Toggle Main Menu Toggle Search

Open Access padlockePrints

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

Lookup NU author(s): Professor Claire Harris



This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND).


© 2018 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.

Publication metadata

Author(s): Zelek WM, Harris CL, Morgan BP

Publication type: Article

Publication status: Published

Journal: Immunobiology

Year: 2018

Volume: 223

Issue: 12

Pages: 744-749

Print publication date: 01/12/2018

Online publication date: 20/07/2018

Acceptance date: 13/07/2018

Date deposited: 19/02/2019

ISSN (print): 0171-2985

ISSN (electronic): 1878-3279

Publisher: Elsevier GmbH


DOI: 10.1016/j.imbio.2018.07.016


Altmetrics provided by Altmetric


Find at Newcastle University icon    Link to this publication