title: Dissociation-independent selection of high-affinity anti-hapten phage antibodies using cleavable biotin-conjugated haptens.
abstract: The engineering of hapten-specific antibodies with affinity constant higher (K(a) values >10(10)M(-1)) than those of conventional antibodies promises hapten immunoassays exhibiting sub-femtomole range sensitivity, based on the conventional competitive assay principle. Here we report a simple method to select phage particles displaying anti-hapten antibody fragments with exceptionally high affinity. 11-Deoxycortisol (11-DC), selected as a model target hapten, was covalently conjugated to biotin via a spacer that included a reductively cleavable disulfide bond. Phage particles displaying high-affinity, single-chain Fv fragments (scFvs) specific for 11-DC (K(a)1.3 x 10(10)M(-1)) were incubated with the "cleavable biotin"-conjugated 11-DC, and the resulting complexes was captured on immobilized NeutrAvidin. Mild reductive conditions that did not decrease phage infectivity easily cleaved the disulfide bond, allowing the recovery of target phage particles; this process is fully independent of the dissociation of the antigen-antibody interaction. Five serial rounds of selection enabled the isolation and enrichment of the anti-11-DC phage (specific phage ratio >90%) from among a 100,000-fold excess of nonspecific phage particles. This method will be applicable for selection of extra-high-affinity phage antibodies against a wide variety of haptens.
authors.affiliation: Kobe Pharmaceutical University, 4-19-1 Motoyama-Kitamachi, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Norihiro
authors.initials: N
authors.lastname: Kobayashi
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Karibe
authors.forename: Junichi
authors.initials: J
authors.lastname: Goto
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000302:isolation & purification*; D000915:Antibody Affinity; D001483:Base Sequence; D001710:Biotin; D002623:Chemistry Techniques, Analytical; D003350:Cortodoxone / Q000276:immunology; D004274:DNA, Recombinant / Q000235:genetics; D006241:Haptens / Q000276:immunology*; D019151:Peptide Library; D015202:Protein Engineering; D019809:Streptavidin
chemical_list: D000906:Antibodies; D004274:DNA, Recombinant; D006241:Haptens; D019151:Peptide Library; D001710:Biotin; D019809:Streptavidin; D003350:Cortodoxone