A novel sandwich immunosensing method for measuring cardiac troponin I in sera.

Common methods for monitoring human cardiac troponin I (cTn I) are based on using antibodies against cTn I labeled with horseradish peroxidase, radioactive isotopes, or other labels. In this study, a novel label-free sandwich immunosensing method for measuring cTn I was developed. Three monoclonal antibodies (mAbs 9F5, 2F11, and 8C12) against human cTn I were generated by the commonly used hybridoma technique and characterized by a surface plasmon resonance (SPR) biosensor. An optimal pair of mAbs for measuring human cTn I was selected, as both mAbs have high affinities for cTn I and do not compete against each other for cTn I binding. An optical immunosensor for measuring cTn I in sera based on SPR was developed by using avidin as an intermediate layer and biotinylated-2F11 as the capturing antibody. Two detection methods for cTn I with the immunosensor were performed: (1) the direct detection of cTn I with a detection range of 2.5 to 40 microg/L and (2) the sandwich immunosensing method. In the sandwich assay mode, the second antibody 9F5 biologically amplified the sensor response. As a result, the sandwich assay showed a sensitivity of 0.25 microg/L and a detection range of 0.5 to 20 microg/L with within-run variation of 4.9 to 6.7% and between-run variation of 5.2 to 8.4%. This method has greatly enhanced the sensitivity for detection compared to that previously reported in the literatures.