Signal amplification in flow cytometry using biotin tyramine

✍ Scribed by John C. Earnshaw; Jane K. Osbourn

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 68 KB
• Volume: 35
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(19990201)35:2<176::aid-cyto10>3.0.co;2-s

✦ Synopsis

Background: Catalysed reporter deposition (CARD) has been successfully used as a means of signal amplification in solid-phase immunoassays. The procedure relies on the use of horseradish peroxidase (HRP)-conjugated reagents-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 generate 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 lymphocytes or mixed mononuclear cells.

Results: Optimum biotin tyramine concentrations were evaluated, and a fivefold increase in signal was observed over standard detection of the anti-human CD4 antibody with anti-mouse-fluorescein isothiocyanate (FITC). In the example using the anti-CD36 antibody, the biotin tyramine treatment was repeated, resulting in an additional 2.5-fold signal amplification.

Conclusions:

The technique described in this report provides a method of amplifying the signals achieved by standard flow cytometry detection reagents.