Simultaneous determination of two phosphorylated p53 proteins in SCC-7 cells by an ICP-MS immunoassay using apoferritin-templated europium(III) and lutetium(III) phosphate nanoparticles as labels

Phosphorylated p53 proteins are biomarkers with clinical utility for early diagnosis of cancer, but difficult to quantify. An inductively coupled plasma mass spectrometry (ICP-MS) based immunoassay is described here that uses uniform lanthanide nanoparticles (NPs) as elemental tags for the simultaneous determination of two phosphorylated p53 proteins. Apoferritin templated europium (Eu) phosphate (AFEP) NPs and apoferritin templated lutetium (Lu) phosphate (AFLP) NPs with 8nm in diameter were used to label two phosphorylated p53 proteins at serine 15 and serine 392 sites (p-p53(15) and p-p53(392)), respectively. The assay has a sandwich format, and p-p53(15) and p-p53(392) were first captured and then recognized by AFEP and AFLP NPs labelled antibodies, respectively. The Eu and Lu were then released from the immune complexes under acidic condition for ICP-MS measurement. The limits of detection for p-p53(15) and p-p53(392) are 200 and 20pgmL(-1), with linear ranges of 0.5-20 and 0.05-20ngmL(-1), respectively. The method was further applied to study the response of p-p53(15) and p-p53(392) in SCC-7 cells exposed to the natural carcinogen arsenite. A significant up-regulation of p-p53(15) and p-p53(392) can be observed when cells were exposed to arsenite at 5molL(-1) level for 24h.