In vitro selection of a high affinity antibody to oestradiol using a phage display human antibody library.
We have isolated a monoclonal antibody binding to oestradiol with high affinity (3.7 nM), and exhibiting a better than 1000-fold selectivity in binding to other steroids. A high affinity antibody with good specificity is essential for the accurate determination of circulating oestradiol levels. To date, conventional hybridoma technology has not yielded a reagent of sufficiently high affinity and specificity for this ligand. The aim of this study was to investigate whether such a reagent was accessible through the engineering of antibodies on the surface of filamentous phage. Antibodies were isolated from a large repertoire of single chain Fv fragments (scFv) derived from non-immunised human donors, with selection and screening procedures biased to favour those binding to free oestradiol. This resulted in an antibody with nanomolar affinity for oestradiol, while affinities for related steroids are in the micromolar range. The relative lack of reactivity for steroids substituted at either end of the molecule suggests that this antibody is unique among anti-steroid monoclonal antibodies in lacking a 'blind-spot'. Our results demonstrate that phage display can provide solutions to problems that have so far proved intractable using conventional hybridoma technology.
Pope
A
A
Cambridge Antibody Technology Ltd., Melbourn, Cambridgeshire, UK.
Pritchard
K
K
Williams
A
A
Roberts
A
A
Hackett
J R
JR
Mandecki
W
W
Johnson
K S
KS
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000918:Antibody Specificity; D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004958:Estradiol / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D017551:Microdialysis; D013256:Steroids / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D013256:Steroids; D004958:Estradiol