Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8418489 | Journal of Immunological Methods | 2013 | 8 Pages |
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.
Keywords
MBLPBS containing 0.05% Tween-20Ficolin-3Pattern recognition moleculeacetylated bovine serum albuminMASP-2PRMTCCTMBPBS-TTBS-TPBSTBSSPS3,3′,5,5′-tetramethylbenzidineopdEDTAEthylenediaminetetraacetic acidortho-phenylenediamineterminal complement complexTris-buffered salineELISAEnzyme-linked immunosorbent assayComplement systemPhosphate buffered salineMannose-binding lectinlectin pathwayArbitrary Unit
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Authors
Dorottya Csuka, Lea Munthe-Fog, Mikkel-Ole Skjoedt, Estrid Hein, Jakob T. Bay, Lilian Varga, George Füst, Peter Garred,