title: Overcoming antibody expression and screening limitations by smart design: applications to PSA immunoassay development.
abstract: Improving the functional and structural properties of target proteins can often be a challenge for researchers. This paper highlights the importance of antibody construct on screening performance, and ultimately, the clone that is selected. We report the reformatting of phage-selected single chain antibody variable region fragments (scFvs) into single chain antibody fragments (scAbs) for improved screening and binding studies. The generation of a scAb, which had a fused human kappa light chain constant domain (C(k)), was shown to significantly improve expression levels in Escherichia coli. Antibody expression levels were compared between the two antibody constructs (scFv and scAb) by ELISA and a 100-fold improvement was observed. The C(k) domain in the expressed scAb also facilitated high throughput analysis by a Biacore capture assay approach. Individual functional scAbs were ranked on the basis of their remaining binding percentage after 5 min dissociation. Selected antibodies were further characterised by kinetic analysis and a sandwich-based immunoassay developed. The scAb construct enhanced expression levels significantly, facilitating antibody screening and immunoassay development for prostate-specific antigen (PSA), a marker for prostate cancer.
authors.affiliation: Applied Biochemistry Group, School of Biotechnology, Dublin City University, Dublin 9, Ireland.
authors.forename: C J
authors.initials: CJ
authors.lastname: Hayes
authors.forename: P
authors.initials: P
authors.lastname: Leonard
authors.forename: R
authors.initials: R
authors.lastname: O'Kennedy
mesh_terms: D000818:Animals; D000915:Antibody Affinity / Q000235:genetics; D005260:Female; D015723:Gene Library; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D017430:Prostate-Specific Antigen / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012107:Research Design; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D017430:Prostate-Specific Antigen