Type

Journal Article

Authors

R O'Kennedy
P Leonard
C J Hayes

Subjects

Microbiology

Topics
immunoassay humans antibody affinity gene library high throughput screening assays kinetics recombinant proteins female chemistry immunology research design animals genetics mice inbred balb c prostate specific antigen biosynthesis mice single chain antibodies

Overcoming antibody expression and screening limitations by smart design: applications to PSA immunoassay development. (2011)

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.
Collections Ireland -> Dublin City University -> PubMed

Full list of authors on original publication

R O'Kennedy, P Leonard, C J Hayes

Experts in our system

1
R O'Kennedy
Dublin City University
Total Publications: 197
 
2
Paul Leonard
Dublin City University
Total Publications: 27