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Purification and biochemical characterization of functional complement factor H from human plasma fractions.

Vox sanguinis (2012-04-14)
H Brandstätter, P Schulz, I Polunic, C Kannicht, G Kohla, J Römisch
ABSTRACT

Complement factor H (CFH) acts as major regulator of the alternative pathway of complement and its mutations and polymorphisms predispose to various human diseases. We aimed to develop a scalable purification process of CFH from human plasma fractions to supply a pathogen-safe and functional CFH concentrate. Starting with intermediates of cold ethanol fractionation of plasma, CFH purification was performed with three chromatographic steps including solvent detergent treatment and nanofiltration. CFH functionality was tested by a haemolysis assay using sheep erythrocytes, by determining decay acceleration activity on C3 convertases and cofactor activity of C3b cleavage. CFH identity was confirmed by Western blot and mass spectrometry. Three scalable chromatographic steps highly purified full-length and native CFH from human plasma fractions. The purification process enabled the removal of truncated and dysfunctional CFH species, yielding a native CFH concentrate as demonstrated in sensitive functional in vitro assays. This novel process provides a pathogen-safe and functional CFH concentrate that can be produced on an industrial scale and is suitable for pre-/clinical studies.

MATERIALS
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Product Description

Sigma-Aldrich
Anti-Human IgG (γ-chain specific), F(ab′)2 fragment antibody produced in goat, affinity isolated antibody, lyophilized powder

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