An improved method for the detection of peroxidase-conjugated antibodies on immunoblots.

A sensitive method for the detection of antigen-antibody complexes on immunoblots is described which employs a modified substrate for peroxidase-conjugates. Dengue virus proteins were chosen for study and were separated by SDS-PAGE followed by electrophoretic transfer onto sheets of nitrocellulose. Virus-specific antigens bound to the nitrocellulose paper were then probed with both mouse monoclonal antibodies and human convalescent sera. Antigen-antibody complexes were detected with anti-species specific IgG-peroxidase conjugates followed by incubation in five different enzyme substrates. By far the most sensitive substrate was found to be a simple mixture of 4-chloronaphthol and diaminobenzidine (CND). A dot-immunobinding assay employing a purified viral protein and a specific monoclonal antibody was able to detect as little as 0.1 ng of antigen using this mixture. The increased sensitivity obtained involves no additional 'enhancement' steps in the procedure, the substrates are inexpensive and the product of the enzyme reaction is black, making the immunoblots ideal for photographic reproduction. Optimization of additional parameters involved in the immunoblot procedure is also described.