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Related Experiment Videos

Complement activation by C-reactive protein on the HEp-2 cell substrate

P Vaith1, V Prasauskas, L A Potempa

  • 1Department of Rheumatology and Clinical Immunology, Freiburg, Germany.

International Archives of Allergy and Immunology
|October 1, 1996
PubMed
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This study introduces HEp-2 cells as a novel substrate to visualize complement activation by C-reactive protein (CRP). This method helps understand complement (C) pathways and diagnose conditions like bacterial endocarditis.

Area of Science:

  • Immunology
  • Biochemistry
  • Cell Biology

Background:

  • Complement (C) activation by C-reactive protein (CRP) is crucial in acute-phase responses.
  • Current indirect immunofluorescence (C3-IFT) methods using rat kidney sections have limitations in visualizing early complement components (C1 complex) and CRP.
  • Unexpectedly negative C3-IFT results in certain conditions, such as bacterial endocarditis, necessitate improved diagnostic tools.

Purpose of the Study:

  • To investigate the mechanisms behind negative C3-IFT results.
  • To develop a more effective method for visualizing CRP-mediated complement activation.
  • To analyze the role of HEp-2 cells as a substrate for studying complement activation by CRP.

Main Methods:

  • Utilized HEp-2 cell monolayers as a substrate for CRP binding and complement activation.

Related Experiment Videos

  • Incubated purified native CRP with normal human serum to detect complement components (C1q, C1r, C1s, C4, C3) via immunofluorescence.
  • Tested C3-IFT-positive and negative acute-phase sera on HEp-2 cells to monitor complement activation pathways.

    • Main Results:

    • Native CRP bound to HEp-2 cell nuclei, leading to the deposition of C1q, C1r, C1s, C4, and C3 in a speckled pattern.
    • Serial complement activation steps were successfully visualized on HEp-2 cells using C3-IFT-positive sera.
    • Some C3-IFT-negative sera showed an activation arrest between C1s and C4, indicating specific pathway defects.

    Conclusions:

    • HEp-2 cells serve as a suitable substrate for monitoring autologous complement activation initiated by endogenous CRP in patient sera.
    • This method allows for the visualization of early complement cascade components, overcoming limitations of previous techniques.
    • The HEp-2 cell assay can identify specific blocks in the complement activation pathway, aiding in the diagnosis of related conditions.