Determination of alternate pathway complement kinetics by electron spin resonance spectroscopy

We describe a technic that measures the kinetics of the metabolic burst of peripheral blood neutrophils (PMN) by electron spin resonance (ESR) spin trapping. Using this technic, a functional assay for the kinetics of the alternate pathway complement cascade is developed. PMN were stimulated by phagocytosis of opsonized zymosan (OpZym) to undergo the metabolic burst and the resulting free hydroxyl radicals generated during the burst were trapped using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) to form a stable spin adduct. Rapid sequential measurements of the spin adduct concentration were made using a computer-controlled ESR spectrometer to follow the time course of the metabolic burst. Differences in the results obtained with OpZym and with PMN incubated with unopsonized zymosan (Zym) and serum yields information about the kinetics of opsonization. The kinetics of the alternate complement pathway opsonization in sera from normal and C8-deficient patients were compared and found not to be affected significantly by the absence of C8 complement protein.