Development of a new immunoassay for the detection of ethyl glucuronide (EtG) in meconium: validation with authentic specimens analyzed using LC-MS/MS. Preliminary results
-
Simona Pichini
,
Luca Morini
Abstract
Background: Ethyl glucuronide (EtG) measurement in neonatal meconium has emerged as a reliable marker to objectively assess prenatal exposure to maternal ethanol complementary to fatty acid ethyl ester (FAEEs) measurement. The detection of EtG in meconium is currently a lengthy, difficult and expensive process using liquid chromatography tandem mass spectrometry (LC-MS/MS) as the analytical procedure. An enzyme-linked immunosorbent assay (ELISA) for the identification of EtG in meconium was developed, validated and applied to authentic meconium specimens from newborns collected in Europe.
Methods: The ELISA procedure was calibrated using 0.45, 0.9, 1.35 and 1.8 nmol/g (100, 200 300 and 400 ng/g) standards. Meconium (0.25 g) was mixed thoroughly, with extraction buffer (pH 7.3; 0.5 mL). The tube was capped, sonicated, centrifuged and the supernatant was decanted. An aliquot of the extract (50 μL) was placed in the well of the microplate followed by enzyme conjugate (150 μL). The plate was incubated for 1 h, washed with deionized water, dried and substrate (200 μL) was added. After 30 min incubation, stop solution was added and the plate was read at 450 nm and 650 nm. Samples were also analyzed for EtG and FAEEs by validated LC-MS/MS assays.
Results: Using an EtG cut-off of 0.9 nmol/g for both ELISA screening test and confirmatory LC-MS/MS, immunoassay sensitivity was 100%; specificity 78%; positive-predictive value (PPV) 29% and negative-predictive value (NPV) 100%.
Conclusions: The assay is proposed as a preliminary screening test for the meconium of newborns suspected of being born to mothers drinking alcohol during pregnancy.
Acknowledgments
The authors thank Emilia Marchei, Laura Martucci and Rita di Giovannandrea from Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanitá, Rome, Italy for technical help.
Conflict of interest statement
Authors’ conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding played no role in thestudy design; in the collection, analysis, and interpretationof data; in the writing of the report; or in the decision tosubmit the report for publication.
Research Funding: Intramural fundings from Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanitá, Rome, Italy and from Immunalysis Corporation.
Employment or leadership: Christine Moore, James Tuyay and Warren Rodrigues are employed by Immunalysis Corporation, the company which manufactures the ELISA assay described here.
Honorarium: None declared.
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©2014 by Walter de Gruyter Berlin/Boston
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Articles in the same Issue
- Frontmatter
- Editorial
- Colorectal cancer and screening programs: not only analytical issues
- Reviews
- Laboratory diagnostics of inherited platelet disorders
- Reticulated platelets: analytical aspects and clinical utility
- Genetics and Molecular Diagnostics
- Advanced tools for BRCA1/2 mutational screening: comparison between two methods for large genomic rearrangements (LGRs) detection
- General Clinical Chemistry and Laboratory Medicine
- Establishing, harmonizing and analyzing critical values in a large academic health center
- Standardization of DiaSorin and Roche automated third generation PTH assays with an International Standard: impact on clinical populations
- First fully automated immunoassay for anti-Müllerian hormone
- A multicenter evaluation of dysthyroxinemia in a defined patient cohort
- New biomarkers in diagnosis of early onset preeclampsia and imminent delivery prognosis
- Soluble Fms-like tyrosine kinase-1 to placental growth factor ratio in mid-pregnancy as a predictor of preterm preeclampsia in asymptomatic pregnant women
- Development of a new immunoassay for the detection of ethyl glucuronide (EtG) in meconium: validation with authentic specimens analyzed using LC-MS/MS. Preliminary results
- Optimizing centrifugation of coagulation samples in laboratory automation
- Evaluation of the automated coagulation analyzer CS-5100 and its utility in high throughput laboratories
- A new sampling device for faecal immunochemical testing: haemoglobin stability is still an open issue
- Reference Values
- Faecal haemoglobin concentrations vary with sex and age, but data are not transferable across geography for colorectal cancer screening
- Cancer Diagnostics
- Enhanced miR-182 transcription is a predictor of poor overall survival in colorectal adenocarcinoma patients
- Importance of promoter methylation of GATA4 and TP53 genes in endometrioid carcinoma of endometrium
- Frequent methylation of HOXA9 gene in tumor tissues and plasma samples from human hepatocellular carcinomas
- Letters to the Editor
- Further comments on “Critical review of laboratory investigations in clinical practice guidelines: proposals for the description of investigation”
- A questionnaire study among nurses: awareness of blood and urine sample collection procedures
- Measurement uncertainty and clinical interpretation of measurement results
- Laboratory automation: how will you select the boarding assays?
- Improvement and evaluation of a 1,2-dioleoylglycerol method for measuring pancreatic lipase catalytic activity in serum
- The novel variant p.Ser465Leu in the PCSK9 gene does not account for the decreased LDLR activity in members of a FH family
- 1,5 Anhydroglucitol serum concentration as a biomarker for screening gestational diabetes in early pregnancy
- A rare condition: IgE type monoclonal gammopathy of undetermined significance
- Laboratory analysis of intraosseous blood: bad to the bone?
