A novel signal amplification technology based on catalyzed reporter deposition and its application in a Dot-ELISA...

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.