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.