Overcoming antibody expression and screening limitations by smart design: Applications to PSA immunoassay development

摘要

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.