Comparison of techniques and immunoreagents used for indirect immunofluorescence and immunoperoxidase identification...

Peroxidase-conjugated antibodies were found to be as sensitive as those conjugated to fluorescein-isothiocyanate (FITC) for identification of selected enterovirus types by the indirect technique. Peroxidase conjugates, however, were found to give fewer nonspecific reactions. The reasons for the higher specificity of the immunoperoxidase technique appear to be related to the relative size, charge, and uniformity of the preparations. Monomers of peroxidase-conjugated globulin are larger than those of FITC conjugates, but the latter readily form aggregates. This was shown by chromatography on Sepharose 6B columns: various FITC-conjugated globulins eluted before those conjugated to peroxidase and before (125)I-labeled immunoglobulin G. The net charge of the conjugates was determined by adsorption to ion-exchange columns. FITC-labeled globulins had a negative net charge, eluting at pH 5.1 from diethylamino-ethyl-Sephadex A-50 columns. Peroxidase conjugates were not retained by either cationic or anionic exchangers at pH values ranging from 4.0 to 10.5. Further, the fluorescein/protein ratios of FITC conjugates from different commercial sources and of those prepared in the laboratory were found to be variable; higher fluorescein/protein ratios (>2:1) give a higher degree of nonspecific reactions, whereas the peroxidase/protein ratio does not appear to affect specificity. These characteristics of peroxidase conjugates make the immunoperoxidase technique easier to standardize and more reliable for enterovirus identification than the immunofluorescence technique.