Ficolins are a group of proteins containing both a collagen-like domain and a fibrinogen-like domain. Three forms of Ficolin have been identified in humans: Ficolin-1 (M-ficolin), Ficolin-2 (L-ficolin) and Ficolin-3 (H-ficolin 3). Ficolin-3, also known as Hakata-antigen or H-ficolin, is composed by a collagen-like strand and three C-terminal recognition domains which bind to acetyl groups on microbial surfaces such as GlcNAc or GalNAc. Ficolin-3 is synthesized both in the liver, from where it is secreted into the blood circulation, and in the lung. Similar to MBL and Ficolin-2, Ficolin-3 relies on MBL-associated serine protease 2 (MASP-2) for activation of the complement system. After binding of Ficolin-3/MASP-2 complexes to microbial surfaces, MASP-2 sequentially cleaves C4 and C2, thereby generating the C3 convertase C4bC2b, which finally leads to opsonization and direct lysis of pathogens and recruitment of inflammatory cells.
Ficolin-3 is present in serum at mean concentration of 15 µg/ml, with only minor variations. Ficolin-3 was present in all sera from more than 150,000 individuals tested, except in some systemic lupus erythromatosis patients. Approximately 5% of systemic lupus erythromatosis patients were found to be Ficolin-3 negative, probably owing to the presence of anti-Ficolin-3 autoantibody. In 398 patients with other autoimmune diseases, Ficolin-3 was always present. In liver disease the serum levels decreased with increasing severity of cirrhosis.
Ficolin-3 bound to a population of late apoptotic cells, while a strong and uniform binding to necrotic cells was observed. The binding properties differed from those of MBL and Ficolin-2. Ficolin-3 binding to late apoptotic cells resulted in a significant increase in adhesion/uptake by macrophages.
The human Ficolin-3 ELISA kit is to be used for the in vitro quantitative determination of human Ficolin-3 in serum, plasma and cell culture supernatant samples.
The human Ficolin-3 ELISA is a ready-to-use solid-phase enzyme-linked immunosorbent assay based on the sandwich principle with a working time of 3½ hours.
The efficient format of 2 plates with twelve disposable 8-well strips allows free choice of batch size for the assay.
Samples and standards are captured by a solid bound specific antibody.
Biotinylated tracer antibody will bind to captured Ficolin-3.
Streptavidin-peroxidase conjugate will bind to the biotinylated tracer antibody.
Streptavidin-peroxidase conjugate will react with the substrate, tetramethylbenzidine (TMB).
The enzyme reaction is stopped by the addition of oxalic acid.
The absorbance at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the Ficolin-3 standards (log).
The human Ficolin-3 concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.
The linearity of the assay was determined by serially diluting a sample containing 98.1 ng/ml human Ficolin-3. The diluted samples were measured in the assay. The line obtained a slope of 0.883 and a correlation coefficient of 1.0.
Normal human blood samples (plasma), containing baseline levels of 500 ng/ml were spiked with recombinant Ficolin-3 in concentrations of 6.5 and 100 ng/ml. Samples with and without Ficolin-3 were incubated for 1 hour at room temperature. Samples were measured using the ELISA. Values for Ficolin-3 ranged between 94 % and 115%.