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-thro ughput analyses of sentinel proteins in functional pathways, The cytokine T NF-alpha is upregulated during inflammatory reactions associated with auto- immune 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 t o >50 ng/mL, as determined by detection of I-125-labeled TNF-alpha. The bea d capture of TNF-alpha can be combined with direct detection by MALDI-MS fo r sample concentrations of >10 ng/mL. TNF-alpha can be captured and detecte d from diluted mouse serum, with minimal interferences observed in the MALD I spectrum. This method-is adaptable to high-throughput sample handling wit h microfluidic devices and automated mass spectrometric analysis.