Rapid isolation of pure Complement Factor H from serum for functional studies by the use of a monoclonal antibody that discriminates FH from all the other isoforms

Mol Immunol. 2016 Apr:72:65-73. doi: 10.1016/j.molimm.2016.03.001. Epub 2016 Mar 9.

Abstract

Several mutations have been identified in the gene coding for Complement Factor H (FH) from patients with atypical Hemolytic Uraemic Syndrome (aHUS), Age-related Macular Degeneration (AMD) and Membranoproliferative Glomerulonephritis (MPGN). These data allow for a precise description of the structural changes affecting FH, but a simple test for specifically assessing FH function routinely is not yet of common use. We have produced and characterised a monoclonal antibody (5H5) which discriminates between FH and the smaller FH-like 1 and FH-related proteins and show here that it specifically binds to FH without detecting the smaller isoforms. We therefore used this mAb for a quick, one-step micro-purification of FH directly from control sera and showed that this affinity chromatography procedure is not disruptive of its cofactor function. We also developed a modified sheep erythrocytes haemolysis test using our antibody and affinity-purified FH. These tests can be used in conjunction for assessing the function of FH purified from patients affected by FH-related diseases. Moreover we used this mAb to develop a FH-specific ELISA test.

Keywords: Affinity chromatography; Complement Factor H; Functional assays; Immunoassays; Purification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibody Specificity
  • Cell Line, Tumor
  • Chickens
  • Complement Factor H / immunology
  • Complement Factor H / isolation & purification*
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Hemolysis
  • Humans
  • Immunoprecipitation
  • Mice
  • Protein Isoforms / immunology
  • Protein Isoforms / isolation & purification
  • Sheep

Substances

  • Antibodies, Monoclonal
  • Protein Isoforms
  • Complement Factor H