Analysis for TNF-α Using Solid-Phase Affinity Capture with Radiolabel and MALDI-MS Detection

• Published in: Analytical chemistry (Washington) Vol. 71; no. 20; pp. 4727 - 4733
• Main Authors: Hurst, Gregory B, Buchanan, Michelle V, Foote, Linda J, Kennel, Stephen J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15-10-1999
• Subjects: AFFINITY; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; CHEMISTRY; DETECTION; Fundamental and applied biological sciences. Psychology; Indicators and Reagents; Mice; Protein hormones. Growth factors. Cytokines; Proteins; RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEAR CHEMISTRY; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tumor Necrosis Factor-alpha - analysis; Vertebrata; Mammalia; Mouse; Cytokine; Rodentia; Analytical method; Autoimmune disease; Inflammation; Mass spectrometry; Laser desorption; Tumor necrosis factor α; Quantitative analysis
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac9905423

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 autoimmune 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.