An enzyme-linked immunosorbent assay (ELISA) for quantification of human collectin 11 (CL-11, CL-K1)

Collectin 11 (CL-11), also referred to as collectin kidney 1 (CL-K1), is a pattern recognition molecule that belongs to the collectin group of proteins involved in innate immunity. It interacts with glycoconjugates on pathogen surfaces and has been found in complex with mannose-binding lectin-associated serine protease 1 (MASP-1) and/or MASP-3 in circulation. Mutation in the CL-11 gene was recently associated with the developmental syndrome 3MC. In the present study, we established and thoroughly validated a sandwich enzyme-linked immunosorbent assay (ELISA) based on two different monoclonal antibodies. The assay is highly sensitive, specific and shows excellent quantitative characteristics such as reproducibility, dilution linearity and recovery (97.7–104%). The working range is 0.15–34 ng/ml. The CL-11 concentration in two CL-11-deficient individuals affected by the 3MC syndrome was determined to be below 2.1 ng/ml. We measured the mean serum CL-11 concentration to 284 ng/ml in 100 Danish blood donors, with a 95% confidence interval of 269–299 ng/ml. There was no significant difference in the CL-11 concentration measured in matched serum and plasma samples. Storage of samples and repeated freezing and thawing to a certain extent did not influence the ELISA. This ELISA offers a convenient and reliable method for studying CL-11 levels in relation to a variety of human diseases and syndromes

Functional Analysis of Ficolin-3 Mediated Complement Activation

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

Association of low ficolin-lectin pathway parameters with Cardiac Syndrome X

In patients with typical angina pectoris, inducable myocardial ischaemia and macroscopically normal coronaries (Cardiac Syndrome X, CSX) significantly elevated plasma level of terminal complement complex (TCC), the common end-product of complement activation, has been observed without subsequent activation of the classical or the alternative pathways. Therefore, our aim was to clarify the role of the ficolin-lectin pathway in CSX. Eighteen CSX patients, 37 stable angina patients with significant coronary stenosis (CHD) and 54 healthy volunteers (HC) were enrolled. Serum levels of ficolin-2, ficolin-3, ficolin-3/MASP-2 complex and ficolin-3 mediated TCC deposition (FCN3-TCC) were determined. Plasma level of TCC was significantly higher in CSX than in HC or in CHD groups (5.45 vs. 1.30 vs. 2.04AU/ml, p<0.001). Serum levels of ficolin-2 and ficolin-3 were significantly lower in CSX compared to HC or to CHD groups (3.60 vs. 5.80 or 5.20mug/ml, p<0.05; 17.80 vs. 24.10 or 26.80mug/ml, p<0.05). The ficolin-3/MASP-2 complex was significantly lower in CSX group compared to HC (92.90 vs. 144.90AU/ml, p=0.006). FCN3-TCC deposition was significantly lower in the CSX group compared to HC and to CHD (67.8% vs.143.3% or 159.7%, p<0.05). In the CSX group, significant correlation was found between TCC and FCN3-TCC level (r=0.507, p=0.032) and ficolin-3/MASP-2 complex level and FCN3-TCC deposition (r=0.651, p=0.003). In conclusion, in patients with typical angina and myocardial ischemia despite macroscopically normal coronary arteries, low levels of several lectin-pathway parameters were observed, indicating complement activation and consumption. Complement activation through the ficolin-lectin pathway might play a role in the complex pathomechanism of CSX. This article is protected by copyright. All rights reserved

Protease inhibitor plasma concentrations associate with COVID-19 infection

Protease inhibitors influence a range of innate immunity and inflammatory pathways. We quantified plasma concentrations of key anti-inflammatory protease inhibitors in chronic haemodialysis patients with COVID-19. The samples were collected early in the disease course to determine whether plasma protease inhibitor levels associated with the presence and severity of COVID-19. We used antibody-based immunoassays to measure plasma concentrations of C1 esterase inhibitor (C1-INH), alpha2-macroglobulin (α2M), antithrombin, and inter-alpha-inhibitor heavy chain 4 (ITIH4) in 100 serial samples from 27 haemodialysis patients with COVID-19. ITIH4 was tested in two assays, one measuring intact ITIH4 and another also detecting any fragmented ITIH4 (total ITIH4). Control cohorts were 32 haemodialysis patients without COVID-19 and 32 healthy controls. We compared protease inhibitor concentration based on current and future COVID-19 severity and with CRP. Results were adjusted for repeated measures and multiple comparisons. Analysis of all available samples demonstrated lower plasma C1-INH and α2M and higher total ITIH4 in COVID-19 compared to dialysis controls. These differences were also seen in the first sample collected after COVID-19 diagnosis, a median of four days from diagnostic swab. Plasma ITIH4 levels were higher in severe than non-severe COVID-19. Serum CRP correlated positively with plasma levels of antithrombin, intact ITIH4, and total ITIH4. In conclusion, plasma protease inhibitor concentrations are altered in COVID-19