Antibody affinity maturation in vitro using unconjugated peptide antigen

Abstract

Selection of antibody library in vitro is almost always performed on a certain solid-phase with immobilized antigen. However, for the selection of small molecule binders, conjugation of the antigen to a carrier molecule is indispensable, which often leads to the selection of unwanted binders such as conjugate-binders or those with insufficient specificity. Here we describe a rapid and efficient way to improve the affinity of an anti-small molecule antibody without antigen derivatization. The method is based on the open-sandwich (OS) principle, which utilizes the antigen-dependent stabilization of antibody variable domain Fv. We used an anti-osteocalcin C-terminal peptide Fv that showed a good response but with moderate sensitivity in OS ELISA as a model. By selecting PCR-randomized V(H)-displaying phages for superior binders to the immobilized V(L) fragment in the presence of limited amount of antigen peptide, V(H) mutants that show superior detection sensitivity in OS ELISA were obtained, and were characterized to retain improved antigen-binding affinity. Furthermore, saturation mutagenesis of a mutant resulted in further improvement in sensitivity. This 'OS-selection' will be the first to select anti-small molecule antibodies without using conjugated antigens, and especially useful in the affinity maturation of antibodies whose Fv has limited stability in the absence of antigen.

Isolation of a Monoclonal Antibody and its Derived Immunosensor for Rapid and Sensitive Detection of 17β-Estradiol

“…The affinity of our antibodies was equal to or slightly better than that of the aforementioned antibodies. In our further studies, we may perform artificial evolution of ESC9 using approaches such as molecular breeding ( Oyama et al, 2013 ) or open-sandwich selection ( Iwai et al, 2010 ) to further improve the affinity of the antibody and sensitivity of the assay. Although ESC9 does not cross-react with dehydroepiandrosterone, pregnenolone acetate, cortisol, and diethylstilbestrol, it does react with TS.…”

An Open Sandwich Immunochromatography for Non-competitive Detection of Small Antigens

“…9 This is possibly due to the slow association kinetics of the VH/VL fragments used in this study. 7 Use of sensitive fluorescent labels, such as quantum dots (QDs), improves the sensitivity of IC. By using QDs, as compared to gold colloid label, the working range of competitive chloramphenicol IC was improved from 0.5-6 ng/mL (gold colloid) to 0.3-20 ng/mL (quantum dot).…”

Protein-based Open Sandwich Immuno-PCR for Sensitive Detection of Small Biomarkers

“…Phagemid pIT2-VH (O2AG2), which encodes the VH gene of a higher affinity mutant of KTM219, O2AG2, was previously isolated from a PCR-randomized VH library by open-sandwich selection. 17 The O2AG2 VH gene was amplified and cloned into the SfiI-NotI region of the pET-MBPp-VH (BPA) vector 18 to make pET-MBPp-VH (O2AG2). E. coli BL21 (DE3, pLysS) cells were transformed with this vector and cultured.…”

“…16 Furthermore, an affinitymatured antibody O2AG2 with higher affinity than KTM219 was made by Iwai et al through a phage-based method. 17 In this study, fragments of these antibodies were used for the detection of BGP-C7.…”