Analysis for TNF-alpha using solid-phase affinity capture with radiolabel and MALDI-MS detection. |
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| Authors: | G B Hurst M V Buchanan L J Foote S J Kennel |
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| Affiliation: | Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Tennessee 37831, USA. hurstgb@ornl.gov |
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| Abstract: | 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. |
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