An inductively coupled plasma-mass spectrometry (ICP-MS) linked immunoassay by means of iodinated antibodies for transferrin quantitative analysis in breast cancer cell lines.

The use of labelled antibodies in inductively coupled plasma-mass spectrometry (ICP-MS) linked immunoassays permits the accurate and sensitive determination of target analytes of biochemical importance. In this regard, the determination of the biomolecules responsible for iron homeostasis in cell cultures is crucial to understand the dysregulation of this element in breast cancer. For this aim, fast, simple, sensitive and accurate analytical strategies have to be developed. In this work, iodinated antibodies have been applied for the sensitive determination of transferrin in breast cancer cell lines using a novel immunoassay coupled to ICP-MS detection of iodine. Firstly, a study on the antibody iodination efficiency revealed an iodine: transferrin molar ratio of 27:1 which corresponds to the iodination of all the tyrosine residues present in the antibody. Secondly, the reactivity of the antibody after labelling was assessed in a quantitative immune subtraction experiment showing that the labelled species maintained their recognition capabilities by capturing 95% of the soluble transferrin. Finally, implementation of the iodinated antibody in a sandwich ICP-MS linked immunoassay was conducted in combination with a secondary biotinylated antibody and the use of streptavidin coated magnetic microparticles. Such set up was used for the determination of transferrin in cell cultures of breast cancer cells of different malignancy. Such determination revealed significant differences among lines with higher transferrin concentration in the line exhibiting higher iron levels, stressing the key aspects of this protein as importer of iron in cells.