Biochemical analysis
Plasma total cholesterol, HDL and triglyceride levels were measured
using an Aeroset automated analyser (Abbott Diagnostics, Maidenhead,
UK). Insulin levels were assessed using an immunometric assay specific
for human insulin (Invitron, Monmouth, UK), and glucose was measured
using the Aeroset chemistry system (Abbott Diagnostics). Total
testosterone was measured by liquid chromatography-tandem mass
spectrometry (Quattro™ Premier XE triple quadrupole
tandem mass spectrometer; Waters Ltd, Watford, UK). Plasma C3 and C4
levels were quantified by nephelometry on a Beckman BN11 nephelometer in
the University Hospital of Wales Clinical Immunology laboratory using
commercial standards. The assay working range for C3 was 0.02–4.1 g/l,
and for C4 was 0.01–1.9 g/l. C5a(desArg), C3a(desArg), factor D and
properdin were quantified using their respective commercial assays from
Hycult Biotech, as instructed by the manufacturer. Plasma C5, TCC and
factor H were all measured using in-house ELISA24-26.
All assays used purified protein (either C5, TCC or factor H) as a
standard. For C5 ELISA, plates were coated with an in-house polyclonal
rabbit anti-human C5 antibody (8 μg/ml, 100 µl/well, in bicarbonate
buffer (pH 9.6)), blocked in 2% (w/v) bovine serum albumin (BSA;
blocking buffer), and then incubated with plasma samples diluted 1 in
600 in blocking buffer. Mouse monoclonal anti-human C5 (MBI-C5-3; 5
μg/ml) was used to detect bound C5, followed by donkey anti-mouse IgG
horseradish peroxidase (HRP; 1 in 2500).
For TCC quantification, plates were coated with an in-house anti-C9
neo-antibody (B7), used at 4µg/ml, 100 µl/well. After blocking with 2%
(w/v) BSA, plasma samples were diluted 1:6 in blocking buffer.
HRP-conjugated monoclonal mouse anti-human C8 (clone E2, in-house) was
added to wells for detection of TCC (100 µl; 2µg/ml). For total factor H
ELISA, plates were coated with affinity-purified rabbit anti-factor H
IgG diluted in bicarbonate coating buffer (pH 9.6) at 5 μg/well. After
blocking with 1% (w/v) BSA, plasma samples were diluted 1:6000 in
blocking buffer. HRP-labelled affinity-purified rabbit anti-human factor
H (100 µl; 1 mg/l) was used to detect total factor H. Of note, many
papers quote higher levels of serum factor H; the extinction coefficient
for factor H standards on which our normal range is based has been
validated previously26.