| 1660059 |
White SR, Kulp GV, Spaethe SM, Van Alstyne E, Leff AR: A kinetic assay for eosinophil peroxidase activity in eosinophils and eosinophil conditioned media. J Immunol Methods. 1991 Nov 22;144(2):257-63.
The activity of eosinophil peroxidase (EPO) is commonly employed as a measure of eosinophil activation in biologic fluids. Determination of product formation by this enzyme by end-point measurement may be affected profoundly by substrate concentrations, reaction time and degradation of end-product and enzyme. To determine more accurately EPO concentrations in media conditioned by isolated, purified eosinophils, we have developed a kinetic, colorimetric assay to measure EPO concentration as a function of maximum velocity of reaction (Vmax). An automated method for determining Vmax in a 96-well microplate colorimetric assay was utilized over a wide range of substrate concentrations. Concentrations greater than or equal to 3 x 10 (-8) g/ml could be determined reliably with this assay. Peroxidase activity was inhibited in a concentration-dependent manner by the addition of 3-amino-1,2,4-triazole (AMT). The EPO concentration in eosinophils determined by this kinetic method was approximately 1.1 x 10 (-5) g/10 (6) eosinophils. Eosinophil activation with 10 (-6) M f-Met-Leu-Phe (fMLP) caused substantial EPO secretion (9.0 +/- 1.7% vs. 2.9 +/- 0.6% total EPO content for control, P = 0.05) and decrease in eosinophil EPO concentration (92.3 +/- 4.2% of control, P = 0.038). Secretion was enhanced by the addition of 5 micrograms/ml cytochalasin B to 10 (-6) M fMLP (25.9 +/- 12.7% total EPO content, P = 0.043 vs. control); similar decreases were noted in eosinophil EPO concentration (71.7 +/- 16.1% of control, P = 0.043). These data demonstrate that determination of EPO secretion by measurement of Vmax is a reliable, accurate method for precise quantification of this enzyme in media containing purified eosinophils or eosinophil products in the absence of other forms of peroxidase activity. |
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