Signal amplification in flow cytometry using biotin tyramine

Background: Catalysed reporter deposition (CARD) has been successfully used as a means of signal amplification in solid-phase immunoassays. The proced ure relies on the use of horseradish peroxidase (HRP)-conjugated re-agents- normally antibodies-in conjunction with substituted phenolic compounds such as biotin tyramine. The HRP catalyses deposition of biotin tyramine around the site of enzyme activity, and streptavidin-HRP can then be added to gen erate an amplified HRP signal. The possibility of using this technique for solution-phase amplifications has been suggested but not yet demonstrated. Methods: This paper describes the application of CARD to signal enhancement in flow cytometry. The specific examples described here are those of anti- human CD4 and anti-human CD36 antibodies binding to either human lymphocyte s or mixed mononuclear cells. Results: Optimum biotin tyramine concentrations were evaluated, and a fivef old increase in signal was observed over standard detection of the anti-hum an CD4 antibody with anti-mouse-fluorescein isothiocyanate (FITC). In the e xample using the anti-CD36 antibody, the biotin tyramine treatment was repe ated, resulting in an additional 2.5-fold signal amplification. Conclusions: The technique described in this report provides a method of am plifying the signals achieved by standard flow cytometry detection reagents . (C) 1999 Wiley-Liss, Inc.