title: An ELISA assay with two monoclonal antibodies allows the estimation of free factor H and identifies patients with acquired deficiency of this complement regulator.
abstract: Complement factor H (FH) serum levels can be affected by the presence of immune complexes of FH with autoantibodies like in autoimmune forms of atypical haemolytic uraemic syndrome (aHUS) or with C3b in homozygous factor I (FI) deficiency. These complexes reduce the amount of free functional circulating FH. In this study we aimed to determine whether FH levels measurement is disturbed in some pathological conditions and to establish a method for quantifying free and total FH in serum. For that purpose, FH levels were measured in serum samples from aHUS patients having anti-FH autoantibodies or mutations in FH gene, in patients with homozygous FI deficiency, and in healthy controls. Two anti-FH monoclonal antibodies, OX24 and A229, recognizing different functional regions in FH, were used as capture antibodies in an ELISA assay. In the control group and in the group of patients with FH mutations, the FH levels obtained with the two monoclonal antibodies were similar. In patients with anti-FH autoantibodies or with homozygous FI deficiency, however, FH levels measured with both antibodies were significantly different. As these patients had complexes of FH with autoantibodies or C3b, we interpreted that OX24 was detecting total FH and A229 was recognising free FH. Therefore, quantification of FH in plasma using these two monoclonal antibodies provides not only total FH level but also gives an estimation of how much FH circulates free and is thus available to properly control complement activation.
authors.affiliation: Unidad de Inmunología, IdiPAZ, Hospital Universitario La Paz, Madrid 28046, Spain; Centro de Investigación Biomédica en Red (CIBERER U754), Madrid, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Nozal
authors.affiliation: Unidad de Inmunología, IdiPAZ, Hospital Universitario La Paz, Madrid 28046, Spain; Centro de Investigación Biomédica en Red (CIBERER U754), Madrid, Spain.
authors.forename: Sofía
authors.initials: S
authors.lastname: Garrido
authors.affiliation: Unidad de Inmunología, IdiPAZ, Hospital Universitario La Paz, Madrid 28046, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Alba-Domínguez
authors.affiliation: Servicio de Nefrología Pediátrica, Hospital Universitario La Paz, Madrid 28046, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Espinosa
authors.affiliation: Servicio de Nefrología Pediátrica, Hospital Universitario La Paz, Madrid 28046, Spain.
authors.forename: Antonia
authors.initials: A
authors.lastname: Peña
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CIB-CSIC), Centro de Investigación Médica en Red (CIBERER U738), Madrid 28040, Spain.
authors.forename: Santiago Rodríguez de
authors.initials: SR
authors.lastname: Córdoba
authors.affiliation: Unidad de Investigación, IdiPAZ, Hospital Universitario La Paz, Madrid 28046, Spain; Centro de Investigación Biomédica en Red (CIBERER U754), Madrid, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Sánchez-Corral
authors.affiliation: Unidad de Inmunología, IdiPAZ, Hospital Universitario La Paz, Madrid 28046, Spain; Centro de Investigación Biomédica en Red (CIBERER U754), Madrid, Spain. Electronic address: mltrascasa@salud.madrid.org.
authors.forename: Margarita
authors.initials: M
authors.lastname: López-Trascasa
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D065766:Atypical Hemolytic Uremic Syndrome; D016022:Case-Control Studies; D002648:Child; D003176:Complement C3 / Q000172:deficiency* / Q000235:genetics; D017242:Complement Factor H / Q000032:analysis* / Q000172:deficiency* / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D030342:Genetic Diseases, Inborn / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D006463:Hemolytic-Uremic Syndrome / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D000081208:Hereditary Complement Deficiency Diseases; D006801:Humans; D008297:Male; D009154:Mutation
chemical_list: D000911:Antibodies, Monoclonal; D003176:Complement C3; D017242:Complement Factor H
keywords: Anti-factor H autoantibodies; Atypical haemolytic uremic syndrome; CFHRs; Complement factor H; Complement factor I deficiency.; FH; FI; aHUS; atypical haemolytic uremic syndrome; factor H; factor H related proteins; factor I