A very sensitive coupled luminescent assay for cytotoxicity and complement-mediated lysis

J Immunol Methods. 1997 Aug 22;207(1):43-51. doi: 10.1016/s0022-1759(97)00098-7.

Abstract

The demand for convenient and sensitive means of measuring cytotoxicity and complement-mediated killing is likely to be increased by the recent identification of Complement Factor H, an important regulatory protein of both the classical and alternate pathways of complement, as a tumor-associated antigen. Here we describe a simple luminometric assay capable of detecting the death of approximately 0.03 nucleated human-cell equivalent or approximately 1 rabbit-erythrocyte equivalent. The assay measures the release of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) from dead or damaged cells by coupling its enzymatic activity to production of ATP, which in turn is measured by well-known methods involving firefly luciferase. This is accomplished by means of a reaction series in which the activity of G3PDH is coupled with that of phosphoglycerate kinase, the next enzyme in the glycolytic pathway. As described, the assay uses inexpensive, commercially available reagents. This coupled assay was used to demonstrate that an anti-factor-H antibody is capable of enhancing complement-mediated killing of the Raji cancer cell line by > 1000%.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Antibodies / immunology
  • Cell Death / immunology
  • Cells, Cultured
  • Complement Factor H / immunology
  • Complement Pathway, Alternative
  • Complement Pathway, Classical
  • Complement System Proteins / immunology*
  • Cytotoxicity Tests, Immunologic / methods*
  • Glyceraldehyde-3-Phosphate Dehydrogenases / analysis
  • Glyceraldehyde-3-Phosphate Dehydrogenases / metabolism
  • L-Lactate Dehydrogenase / metabolism
  • Luciferases / analysis
  • Luciferases / metabolism
  • Octoxynol
  • Phosphoglycerate Kinase / metabolism
  • Polyethylene Glycols / pharmacology
  • Tumor Cells, Cultured

Substances

  • Antibodies
  • CFH protein, human
  • Polyethylene Glycols
  • Complement Factor H
  • Adenosine Triphosphate
  • Octoxynol
  • Complement System Proteins
  • Nonidet P-40
  • L-Lactate Dehydrogenase
  • Luciferases
  • Glyceraldehyde-3-Phosphate Dehydrogenases
  • Phosphoglycerate Kinase