Analysis for TNF-α 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-α is upregulated during inflammatory reactions associated with antoimmune diseases. We have developed a method to monitor the concentration of TNF-α under physiological conditions. TNF-α 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-α. The bead capture of TNF-α can be combined with direct detection by MALDI-MS for sample concentrations of > 10 ng/mL. TNF-α 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.