Simultaneous detection of whole blood chemiluminescence in microtitre plates

The measurement of reactive oxygen species provides a simple method for monitoring the degree of activation of leukocytes in various disorders, and for determining the effects of drugs on this activation. The present report describes the determination of luminol- or lucigenin-amplified chemiluminescence of whole blood in a microtitre plate assay with a 96-well luminometer (HAMAMATSU MTP reader). Using heparinized venous human blood from healthy donors, optimal chemiluminescence intensities were determined at a blood dilution of 1/100 in a total volume of 0.25 ml of Hank's balanced salt solution, containing 0.4 mmol/l luminol as enhancer and either opsonized zymosan (1 milligram) or the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (10(-6) mol/l), as stimuli. The in vitro effects of nordihydroguaiaretic acid, diphenylene iodonium, and diclofenac were tested. After preincubation of the diluted whole blood with these drugs for 15 min, the zymosan-stimulated chemiluminescence was diminished in all cases. The specific NADPH oxidase inhibitor, diphenylene iodonium, was most effective (half maximal inhibition at 1.5 x 10(-8) mol/l), whereas higher concentrations of the antioxidant, nordihydroguaiaretic acid (1.6 x 10(-6) mol/l), or the nonsteroidal antiinflammatory drug, diclofenac (about 10(-5) mol/l), were needed to achieve half maximal inhibition. In addition to its usefulness in the rapid screening of drug effects this assay system seems to be very beneficial for the clinical diagnosis of congenital disorders. Furthermore, it is suited as an effective and simple method for the continuous determination of the phagocyte functional state in patients in pathophysiological situations and during therapy.