Microplate wells can be coated with silver ions using glutaraldehyde as a spacer molecule and thiourea as a complexing ligand. Microwells containing surface silver ions are shown to immobilize biotin-labeled horseradish peroxidase (HRP) in active form, while showing very little affinity for the unlabeled enzyme. These plates can also immobilize biotin-labeled antibodies that exhibit bioactivity after immobilization. Silver ions are needed for the complexation of the biotinylated enzyme or antibody because microwells modified to contain surface amine or thiourea molecules do not immobilize appreciable amounts of the labeled proteins. A maximum surface coverage for biotin-labeled HRP of 40 ng/cm2 and an immobilization binding constant of Km = 8 × 109/M are determined from serial dilutions in a microplate. Detection of as little as 6.7 fmol HRP is achieved using antibodies immobilized on the silver ion-modified microplates. Active antibody surface densities were estimated to be between 130 and 260 nm2/antibody molecule. Background binding of HRP to the modified silver ion microplates was very low, allowing for reasonably accurate detection between 10-14 and 10-11 mol HRP.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)