Quantification of mannan-binding lectin.
Mannan-binding lectin (MBL) is attracting considerable interest due to its role in the immune defense. The high frequency of congenital MBL deficiency makes it feasible to evaluate clinical relevance through epidemiological investigations on fairly limited numbers of patients. MBL deficiency is determined by three mutant allotypes termed B, C and D in the coding region as well as mutations in the promoter region. It has been suggested that individuals, with deficiency-associated allotypes, may present significant amounts of low molecular weight MBL. We have compared the quantification of MBL by four commercially available assays with results obtained by our own in-house assays. Most assays are selectively sensitive for the wild type MBL (allotype A), but special combinations of antibodies also detect mutant forms of MBL. Thus a sandwich-type time-resolved immunoflourometric assay (TRIFMA), with a mouse monoclonal antibody (93C) as the catching and detecting antibody, shows B/B and D/D homozygous individuals to present signals corresponding to up to 500 ng MBL per ml (with plasma from an A/A individual as standard) as compared to less than 50 ng/ml and 200 ng/ml, respectively, when measured in other assays. In GPC at isotonic conditions the MBL in B/B and D/D individuals showed a Mr of 450 kDa. This MBL cannot bind to mannan. We further present a new method for quantifying the amount of MBL polypeptide chain. By applying plasma samples on SDS-PAGE at reducing conditions followed by Western blotting and quantification by chemiluminescense, this approach presents single polypeptide chains to the antibody independent of allotype differences in the collagen-like region. Titrations of recombinant MBL served as standard. In sera from homozygous mutants (O/O) the MBL concentrations estimated on Western blot were in the range of 100 to 500 ng/ml and correlated with that measured in the 93C-based TRIFMA.
Frederiksen
Pernille D
PD
Department of Medical Microbiology and Immunology, University of Aarhus, Wilhelm Meyers Allé, 8000 Aarhus, DK, Denmark. pdf@microbiology.au.dk
Thiel
Steffen
S
Jensen
Lisbeth
L
Hansen
Annette G
AG
Matthiesen
Finn
F
Jensenius
Jens Chr
JC
D015153:Blotting, Western / Q000379:methods*; D002850:Chromatography, Gel; D014644:Genetic Variation; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D037601:Mannose-Binding Lectin / Q000032:analysis* / Q000097:blood / Q000276:immunology
D037601:Mannose-Binding Lectin