Earnshaw J C, Osbourn J K
Cambridge Antibody Technology, Melbourn, Cambridgeshire, United Kingdom.
Cytometry. 1999 Feb 1;35(2):176-9. doi: 10.1002/(sici)1097-0320(19990201)35:2<176::aid-cyto10>3.0.co;2-s.
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.
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.
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.
The technique described in this report provides a method of amplifying the signals achieved by standard flow cytometry detection reagents.
催化报告沉积（CARD）已成功用作固相免疫测定中的信号放大手段。该过程依赖于使用辣根过氧化物酶（HRP）偶联试剂——通常是抗体——与生物素酪胺等取代酚类化合物结合使用。HRP催化生物素酪胺在酶活性位点周围沉积，然后可以添加链霉亲和素-HRP以产生放大的HRP信号。有人提出了将该技术用于溶液相放大的可能性，但尚未得到证实。
本文描述了CARD在流式细胞术中用于信号增强的应用。此处描述的具体示例是抗人CD4和抗人CD36抗体与人类淋巴细胞或混合单核细胞结合的示例。
评估了生物素酪胺的最佳浓度，与用抗小鼠异硫氰酸荧光素（FITC）对抗人CD4抗体进行标准检测相比，观察到信号增加了五倍。在使用抗CD36抗体的示例中，重复进行生物素酪胺处理，导致信号额外放大2.5倍。
本报告中描述的技术提供了一种放大标准流式细胞术检测试剂所获得信号的方法。
