Stepwise silver-staining-based immunosorbent assay for amyloid-β autoantibody detection

Aims: To detect amyloid-beta (A beta) autoantibodies in a reliable and high-throughput fashion, we developed a stepwise silver-staining-based immunosorbent assay in a 96-well-plate platform. Materials & methods: A beta autoantibodies were incubated in an A beta-immobilized 96-well microplate. Antihuman IgG-modified gold nanoparticle probes were then used to bind to the autoantibodies and signal enhancement was carried out with stepwise silver-staining on immobilized gold nanoparticle probes. A microplate reader was used to quantify silver-stained gold nanoparticles on a well-plate surface. Results & discussion: Stepwise silver-staining at low temperature enables long-term silver-staining with minimal increase of background signal. This stepwise staining method helps solve the problems of one-step staining, such as nonspecific binding or nonuniformity in silver precipitation after prolonged silver-staining for signal enhancement. Conclusion: A stepwise silver-staining strategy could be useful in minimizing nonspecific background signals. This 96-well-plate-based A beta antibody detection assay could be useful in studying and diagnosing Alzheimer's disease.