Bhattacharya R, Bhattacharya D, Dhar T K
Indian Institute of Chemical Biology, Jadavpur, Calcutta.
J Immunol Methods. 1999 Jul 30;227(1-2):31-9. doi: 10.1016/s0022-1759(99)00067-8.
A novel strategy to improve significantly antigen detection sensitivity of Dot-ELISA by catalyzed reporter deposition (CARD) method of signal amplification has been developed. The method, termed Super-CARD, utilizes synthesized electron rich proteins having multiple binding sites as blocking agents. After completion of conventional Dot-ELISA, the solid phase bound horseradish peroxidase (HRP) oxidises the added labeled substrate, which deposits onto the solid phase. This deposition is markedly increased in the presence of immobilized electron rich proteins, which not only amplifies the signal but also increases the sensitivity. The high specificity of the amplification reaction avoids the generation of any false positive signal. The extremely high sensitivity of Super-CARD technology permits visual detection of as few as 800 rabbit IgG molecules (1.33 x 10(-21) mol). The method is approximately 10(5)-fold more sensitive than conventional Dot-ELISA. Direct comparison with existing CARD methods demonstrates approximately 1.6 x 10(4)-fold enhancement in detection sensitivity which is much higher than that of any other existing methods. The Super-CARD technology is specific, flexible and may be applied to clinical diagnostics.
已开发出一种新策略，通过催化报告分子沉积（CARD）信号放大方法显著提高斑点酶联免疫吸附测定（Dot-ELISA）的抗原检测灵敏度。该方法称为超级CARD，利用具有多个结合位点的合成富电子蛋白作为封闭剂。在完成常规Dot-ELISA后，固相结合的辣根过氧化物酶（HRP）氧化添加的标记底物，使其沉积在固相上。在固定化富电子蛋白存在的情况下，这种沉积会显著增加，这不仅放大了信号，还提高了灵敏度。扩增反应的高特异性避免了任何假阳性信号的产生。超级CARD技术的极高灵敏度允许肉眼检测低至800个兔免疫球蛋白G（IgG）分子（1.33×10⁻²¹摩尔）。该方法的灵敏度比传统Dot-ELISA高约10⁵倍。与现有CARD方法的直接比较表明，检测灵敏度提高了约1.6×10⁴倍，远高于任何其他现有方法。超级CARD技术具有特异性、灵活性，可应用于临床诊断。
