A NONHEMOLYTIC ASSAY FOR THE ACTIVATION OF THE ALTERNATIVE PATHWAY OF BOVINE COMPLEMENT

An assay for assessing activation of the bovine alternative pathway of complement was developed. The assay focused on events on the surface of yeast. Yeast cells were incubated with EGTA-Mg2+ plasma, washed and the yeast-bound complement proteins eluted by 100 mM methylamine. Detection of eluted proteins was achieved by Western blot and ELISA. An ELISA for the quantification of the Bb fragment of factor B was chosen to measure activation of the alternative pathway of complement. Using this system, it was possible to demonstrate the kinetics of deposition of Bb on yeast incubated with plasma samples from individual cattle and to show differences between cattle. We were able to categorize cattle into 'fast or slow amplifiers' of the alternative pathway of complement. We suggest that this classification has implications for host protection against invading microorganisms.