Enhanced chemiluminescence ELISA for the detection of antibody to hepatitis B virus surface antigen

Abstract

A chemiluminescent enzyme linked immunosorbent assay (ELISA) for the detection of antibody to hepatitis B virus surface antigen (anti-HBs) in human serum has been developed. Polystyrene microtitre plates were coated with recombinant, yeast-derived hepatitis B surface antigen (rec-HBsAg). Patient serum samples and appropriate controls were added to the rec-HBsAg-coated wells and incubated to bind anti-HBs. The wells were then washed and a fluorescein isothiocyanate (FITC) conjugate of a human plasma-derived hepatitis B surface antigen (HBsAg) was added. Following incubation and further washing, the bound FITC-labelled HBsAg was detected after addition of a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody and assaying for the enzyme. The activity of the HRP was measured using luminol and hydrogen peroxide as substrates and iodophenol as a chemiluminescence enhancer. The luminescence was recorded using a camera luminometer. Preliminary tests have shown the assay to be suitable for the detection of antibody in sera from both vaccinees and also from individuals with a past hepatitis B virus infection. The use of the FITC-anti-FITC system together with the measurement of a chemiluminescence signal makes possible the completion of this assay in a few hours. The assay has been shown to be both specific and sensitive and provides a permanent photographic record.

An Enzyme Immunoanalytical System Based on Sequential Cross-Flow Chromatography

“…As exemplified in ELISA, enzymes are an alternative tracer that has been used over the past two decades . The tracer produces an amplified signal resulting from the catalytic action and also offers different types of signals that are measurable by comparatively simple detectors (e.g., based on photometry, , chemiluminometry, and electrochemistry), depending on the substrate as well as the enzyme used. The enzyme reaction, as a unique property of this tracer, should be carried out separately for signal generation following the completion of the antigen−antibody bindings.…”