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The measurement of proteins with a limited number of available non-overlapping epitopes recognizable by antibodies represents a common challenge for the development of drug-tolerant clinical biomarker assays. For target proteins with two dominant epitopes, only one epitope remains when the other is occupied by the therapeutic antibody. Alternative strategies for overcoming this obstacle have been described in the literature; however, these methods have potential limitations. We have developed a novel method for measuring target engagement when only one epitope remains after therapeutic antibodies bind their analytes. The method combines Affinity Capture Elution (ACE) followed by simultaneous capture and detection of the protein of interest. This novel method has been named ACE-Sandwich. The application of this method is not dependent on the immunoglobulin G subclass of the therapeutic antibody, nor does this method require sample pretreatment. Furthermore, the ACE-Sandwich method is highly sensitive, reproducible, and tolerant to high concentrations of therapeutic antibody., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Selection from phage antibody libraries can be considered to be an in vitro immune system in which the antibody response is reduced to the bare minimum of antigen recognition. Using selections of antibodies on peptides from a phage antibody library, we investigated what constitutes peptide antigenicity in the context of the antibody-protein binding site. We selected polyclonal antibodies in a high throughput format against 44% of 90 overlapping peptides derived from three different proteins. Of these, 33% of peptides (epitopic peptides) were able to select antibodies that recognized the protein from which the peptides were derived. Although no algorithm was able to predict all epitopic peptides, solvent accessibility was the best predictor in this cell-free antibody selection context. We subsequently applied solvent accessibility to successfully predict epitopic peptides from p53 and Znf217, and showed that such peptide selected single-chain antibodies were able to recognize soluble p53 in ELISA and Znf217 in a western blot. This is likely to have considerable utility in functional genomics and proteomics where it should be possible to select antibodies against gene products on the basis of deduced amino acid sequence in a high throughput fashion.

In order to more productively utilize the rich source of antigen-specific reagents present in the previously described non-immune single chain fragment variable (scFv) yeast display library [Nat. Biotechnol. 21 (2003) 163], one must be able to efficiently isolate and characterize clones within the library. To this end, we have developed and validated a magnetic bead sorting technique utilizing the Miltenyi Macs™ system to recover greater than 90% of the antigen-specific clones present in the library. In combination with flow cytometry, we rapidly reduced diversity and enriched for antigen-specific clones in three rounds of selection. Furthermore, we demonstrate the use of pre-existing monoclonal antibodies (mAbs) for antigen labeling and subsequent flow cytometric sorting and characterization of epitope-specific scFv. Combining these two improvements in library screening allowed isolation and characterization of three epitope-specific scFv, including a previously uncharacterized epitope to a 6-kDa protein, epidermal growth factor. [Copyright &y& Elsevier]