title: A novel assay to quantitate MASP-2/ficolin-3 complexes in serum.
abstract: Ficolin-1, -2 and -3 are recognition molecules in the lectin complement pathway and form complexes with serine proteases named MASP-1, -2 and -3 and two nonenzymatic proteins. MASP-2 is the main initiator of lectin pathway activation, while ficolin-3 is the most abundant ficolin molecule in the circulation. The significance of lectin pathway complexes in the circulation is unknown. Thus, we established an assay for the measurement of circulating MASP-2/ficolin-3 complexes. A quantitative sandwich ELISA was developed for the measurement of the MASP-2/ficolin-3 complexes in serum based on monoclonal antibodies against MASP-2 for coating and anti-ficolin-3 for detection. In addition, we assessed the serum concentrations of ficolin-3 and MASP-2 and the extent of ficolin-3 mediated C4 deposition on acetylated BSA in samples from 97 healthy donors. The median concentration of MASP-2/ficolin-3 complexes was found to be 119.7 AU/ml (range: 2.9-615.5 AU/ml). Significant correlations were found between the level of MASP-2/ficolin-3 complexes and the concentration of ficolin-3 (Spearman r=0.2532, p=0.0124), and MASP-2 (Spearman r=0.4505, p<0.0001), as well as the degree of C4 deposition (Spearman r=0.671, p<0.0001). When ficolin-3 deficient (homozygous for the rs28357092 polymorphism) and MASP-2 deficient (homozygous for the rs72550870 polymorphism) sera were incubated together, complex formation was induced between MASP-2 and ficolin-3. The complex formation disappeared in the presence of EDTA. An assay allowing quantitative measurement exclusively of MASP-2/ficolin-3 complexes in serum is described. This method may add further insight into the pathophysiology of disorders associated with the deficiency or abnormal activities of MASP-2 and ficolin-3.
authors.affiliation: Laboratory of Molecular Medicine, Department of Clinical Immunology, Section 7631, Rigshospitalet, Faculty of Health Sciences, University Hospital of Copenhagen, Denmark.
authors.forename: Dorottya
authors.initials: D
authors.lastname: Csuka
authors.forename: Lea
authors.initials: L
authors.lastname: Munthe-Fog
authors.forename: Mikkel-Ole
authors.initials: MO
authors.lastname: Skjoedt
authors.forename: Estrid
authors.initials: E
authors.lastname: Hein
authors.forename: Jakob T
authors.initials: JT
authors.lastname: Bay
authors.forename: Lilian
authors.initials: L
authors.lastname: Varga
authors.forename: George
authors.initials: G
authors.lastname: Füst
authors.forename: Peter
authors.initials: P
authors.lastname: Garred
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D003181:Complement C4 / Q000276:immunology / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D046912:Multiprotein Complexes / Q000097:blood / Q000276:immunology* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D012016:Reference Values; D044967:Serum / Q000187:drug effects / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D003181:Complement C4; C109662:FCN3 protein, human; D006023:Glycoproteins; D037102:Lectins; D046912:Multiprotein Complexes; D004492:Edetic Acid; C105296:MASP2 protein, human; D050606:Mannose-Binding Protein-Associated Serine Proteases