Study of antibody-antigen interaction through site-directed mutagenesis of the VH region of a hybrid phage-antibody fragment.
An important aspect of the study of antibody structure-function relationships involves analysis of natural or synthetic mutations of antigen-combining sites. The anti-hen egg lysozyme monoclonal antibody HyHEL-10 has been a focus for antibody structure-function studies. We have displayed on bacteriophage of a hybrid single chain Fv, containing the light chain variable region of HyHEL-10 and the heavy chain variable region of a structurally related but functionally distinct antibody, AS32. By using a combination of site-directed mutagenesis, complementary determining region grafting and molecular modeling, we have identified a number of contact and non-contact residues that are important in the affinity of HyHEL-10 for lysozyme. In particular, the heavy chain variable region framework residue at position 94 was shown to be an important determinant of high-affinity binding. The phage display approach eliminates the need for purification of antibodies and, when used in combination with polymerase chain reaction for variable region sequence mutagenesis, facilitates the rapid generation and characterization of mutant antibodies.
Li
Y
Y
Department of Botany, University of Leicester, UK.
Owen
M R
MR
Cockburn
W
W
Kumagai
I
I
Whitelam
G C
GC
D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology*; D000937:Antigen-Antibody Reactions / Q000235:genetics*; D001666:Binding Sites, Antibody / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005803:Genes, Immunoglobulin; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D010834:Phytochrome / Q000276:immunology; D051942:Phytochrome A; D010940:Plant Proteins / Q000276:immunology; D017422:Sequence Analysis, DNA
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D051942:Phytochrome A; D010940:Plant Proteins; D010834:Phytochrome