KINETIC MEASUREMENT OF THE MEMBRANOLYTIC ACTIVITY OF SERUM COMPLEMENTUSING BIOLUMINESCENT BACTERIA

A method for studying the kinetics of serum complement activity is pre sented. The assay utilises Escherichia coli and Bacillus subtilis cell s which have been made bioluminescent by expressing an insect lucifera se gene from Pyrophorus plagiophthalamus. The diffusion of the lucifer ase enzyme substrate through the cell membranes is very slow, and ther efore a change in membrane permeability is seen as a change of the in vivo luminescence of the cells. Treatment of the bacteria with human s erum resulted in a bell-shaped curve of in vivo luminescence since thi s procedure facilitates passage of the substrate to the cytoplasm. The time point of maximum light emission was dependent on serum dilution. In vivo luminescence also proved to be a good indicator of the viabil ity of bacterial cells. Using Clq deficient serum, or following treatm ent of normal serum with EGTA and Mg2+ it was possible to separate the membranolytic activities of alternative and classical pathways.