Analysis of antibody selection by phage display utilizing anti-phenobarbital antibodies

Abstract

The generation of new antibodies for diagnostic applications using phage display could greatly decrease the time and expense of new assay development but, to be effective, the process must yield antibodies with desired specificity and affinity comparable to those obtained by monoclonal antibody technology. In order to evaluate the ability of the phage selection process to yield antibodies with the desired specificity and affinity, a family of anti-phenobarbital antibodies were cloned as scFvs and Fabs and displayed on M13 gene III fusion proteins. All of the antibodies are derived from similar germline VL and VH genes and exhibit extensive sequence homology except in VH CDR3. Despite these similarities, the range of panning efficiencies was observed to vary by two orders of magnitude for expressed scFvs. Unexpectedly, the scFv with the highest panning efficiency has the lowest affinity. In competitive panning experiments this scFv is preferentially isolated over higher affinity antibodies. This scFv expresses high levels of soluble binding protein while higher affinity scFvs express lower levels of protein or nonfunctional protein. These results suggest that the efficiency of functional expression of scFv proteins can greatly influence the type of antibody selected by phage display. The range of panning efficiency for functional Fabs was significantly less (four-fold) than that observed for scFvs and did not correlate to the expression level of the secreted proteins. Based on the results of the Fabs examined, it is concluded that the expression properties of Fabs may not exhibit the extent of variability observed for scFvs when displayed and use of an Fab architecture may provide an advantage over scFv architecture in the selection process. The feasibility of selecting against undesirable cross-reactivities has also been demonstrated by simple modification of phage panning conditions. These combined results support the concept of obtaining antibodies with desirable specificity and affinity for diagnostic applications through the use of phage display.

On the influence of vector design on antibody phage display

“…It has been reported that scFvphage and Fab-phage have comparable display levels (Chapman et al, 1997), yet Fab-phage have been claimed to be superior for biopanning selection (Malone and Sullivan, 1996). Our study could not assess the impact of antibody format since the scFv-encoding phagemid, pSEX81, differs from the Fab-encoding phagemids by multiple sequence motifs.…”

Early Oligodendrocyte/Myelin Pathology in Alzheimer's Disease Mice Constitutes a Novel Therapeutic Target

“…18 We similarly constructed a control vector (parent scFv construct kindly provided by Dr. Mark A. Sullivan, University of Rochester), 19 which expresses an ER-targeted anti-phenobarbital-specific single-chain IB (pAAV-scFvphe KDEL IB ). Briefly, a phenobarbital-specific IB sequence with an in-frame ER targeting signal (KDEL) and c-myc epitope tag at the C terminus to facilitate immunocytochemical detection was cloned into the pBS-FBR mcs shuttle vector under the transcriptional control of the human cytomegalovirus promoter and a SV40-derived polyadenylation signal was included at the 3Ј end of the transcription unit.…”

Enhanced Phagocytosis ofCandidaSpecies Mediated by Opsonization with a Recombinant Human Antibody Single-Chain Variable Fragment

“…scFv2-18, which recognizes cell surface mannans (data not shown) on blastoconidia and filamentous forms of Candida, and a control scFv, scFv5 (2), which recognizes a protein antigen expressed only on filamentous Candida, were prepared using the method of Kipriyanov et al (11). Because scFv does not contain an Fc region, but does contain the FLAG epitope tag (14), organisms were opsonized with a three-layer complex consisting of 10 g of scFv2-18 per ml, 10 g of M2 anti-FLAG mouse monoclonal antibody per ml (Sigma-Aldrich, St. Louis, Mo. ), and a 1:250 dilution of an immunoglobulin G (IgG) fraction of rabbit anti-mouse polyclonal antiserum (Jackson ImmunoResearch, West Grove, Pa.).…”