Nitrocellulose-enzyme-linked immunosorbent assay (NC-ELISA) — A sensitive technique for the rapid visual detection of both viral antigens and antibodies

Abstract

A modification of the enzyme-linked immunosorbent assay (ELISA) is described for sensitive and rapid direct visual detection of antigens of four adenoviruses (Ad) and anti-Ad antibodies using nitrocellulose (NC) membrane discs as high-capacity solid phase. The NC-ELISA was performed in microtitre plates containing NC-discs. Small amounts of crude supernatants from Ad-infected cells as antigen were bound to the discs. Additional binding capacity was blocked with an excess of bovine serum albumin. The subsequent reaction of virus antigen with specific rabbit antibody was visualized using alkaline phosphatase-conjugated anti-rabbit IgG and histochemical substrates. The sensitivity of the NC-ELISA for the detection of Ad-antigens was found to be 8–10-fold higher than a conventional ELISA using polystyrene solid phase supports. The sensitivity levels were estimated to be similar comparing NC-ELISA and tissue culture assay results. The quantitative determination of anti-Ad antibodies by NC-ELISA showed 8-fold higher sensitivity compared to microneutralization test. The NC-ELISA could detect purified immunoglobulin at a level of 1 ng using a direct test procedure and 10 ng using the indirect method. The detection limits are good compared to other highly sensitive assays. The use of crude antigen combined with high sensitivity and easy technical performance makes the NC-ELISA useful as a tool for rapid viral diagnosis.