Analysis for TNF-alpha using solid-phase affinity capture with radiolabel and MALDI-MS detection.
Screening of mutant mice for subtle phenotypes requires sensitive, high-throughput analyses of sentinel proteins in functional pathways. The cytokine TNF-alpha is upregulated during inflammatory reactions associated with autoimmune diseases. We have developed a method to monitor the concentration of TNF-alpha under physiological conditions. TNF-alpha is captured, purified, and concentrated using monoclonal antibody-coated microbeads. The capture is efficient (> 80%) and can be used in the concentration range < 100 pg/mL to > 50 ng/mL, as determined by detection of 125I-labeled TNF-alpha. The bead capture of TNF-alpha can be combined with direct detection by MALDI-MS for sample concentrations of > 10 ng/mL. TNF-alpha can be captured and detected from diluted mouse serum, with minimal interferences observed in the MALDI spectrum. This method is adaptable to high-throughput sample handling with microfluidic devices and automated mass spectrometric analysis.
Hurst
G B
GB
Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Tennessee 37831, USA. hurstgb@ornl.gov
Buchanan
M V
MV
Foote
L J
LJ
Kennel
S J
SJ
D000818:Animals; D007202:Indicators and Reagents; D051379:Mice; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
D007202:Indicators and Reagents; D014409:Tumor Necrosis Factor-alpha