title: Comparative study of thiolated protein G scaffolds and signal antibody conjugates in the development of electrochemical immunosensors.
abstract: To achieve a high efficiency of analyte capture by a capture antibody attached to an electrochemical immunosensor, we have immobilised an analyte-specific antibody on a self-assembled layer of recombinant Protein G that was thiolated with succinimidyl-6-[3'-(2-pyridyldithio)-propionamido] hexanoate (LC-SPDP). Then two techniques were employed for conjugating a second antigen-specific antibody to alkaline phosphatase (mAb2-AP) using either LC-SPDP or the biotin-streptavidin interaction as the mode of cross-linking the antibody and enzyme. After characterising the two mAb2-AP preparations (mAb2-(LC-SPDP)-AP and mAb2-(Biotin-SA)-AP), they were each used as the signal antibody for immunosensors formatted for two-site immunoassays where the capture antibody was attached to a Protein G-(LC-SPDP) scaffold on gold electrodes. The antibodies and assays were specific for the clinically important hormone, human chorionic gonadotrophin (hCG). Protein G-(LC-SPDP) provided a stable scaffold, while mAb2-(LC-SPDP)-AP and mAb2-(Biotin-SA)-AP performed well as the signal antibodies. Immunosensors with mAb2-(Biotin-SA)-AP were characterised by a limit of detection of 216 I UL(-1) for hCG and a linear response up to approximately 2000 I UL(-1). Conversely, immunosensors with mAb2-(LC-SPDP)-AP exhibited a limit of detection of 240 I UL(-1) and a linear response up to 4000 I UL(-1).
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia.
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Fowler
authors.forename: Margaret C
authors.initials: MC
authors.lastname: Stuart
authors.forename: Danny K Y
authors.initials: DK
authors.lastname: Wong
mesh_terms: D000906:Antibodies; D015374:Biosensing Techniques / Q000295:instrumentation*; D002208:Caproates; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry; D004563:Electrochemistry / Q000295:instrumentation*; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation; D009419:Nerve Tissue Proteins / Q000737:chemistry; D013438:Sulfhydryl Compounds
chemical_list: D000906:Antibodies; D002208:Caproates; C029152:G-substrate; D009419:Nerve Tissue Proteins; D013438:Sulfhydryl Compounds; D017869:Cyclic GMP-Dependent Protein Kinases