A chemiluminescent metalloimmunoassay based on silver deposition on colloidal gold labels

Abstract

A sensitive chemiluminescent (CL) immunoassay of human immunoglobulin (IgG) which combined the inherent high sensitivity of CL analysis with the dramatic signal amplification of silver precipitation on colloidal gold tags was developed. First, the sandwich-type complex was formed in this protocol by the primary antibody immobilized on the polystyrene wells, the analyte in the sample, and the secondary antibody labeled with colloidal gold. Second, the colloidal gold was treated by an Ag + reduction solution, which resulted in the catalytic precipitation of silver on the surface of colloidal gold. Third, a large number of Ag + were oxidatively released in HNO 3 solution from the silver metal anchored on the sandwich-type complexes and then the human IgG was indirectly determined by a sensitive combined CL reaction of Ag +–K 2S 2O 8–Mn 2+– H 3PO 4–luminol. The chemiluminescence intensity depends linearly on the logarithm of the concentration of human IgG over the range of 0.02–50 ng ml −1 and detection limit (3 σ) is 0.005 ng ml −1 (i.e., ∼3 × 10 −14 M, 3 amol in 100-μl sample). This assay has been successfully applied to the determination of human IgG in human serum samples and showed great potential for numerous applications in immunoassay.