Conventional immunization-based antibodies are not always applicable to monitor trace amounts of clinical biomarkers due to insufficient antigen-binding affinity. The development of in vitro affinity maturation for native antibodies has paved the way to solve this problem, but there has been minimal success in obtaining improved antibody mutants for small molecule biomarkers (haptens). We here describe a "molecular breeding" approach, in which a practical anti-hapten antibody mutant has successfully been generated from a prototype antibody with poor binding properties. Random point mutations were introduced by error-prone polymerase chain reaction in the VH and VL genes of a second-generation mutant of a single-chain Fv fragment (scFv) that binds estradiol-17β (E2), whose parent mouse Fab fragment showed poor affinity for E2 (Ka = 5.2 × 107 M-1). The resulting scFv gene library was expressed on filamentous phage particles. Phage clones with strong E2 binding were isolated with dissociation-independent methods using newly developed reagents. The binding characteristics and clinical applicability of the soluble scFvs prepared from the selected clones were examined in a competitive enzyme-linked immunosorbent assay (ELISA). We finally obtained a third-generation scFv mutant, scFv#m3-a18 (11 amino acid substitutions), which showed ∼250-fold greater Ka (1.3 × 1010 M-1) than the parent Fab, and yielded sensitive ELISA dose-response curves for E2 (limit of detection < 0.5 pg/assay) with practical specificity. The average human serum E2 levels, determined after acetonitrile extraction, were compatible with reported reference ranges. The present results will prompt a new era for preparing diagnostic reagents.
ASJC Scopus subject areas
- Analytical Chemistry