Antibody Labeling and Elemental Mass Spectrometry (Inductively Coupled Plasma-Mass Spectrometry) Using Isotope Dilution for Highly Sensitive Ferritin Determination and Iron-Ferritin Ratio Measurements

Ferritin, an iron storage protein, is a sensitive clinical biomarker for iron metabolic disorders. It is mainly accumulated in the liver hepatocytes and is present in human plasma at trace levels (picomolar or nanograms per milliliter). Therefore, highly sensitive analytical methods are required to perform ferritin quantification in plasma with high precision and accuracy. For this purpose, we present a mass spectrometry-based analytical strategy (inductively coupled plasma-mass spectrometry, ICP-MS) combined with antibody labeling in a sandwich assay format for ferritin determination. The developed methodology involves two ferritin monoclonal antibodies, one of them biotinylated and the other one labeled with a ruthenium chelate [Ru(bpy)(3)](2+). The complex formed in solution. between ferritin and the two antibodies is then captured using streptavidin-coated magnetic microparticles and directly introduced into ICP-MS for Ru monitoring. Since the Ru complex also allows one to obtain electrogenerated chemiluminescence (ECL), the combination of both sets of data (ICP-MS and ECL) will permit the establishment of the ferritin:Ru stoichiometry. This serves as a basis for further quantification studies using flow injection analysis with isotopically enriched Ru-99 as a carrier with ICP-MS detection. Such strategy permits absolute ferritin determination at a picomolar level with good precision (below 5%) and accuracy (85-109% recovery in the existing ferritin reference material, NIBSC code 94/572). Furthermore, the development of a new strategy to address ferritin:iron-ferritin ratios by ICP-MS opens the door also to address the potential of such ratios as a new clinical biomarker for Fe metabolic disorders.