Functional analysis of Ficolin-3 mediated complement activation

PLoS One. 2010 Nov 10;5(11):e15443. doi: 10.1371/journal.pone.0015443.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Animals
  • Cattle
  • Complement Activation*
  • Complement C3 / metabolism
  • Complement C4 / metabolism
  • Complement Membrane Attack Complex / metabolism
  • Glycoproteins / blood
  • Glycoproteins / genetics
  • Glycoproteins / metabolism*
  • Humans
  • Lectins / blood
  • Lectins / genetics
  • Lectins / metabolism*
  • Mannose-Binding Lectin / genetics
  • Mannose-Binding Lectin / metabolism
  • Protein Binding
  • Recombinant Proteins / metabolism
  • Serum Albumin, Bovine / metabolism*

Substances

  • Complement C3
  • Complement C4
  • Complement Membrane Attack Complex
  • FCN3 protein, human
  • Glycoproteins
  • Lectins
  • Mannose-Binding Lectin
  • Recombinant Proteins
  • ficolin
  • Serum Albumin, Bovine