Simultaneous immunoassay using electrochemical detection of metal ion labels

The concept of a simultaneous dual analyte immunoassay based on two different metal ion labels is demonstrated. The model system consists of two proteins, human serum albumin (HSA) and immunoglobulin G (IgG). Bismuth and indium ions have been coupled to these proteins through the bifunctional chelating agent diethylenetriamine-pentaacetic acid (DTPA). A maximum molar labeling ratio of 6:1 and 10:1 was obtained for HSA and IgG, respectively. Following a competitive equilibrium between unlabeled and labeled protein for a limited amount of specific antibody immobilized on polystyrene, the bound metal ion labels were released by acidification and detected by differential pulse anodic stripping voltammetry (ASV). Limits of detection for HSA and IgG are 1.8 and 0.6 microgram/mL, respectively. Application of the dual immunoassay to human serum samples gave results that were comparable to those obtained by nephelometry.