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 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.
Earnshaw
J C
JC
Cambridge Antibody Technology, Melbourn, Cambridgeshire, United Kingdom.
Osbourn
J K
JK
D001710:Biotin / Q000032:analysis*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D014439:Tyramine / Q000032:analysis*
C400422:biotin tyramine; D001710:Biotin; D016650:Fluorescein-5-isothiocyanate; D014439:Tyramine