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 8 nm in diameter were used to label two phosphorylated p53 proteins at serine 15 and serine 392 sites (p-p5315 and p-p53392), respectively. The assay has a sandwich format, and p-p5315 and p-p53392 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-p5315 and p-p53392 are 200 and 20 pg·mL-1, with linear ranges of 0.5-20 and 0.05-20 ng·mL-1, respectively. The method was further applied to study the response of p-p5315 and p-p53392 in SCC-7 cells exposed to the natural carcinogen arsenite. A significant up-regulation of p-p5315 and p-p53392 can be observed when cells were exposed to arsenite at 5 µmol·L-1 level for 24 h. Graphical abstract Schematic presentation of the ICP-MS immunoassay using apoferritin templated europium (III) and lutetium (III) phosphate nanoparticles as labels for the simultaneous determination of two phosphorylated p53 proteins. Europium (Eu) phosphate nanoparticles (blue) and lutetium (Lu) phosphate nanoparticles (pink) were synthesized in the size-restricted cavity of apoferritin. They were further coupled with antibodies to prepare Eu and Lu labelled probes for p-p5315 (blue) and p-p53392 (pink), respectively. After formation of a a sandwich, the labelled Eu and Lu were dissociated in acid and then introduced to ICP-MS for the simultaneous determination of two phosphorylated p53 proteins p-p5315 (blue) and p-p53392 (pink).

Complex biological patterns of soluble cytokines and CD163 in childhood necessitating age-specific reference intervals for evidence-based clinical interpretation.

BACKGROUND: Cytokine measurements to support clinical laboratory and research investigations have become increasingly common in pediatrics. However, there is a paucity of accurate pediatric reference intervals (RIs) essential to the interpretation of cytokine results. To address this gap, here, we establish age- and sex-specific pediatric reference values for clinically relevant inflammatory markers including CD163, and the cytokines IL-1ß, IL-6, IL-10, IL-18, TNF-α, IFN-γ, and CXCL-9. METHODS: Healthy children and adolescents (n = 311, 1-19 years) were recruited as part of the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) study. Multi-analyte measurements in plasma and analytical performance verification were conducted on the ProteinSimple® Ella™ automated immunoassay platform (Bio-Techne, MN, USA). Age- and sex-specific RIs were calculated based on Clinical and Laboratory Standards Institute guidelines. Additionally, 75th and 95th percentile cut-offs were determined. RESULTS: Three types of reference value distributions were observed: (a) consistent levels throughout age and sex: IL-6, and IFN-γ, (b) gradual decline in concentration with age: CD163, TNF-α, CXCL-9, and IL-10, (c) significantly higher concentrations during ages 4-14 years than earlier and later ages: IL-1ß and IL-18. Reference values for CXCL-9, IL-10, and TNF-α under 8 years of age differed significantly from older children. CD163, IL-18 and IL-1ß required three age partitions. CD163 demonstrated significant sex differences in ages 8-13 years. CONCLUSION: The circulating profile of cytokines in children is complex and can vary by age and sex. This necessitates careful interpretation of test results based on age and/or sex specific RIs facilitating more accurate clinical decision making.

Dual-label time-resolved fluoroimmunoassay as an advantageous approach for investigation of diethyl phthalate & dibutyl phthalate in surface water.

Due to the extensive presence of phthalate esters (PAEs) in the environment, it is very important to develop highly efficient methods for determining their environmental concentrations and for risk assessment. In this study, europium (Eu3+) and samarium (Sm3+) were employed as fluorescent labels to develop a dual-labeled time-resolved fluoroimmunoassay (TRFIA) for the sensitive detection of diethyl phthalate (DEP) and dibutyl phthalate (DBP) in aquatic environments. Under optimum conditions, the half-maximal inhibition concentration (IC50) and limit of detection (LOD, IC10) of the TRFIA were 28.1 and 4.9ng/mL for DEP, and 33.4 and 3.9ng/mL for DBP, respectively. The cross-reactivities of the TRFIA between these two phthalates and with their analogues were negligible. The proposed method indicated satisfactory accuracy with recoveries of 78.30-120.13% for DEP and 78.58-113.07% for DBP, which were in good agreement with the gas chromatography tandem mass spectrometry analysis results of the same samples. Meanwhile, the results of the immunoassay were used to evaluate the presence and environmental risk of those pollutants in the inner rivers of Zhenjiang city. The concentrations of DEP and DBP ranged from non-detectable to 61.31ng/mL and from non-detectable to 94.57ng/mL, respectively. The results of the potential ecological risk assessment by the risk quotient method showed that there were some sampling points exceeding risk limits, and the total ecological risk of DBP was higher than that of DEP.