Bobrow M N, Shaughnessy K J, Litt G J
Medical Products Department, E.I. Du Pont De Nemours & Co., North Billerica, MA 01862.
J Immunol Methods. 1991 Mar 1;137(1):103-12. doi: 10.1016/0022-1759(91)90399-z.
In a previous publication (Bobrow et al., J. Immunol. Methods (1989) 279-285), we described a novel signal amplification method, catalyzed reporter deposition (CARD), and its application to microplate immunoassays. The method utilizes the analyte-dependent reporter enzyme (ADRE) to catalyze the deposition of additional reporter onto the surface of a solid-phase immunoassay system. In this paper, we describe the utilization of CARD amplification for nonradiometric membrane assays where detection is facilitated by the formation of an insoluble chromogenic product. In the examples described, deposition of reporter is accomplished in two steps: (i) a horseradish peroxidase (HRP) ADRE catalyzes the deposition of either a biotin or fluorescein labeled phenol, and (ii) incubation with either enzyme labeled streptavidin or anti-fluorescein, respectively, results in the deposition of additional enzyme. Using this method, we have improved detection limits from 8- to greater than 200-fold depending on the amplification format and the chromogen used.
在之前的一篇出版物中（Bobrow等人，《免疫学方法杂志》（1989年）第279 - 285页），我们描述了一种新型信号放大方法——催化报告分子沉积（CARD）及其在微孔板免疫测定中的应用。该方法利用分析物依赖性报告酶（ADRE）催化额外的报告分子沉积到固相免疫测定系统的表面。在本文中，我们描述了CARD扩增在非放射性膜测定中的应用，其中通过形成不溶性显色产物来促进检测。在所描述的示例中，报告分子的沉积分两步完成：（i）辣根过氧化物酶（HRP）ADRE催化生物素或荧光素标记的苯酚的沉积，以及（ii）分别与酶标记的链霉亲和素或抗荧光素孵育，导致额外酶的沉积。使用这种方法，根据扩增形式和所使用的显色剂，我们将检测限提高了8倍至200倍以上。
