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Related Concept Videos

Enzyme-Linked Immunosorbent Assay01:33

Enzyme-Linked Immunosorbent Assay

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In 1971, Peter Perlman and Eva Engvall developed an Enzyme-linked immunosorbent assay (ELISA or EIA). ELISA differs from western blot in that the assays are conducted in microtiter plates or in vivo rather than on an absorbent membrane.
There are many different types of ELISAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen.  Enzyme-substrate reaction allows the antigen to be visualized or...
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Related Experiment Video

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Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells
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A Cell-Based Assay to Measure the Activity of the Complement Convertases.

Małgorzata Stasiłojć1, Grzegorz Stasiłojć1, Alicja Kuźniewska1

  • 1Department of Cell Biology and Immunology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Gdańsk, Poland.

Kidney International Reports
|July 31, 2024
PubMed
Summary
This summary is machine-generated.

A new assay can now measure complement convertase activity, aiding in the diagnosis of rare diseases. This tool helps identify overactivity in complement proteins like C3, crucial for understanding disease mechanisms.

Keywords:
C3GaHUScomplement systemconvertaseeculizumab

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Area of Science:

  • Immunology
  • Complement System Biology

Background:

  • The complement system is vital for pathogen defense but its dysregulation causes disease.
  • Complement convertases (classical/lectin pathway and alternative pathway) are central to the cascade.
  • Analyzing convertase activity is challenging due to their instability and complex interactions.

Purpose of the Study:

  • To develop a novel assay for functional evaluation of complement convertase activity.
  • To enable detection of gain-of-function variants in complement proteins.
  • To assess the assay's utility in diagnosing complement-related diseases.

Main Methods:

  • Utilized a sensitized human lymphoma cell line to activate the classical pathway (CP) and alternative pathway (AP).
  • Employed C5 blockers (eculizumab, crovalimab) to halt the cascade at the convertase level.
  • Washed away inhibitors and used guinea pig serum to develop lytic sites on existing convertases.

Main Results:

  • The assay detects recombinant gain-of-function components of both classical/lectin pathway and alternative pathway convertases.
  • Demonstrated utility in analyzing nephrological patients with C3 genetic variants.
  • Successfully distinguished between patients and asymptomatic relatives with the same C3 variant.

Conclusions:

  • Provided proof-of-concept for a new assay detecting convertase overactivity.
  • The assay identifies individuals with pathogenic or unknown significance variants in complement proteins like C3.
  • This assay can aid in diagnosing rare complement-related diseases.