Rapid isolation of pure Complement Factor H from serum for functional studies by the use of a monoclonal antibody that discriminates FH from all the other isoforms

•A monoclonal antibody (5H5) specific for Complement Factor H but unreactive with FHL-1 and FHR-proteins is described.•5H5 recognises both the uncleaved and the cleaved form of FH, useful for quality control.•A fast and specific purification method for Complement Factor H is proposed.•Two functional assays to be used in conjunction with the purification method are proposed.•A specific quantitative assay using mAb 5H5 was developed.

Several mutations have been identified in the gene coding for Complement Factor H (FH) from patients with atypical Hemolytic Uraemic Syndrome (aHUS), Age-related Macular Degeneration (AMD) and Membranoproliferative Glomerulonephritis (MPGN). These data allow for a precise description of the structural changes affecting FH, but a simple test for specifically assessing FH function routinely is not yet of common use. We have produced and characterised a monoclonal antibody (5H5) which discriminates between FH and the smaller FH-like 1 and FH-related proteins and show here that it specifically binds to FH without detecting the smaller isoforms. We therefore used this mAb for a quick, one-step micro-purification of FH directly from control sera and showed that this affinity chromatography procedure is not disruptive of its cofactor function. We also developed a modified sheep erythrocytes haemolysis test using our antibody and affinity-purified FH. These tests can be used in conjunction for assessing the function of FH purified from patients affected by FH-related diseases. Moreover we used this mAb to develop a FH-specific ELISA test.