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

L Selman,M.L Henriksen,J Brandt,Y Palarasah,A Waters,P.L Beales,U Holmskov,T.J.D Jørgensen,C Nielsen,K Skjodt,S Hansen

doi:10.1016/j.jim.2011.10.010

Abstract

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 0.15–34ng/ml. The CL-11 concentration in two CL-11-deficient individuals affected by the 3MC syndrome was determined to be below 2.1ng/ml. We measured the mean serum CL-11 concentration to 284ng/ml in 100 Danish blood donors, with a 95% confidence interval of 269–299ng/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.

Highlights

Collectin 11 (CL-11), known as collectin kidney 1 (CL-K1), belongs to the collectin group of the innate immuneAbbreviations: CL-11, collectin 11;mannose-binding lectin-associated serine protease 1 (MASP-1)/3, Mannose-binding lectin associated serine proteases 1/3;mannosebinding lectin (MBL), mannose-binding lectin;SLE, systemic lupus erythematosus;SP-D, surfactant protein D;ELISA, enzyme-linked immunosorbent assay;CRD, carbohydrate recognition domain;SP-A, surfactant protein A;SP-D, surfactant protein D; Monoclonal antibodies (MAbs), monoclonal antibody;BSA, bovine serum albumin;Optical density (OD), optical density; quality controls (QCs), quality control;Coefficients of variation (CV), coefficient of variation.⁎ Corresponding author at: Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, Winslowparken 21-1, DK-5000 Odense C, Denmark
The CL-11 concentration calculated for each dilution and multiplied by the dilution factor was compared to a mean of the CL-11 concentration that was back-calculated from four dilutions of each sample (1/16–1/128 for serum samples and 1/20–1/160 for plasma samples)
Using the normally distributed data, we found no significant difference in CL-11 levels between males and females

Summary

Introduction

Variations in serum levels of collectins have been implicated in various immunity disorders. Functional mannosebinding lectin (MBL) deficiency, caused by single-nucleotide polymorphisms in the coding region of the MBL2 gene, has been associated with increased susceptibility to infections in young children and immunocompromised individuals (Sumiya et al, 1991; Garred et al, 1997; Summerfield et al, 1997; Neth et al, 2001; Peterslund et al, 2001). MBL deficiency or low MBL serum levels are associated with the occurrence of autoimmune disorders, such as systemic lupus erythematosus (SLE) (Lee et al, 2005). Low serum levels of SP-D, caused by the variant allele Thr, may increase susceptibility to tuberculosis (Floros et al, 2000). Low serum levels of SP-D have been implicated in pathogenesis of SLE (Hoegh et al, 2009)

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