Enhanced chemiluminescence: a high-sensitivity detection system for in situ hybridization and immunohistochemistry

The breakthrough of chemiluminescence in the field of solution immunoassays and transfer membranes prompted us to explore whether a light-based detection system could provide a gain in sensitivity over chromogenic and FITC markers for nucleic acid and protein detection on histological preparations. A Hamamatsu device and an enhanced chemiluminescence (ECL) luminol substrate of the peroxidase were used to detect epithelial and endothelial components by immunohistochemistry (IHC) and for in situ hybridization (ISH) of papilloma virus DNA. The accuracy of the signal was compared to that obtained with DAB-peroxidase, silver-enhanced DAB-peroxidase, NBT-BCIP-alkaline phosphatase, and FITC. Our results demonstrated the feasibility and high sensitivity of luminescence detection for histological preparations. In part due to the ultrasensitive videocamera and photon-counting imaging, interpretable and reproducible results were obtained within counting times shorter than 5 min, and with dilutions of the primary antibodies 100- to 10,000-fold greater than those used for chromogenic and FITC reactions. As for ISH, with identical concentrations of the HPV 18 DNA probe on HeLa cells, labeling was apparent by luminescence but undetectable with the chromogen. The morphological resolution allowed a discriminatory analysis of the signal. Therefore, at the light microscopic level, enhanced chemiluminescence offers an appealing alternative to FITC and chromogenic markers for detection and quantification of low-concentration molecules.