A newly developed immunoassay method based on optical measurement for Protein A detection.

We describe the development of an immunoassay using an antibody-silver nanoparticle (Ab-AgNP) conjugate as a catalyst for the silver enhancement reaction. The immuno-reaction signals that were magnified by silver metal precipitation were quantified using a commercial flatbed scanner. Protein A from Staphylococcus aureus (S. aureus), a common clinical pathogenic bacterium, was used in this research. The ease of infection of S. aureus necessitates the development of a fast detection method. The framework of the method described in this paper is based on the sandwich immunoassay and contains a 1st antibody (immunoglobulin G, IgG), an antigen (Protein A), and a 2nd antibody-colloidal silver conjugate (IgG-AgNPs). The silver enhancement reaction, a signal amplification method in which silver ions are reduced to metallic silver, is used to magnify the immuno-reaction signal. The change in signal, as visualized in grayscale, can be easily observed and analyzed by our optical scanning detection system. The relationship between antigen concentration and grayscale value is discussed. The detectable concentration limit for the antigen was found to be 1 ng/mL with 10 µg/mL of IgG and 300 µM of the IgG-AgNP conjugate. This immunoassay method provides the advantages of low cost, easy operation, and short detection time. Moreover, it has potential applications in clinical diagnoses.