|Title||Prostate-specific antigen immunosensing based on mixed self-assembled monolayers, camel antibodies and colloidal gold enhanced sandwich assays.|
|Publication Type||Journal Article|
|Year of Publication||2005|
|Authors||Huang, L., G. Reekmans, D. Saerens, J-M. Friedt, F. Frederix, L. Francis, S. Muyldermans, A. Campitelli, and C. Van Hoof|
|Date Published||2005 Sep 15|
|Keywords||Adsorption, Animals, Antibodies, Biosensing Techniques, Blood Chemical Analysis, Camels, Coated Materials, Biocompatible, Crystallization, Gold Colloid, Humans, Immunoassay, Prostate-Specific Antigen, Protein Binding, Surface Plasmon Resonance|
Prostate-specific antigen (PSA) is a valuable biomarker for prostate cancer screening. We developed a PSA immunoassay on a commercially available surface plasmon resonance biosensor. Our PSA receptor molecule consists of a single domain antigen-binding fragment, cAbPSA-N7, derived from dromedary heavy-chain antibodies and identified after phage display. It binds PSA with a high k(on) value of 1.9x10(6) M-1 s-1, and was covalently immobilised on a gold substrate via a mixed self-assembled monolayer (SAM) of alkanethiols by using carbodiimide-coupling chemistry in 10mM acetate buffer pH 5.5 to obtain an optimal pre-concentration. The best performing and optimised mixed SAM consisted of (10%) 16-mercapto-1-hexadecanoic acid (16-MHA) for covalent cAbPSA-N7 immobilisation and (90%) 11-mercapto-1-undecanol (11-MUOH) to minimise non-specific adsorption of the analyte. In this way, two advantages are incorporated in a single coupling layer. Up to 28 fmol/mm2 of cAbPSA-N7 could be immobilised and 30% of its binding sites participate actively in PSA interaction. In addition, the optimised layer showed also optimal performance to assess physiological samples. Although PSA concentrations as low as 10 ng/ml could be detected directly, this detection limit could be enhanced to PSA levels in the sub ng/ml range by introducing a sandwich assay involving a biotinylated secondary antibody and streptavidin modified gold nanoparticles. This approach realizes the PSA detection at clinical relevant concentrations.
|Alternate Journal||Biosens Bioelectron|