A comparison of the sensitivity of immunoperoxidase staining methods with high-sensitivity fluorescence flow...

Surface molecules present in low copy numbers can be detected with high-sensitivity fluorescence flow cytometry. Many cells previously thought not to express certain molecules on their surface can now be shown to have these molecules in very low copy numbers by high-sensitivity fluorescent cytometric methods. Detection of molecules by immunoperoxidase staining methods has not previously been compared with high-sensitivity flow cytometry techniques. Computerized video image analysis (VIA) is a method that allows measurement of area and density of the immunostain chromogen reaction product in a standardized fashion analogous to flow cytometry. In this study, we compared immunoperoxidase reaction products measured by VIA methods with high-sensitivity flow cytometric measurements for cells with 10,000 down to 50 antibody molecules bound to their surfaces. Detection of 100-200 surface molecules was possible with heavy metal-enhanced immunoperoxidase methods, whereas standard immunoperoxidase methods were not as sensitive. The sensitivity of the nickel-enhanced immunoperoxidase staining method was confirmed for detection of an epitope (Tac-IL2 receptor alpha-chain) present in low numbers on the surface of peripheral blood lymphocytes.