Extracting the barbs from complement assays: Identification and optimisation of a safe substitute for traditional buffers

Complement assays have for many years utilised buffers based on barbitone (veronal) despite the well-recognised toxicity of this agent and the tight regulations on its use in most countries. The use of barbitone in complement assay buffers is steeped in history, from a time when no other suitable buffers were available. This is no longer the case, encouraging us to explore alternatives to barbitone for complement assays. We compared a simple, non-toxic HEPES buffer with commercially sourced complement fixation test diluent (CFD), the "gold standard" barbitone buffer, in several clinically relevant complement activity assays and across species. In classical pathway haemolysis assays in human and non-human serum, there was no difference in haemolytic curves or calculated haemolytic activity (CH50) between CFD and an optimised HEPES buffer (HBS) supplemented with cations. Alternative pathway haemolysis assays in human serum were also identical in the two buffers. In a complement fixation test for anti-erythrocyte antibodies, complement consumption was identical for the two buffer systems. The data demonstrate that barbitone-based buffers are unnecessary for assays of complement activity and can readily be replaced with safe and simple alternatives.