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- Title
A Novel ELISA for the Quantification of Serum Levels of 2 Glycosylated Variants of Apolipoprotein J: Biomarkers for Myocardial Ischemia.
- Authors
Fernández Encinas, Lola; Lluch, Nuria; Wu, Alan H B; Kaski, Juan Carlos; Badimon, Lina; Cubedo, Judit
- Abstract
Background: Previous studies have pointed out a potential role of ApoJ-Glyc as a biomarker of cardiac ischemia. The aim of this study was to validate the analytical performance of 2 novel ELISAs against 2 different glycosylated ApoJ variants, ApoJ-GlycA2 and ApoJ-GlycA6. Methods: The analytical measuring range, limit of blank (LoB), lower limit of quantification (LoQ), precision, accuracy, recovery, cross-reactivity, and stability were evaluated in serum samples. Results: The analytical measuring range was 500–16 000 ng/mL for ApoJ-GlycA2 and 125–4000 ng/mL for ApoJ-GlycA6, with a LoB of 455 ng/mL and 121 ng/mL for ApoJ-GlycA2 and ApoJ-GlycA6, respectively. The LoQ was 500 ng/mL for ApoJ-GlycA2 and 125 ng/mL for ApoJ-GlycA6. The assay performance fulfills the acceptance criteria established in the European Medicines Agency Guideline on bioanalytical method validation. Specifically, the calibration range variability is <15% for ApoJ-GlycA2 and ApoJ-GlycA6; the accuracy is <15% for ApoJ-GlycA2 and ApoJ-GlycA6; the between- and within-run precision is <15% for ApoJ-GlycA6 and ≤20% for ApoJ-GlycA2; and the total allowable error is <30% for ApoJ-GlycA2 and ApoJ-GlycA6. Cross-reactivity studies revealed the absence of cross-reactivity with endogenous components of the matrix (using ApoJ-depleted serum), with nonglycosylated ApoJ and with transferrin (as a high abundant N-glycosylated serum protein). Both ApoJ-GlycA2 and ApoJ-GlycA6 measurements were stable after storage of serum samples at −80°C for 24 months. Conclusions: The newly developed ELISAs to quantify ApoJ-GlycA2 and ApoJ-GlycA6 serum levels showed an acceptable analytical performance according to European Medicines Agency guidelines on bioanalytical method validation in terms of precision, accuracy, recovery, cross-reactivity, and stability.
- Subjects
MYOCARDIAL ischemia; BLOOD proteins; BIOMARKERS; CROSS reactions (Immunology); GLYCOSYLATED hemoglobin; TRANSFERRIN
- Publication
Journal of Applied Laboratory Medicine, 2023, Vol 8, Issue 5, p917
- ISSN
2475-7241
- Publication type
Article
- DOI
10.1093/jalm/jfad034