Distribution and determinants of circulating complement factor H concentration determined by a high-throughput immunonephelometric assay

• Published in: Journal of immunological methods Vol. 390; no. 1-2; pp. 63 - 73
• Main Authors: Sofat, Reecha, Mangione, P. Patrizia, Gallimore, J. Ruth, Hakobyan, Svetlana, Hughes, Timothy R., Shah, Tina, Goodship, Tim, D'Aiuto, Francesco, Langenberg, Claudia, Wareham, Nick, Morgan, B. Paul, Pepys, Mark B., Hingorani, Aroon D.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 30.04.2013
• Subjects: Adult; binding sites; Blood Specimen Collection - methods; Body fat; C-Reactive Protein - analysis; Cholesterol - blood; Cholesterol, HDL - blood; complement; Complement factor H; Complement Factor H - analysis; Cryopreservation - methods; epidemiological studies; freeze-thaw cycles; Freeze-thawing; High throughput assay; human diseases; Humans; Immunoassay - methods; Immunoassays; Immunodiffusion; Immunodiffusion - methods; Immunonephelometric; Inflammation; Lipids; Middle Aged; Nephelometry and Turbidimetry - methods; Population based study; Population levels; Population studies; Protein Stability; Reproducibility of Results; Triglycerides - blood; volunteers; High throughput assay; fHL-1; fHR; fH; Complement factor H; Population based study; Immunonephelometric
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2013.01.009

Research on complement factor H (fH) in human disease is hampered by lack of an assay suitable for use in large-scale epidemiological studies. We describe the development and validation of a high throughput nephelometric assay for fH. Reagents from a commercial radial immunodiffusion (RID) assay (The Binding Site) were adapted for use on the Siemens BNII high throughput nephelometric instrument. The assay was calibrated with a highly purified human fH preparation with rigorously determined concentration, and assay performance was comprehensively evaluated using samples from healthy human volunteers, with the commercial RID assay as a comparator. The distribution and determinants of circulating fH concentration in humans were then investigated in a large representative population sample. The nephelometric assay had recovery close to 100%, was reproducible with intra- and inter-assay CV's of 11% and 5–15% respectively, and had a wider operating range than the RID assay. fH values were unaffected after multiple freeze-thaw cycles demonstrating that it is evidently a stable analyte for immunoassay. fH concentration was unaltered by an acute inflammatory stimulus. The population study showed that plasma fH concentration is associated with circulating lipids and indices of body fat. We present the first high throughput assay for circulating fH; the assay is accurate and reliable with reproducible measures from stored samples. It has established the distribution of fH values at a population level and demonstrated important associations with circulating lipids and indices of body fat, thus providing an important reference for future clinical and epidemiological investigations.