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.