Adaptation of the Ngo-Lenhoff peroxidase assay for solid phase ELISA

A modification of the Ngo-Lenhoff peroxidase assay has been developed. By this method, peroxidase is detectable at 5 fmoles of peroxidase per ml. The assay is easy to perform and the reagents are inexpensive. We have modified the buffer by the addition of citric acid and sodium azide to reduce background color development and to inhibit the activity of catalase, respectively. Production of the indamine dye by the peroxidase reaction is rapid but it may be stopped by lowering the pH to 3.0. The pH change of 7.0 to 3.0 is accompanied by a color change from purple-blue to blue and a shift in the maximum absorbance from 590 nm to 595 nm. The sensitivity of the assay was unaffected by these modifications.