Determination of binding constants of diabodies directed against prostate-specific antigen using electrochemiluminescence-based immunoassays

Journal of Molecular Recognition : JMR
R AbrahamM Darsley

Abstract

Two diabody molecules directed against the prostate-specific antigen (PSA) were generated from a combinatorial phage display library. The C-termini of diabodies incorporated the FLAG peptide epitope (P008D diabody) or the myc epitope (P001D). Both diabodies have the same antigen-binding site. Equilibrium-binding constants of these molecules were determined in two immunoassays using the ORIGEN detection system based on electrochemiluminescence. The binding of diabodies to biotinylated PSA was detected with either polyclonal antimouse Fab F(ab')2 or a monoclonal antibody directed against the FLAG epitope. Both detecting antibody preparations were covalently labeled with a ruthenium (II) tris (bipyridyl) moiety, Ru(bpy)3(2+), which allows quantification via an electrochemically triggered light reaction in an ORIGEN analyzer. The binding constants obtained by Scatchard analysis of non-linear curve fitting calculations from electrochemiluminescence immunoassays were compared with data derived from kinetic-binding studies using the BIAcore technology based on surface plasmon resonance. Depending on the detecting antibody, the dissociation constants KD determined at equilibrium with the ORIGEN technology are between 0.1 and 0.4 nM for...Continue Reading

References

Dec 1, 1992·Immunological Reviews·H R HoogenboomG Winter
Mar 20, 1992·Journal of Molecular Biology·J Foote, G Winter
Aug 15, 1991·Nature·T ClacksonG Winter
Jan 24, 1991·Nature·G Winter, C Milstein
Oct 24, 1985·Journal of Immunological Methods·C J StanleyC H Self
Jan 1, 1994·Journal of Clinical Laboratory Analysis·J T Wu
Jan 1, 1994·The Prostate. Supplement·G R Blackledge, K Lowery
Jun 1, 1993·Disease Markers·J H Howanitz
Feb 1, 1994·Molecular Immunology·D P McGregorW J Harris
Jul 15, 1993·Proceedings of the National Academy of Sciences of the United States of America·P HolligerG Winter

❮ Previous
Next ❯

Citations

Jul 17, 2015·Journal of Biomolecular Screening·Daniela Della DucataBodo Brocks
Jun 13, 2018·Toxins·Line LedsgaardAndreas H Laustsen

❮ Previous
Next ❯