title: 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.
abstract: 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.
authors.affiliation: "L. Sacco" Department of Biomedical and Clinical Sciences (DIBIC), University of Milan, Italy. Electronic address: silvia.berra@unimi.it.
authors.forename: Silvia
authors.initials: S
authors.lastname: Berra
authors.affiliation: "L. Sacco" Department of Biomedical and Clinical Sciences (DIBIC), University of Milan, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Clivio
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D002645:Chickens; D017242:Complement Factor H / Q000276:immunology / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006461:Hemolysis; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D020033:Protein Isoforms / Q000276:immunology / Q000302:isolation & purification; D012756:Sheep
chemical_list: D000911:Antibodies, Monoclonal; D020033:Protein Isoforms; D017242:Complement Factor H
keywords: Affinity chromatography; Complement Factor H; Functional assays; Immunoassays; Purification