title: Functional analysis of Ficolin-3 mediated complement activation.
abstract: The recognition molecules of the lectin complement pathway are mannose-binding lectin and Ficolin -1, -2 and -3. Recently deficiency of Ficolin-3 was found to be associated with life threatening infections. Thus, we aimed to develop a functional method based on the ELISA platform for evaluating Ficolin-3 mediated complement activation that could be applicable for research and clinical use. Bovine serum albumin (BSA) was acetylated (acBSA) and chosen as a solid phase ligand for Ficolins in microtiter wells. Binding of Ficolins on acBSA was evaluated, as was functional complement activation assessed by C4, C3 and terminal complement complex (TCC) deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition on acBSA were dependent only on Ficolin-3 in appropriate serum dilutions. Deposition of down stream complement components correlated highly significantly with the serum concentration of Ficolin-3 but not with Ficolin-2 in healthy donors. To make the assay robust for clinical use a chemical compound was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides the possibility to diagnose functional and genetic defects of Ficolin-3 and down stream components in the lectin complement pathway.
authors.affiliation: Laboratory of Molecular Medicine, Department of Clinical Immunology, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Estrid
authors.initials: E
authors.lastname: Hein
authors.forename: Christian
authors.initials: C
authors.lastname: Honoré
authors.forename: Mikkel-Ole
authors.initials: MO
authors.lastname: Skjoedt
authors.forename: Lea
authors.initials: L
authors.lastname: Munthe-Fog
authors.forename: Tina
authors.initials: T
authors.lastname: Hummelshøj
authors.forename: Peter
authors.initials: P
authors.lastname: Garred
mesh_terms: D000107:Acetylation; D000818:Animals; D002417:Cattle; D003167:Complement Activation; D003176:Complement C3 / Q000378:metabolism; D003181:Complement C4 / Q000378:metabolism; D015938:Complement Membrane Attack Complex / Q000378:metabolism; D006023:Glycoproteins / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D037102:Lectins / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D037601:Mannose-Binding Lectin / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D000097604:Ficolins
chemical_list: D003176:Complement C3; D003181:Complement C4; D015938:Complement Membrane Attack Complex; D006023:Glycoproteins; D037102:Lectins; D037601:Mannose-Binding Lectin; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; D000097604:Ficolins; C109662:FCN3 protein, human