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Analysis for TNF-alpha using solid-phase affinity capture with radiolabel and MALDI-MS detection.

G B Hurst·M V Buchanan·L J Foote·S J Kennel
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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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AnimalsIndicators and ReagentsMiceSpectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationTumor Necrosis Factor-alphaJournal ArticleResearch Support, U.S. Gov't, Non-P.H.S.

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G B Hurst, M V Buchanan, L J Foote, S J Kennel. 1999-10-15. Analysis for TNF-alpha using solid-phase affinity capture with radiolabel and MALDI-MS detection.. https://doi.org/10.1021/ac9905423

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