expression for analysis of recognition sites of human autoantibodies: comparison of single chain Fv and Fab.
Human autoantibodies can mediate the pathology of autoimmune disease. To plan therapeutic intervention based on inhibition of binding to autoantigen, it is desirable to map the amino acid residues involved in recognition. Bacterial expression provides a simple and rapid system for production and mutagenesis of recombinant antibody fragments, but there are problems with reproducible folding and dispersity of soluble products. Expression at the surface of phage particles avoids both these problems, and allows variable region gene sequences to be expressed as functional monomers. To apply this technology, we have expressed a human anti-DNA monoclonal antibody, derived from a hybridoma, at the surface of the phage. The presence of the phage particle allowed highly sensitive detection of antibody activity by ELISA, so that minimal levels of expression were required. By measuring the amount of expressed antibody protein, it was possible to analyze the antibody activity/microgram. We expressed the antibody as FAB or single chain (sc) Fv, and found binding activities to be closely similar. The parental anti-DNA antibody utilizes the human V4-34 gene and displays the associated conformation-dependent 9G4 idiotope. Both the Fab and scFv expressed the idiotope, confirming similar molecular folding. This technology can be applied rapidly and efficiently to investigations of human autoantibody structure, with the option for expression as either Fab or scFv, and the only requirement being knowledge of VH and VL sequences.
Chapman
C J
CJ
Molecular Immunology Group, Tenovus Laboratory, Southampton University Hospitals, UK.
Mockridge
C I
CI
Spellerberg
M B
MB
Zhu
D
D
Stevenson
F K
FK
D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000974:Antibodies, Antinuclear / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics*; D001666:Binding Sites, Antibody / Q000235:genetics*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*
D000888:Antibodies, Anti-Idiotypic; D000974:Antibodies, Antinuclear; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; C055966:immunoglobulin Fv