Validation of a multiplex electrochemiluminescent immunoassay platform in human and mouse samples.
Pubmed ID: 24768796
Pubmed Central ID: PMC4120713
Journal: Journal of immunological methods
Publication Date: 06/01/2014
Affiliation: Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University School of Medicine, T-1218 MCN, Nashville, TN 37232-2650, United States.
MeSH Terms: Humans, Animals, Enzyme-Linked Immunosorbent Assay, Species Specificity, Predictive Value of Tests, Reproducibility of Results, Mice, Inflammation Mediators, Bronchoalveolar Lavage Fluid, Reagent Kits, Diagnostic, Interleukin-6, Interleukin-8, Mice, Inbred C57BL, Luminescent Measurements, Electrochemical Techniques, Interleukins, Limit of Detection, Biomarkers
Grants: HL081332, HL103836, K08 HL090785, K24 HL103836, R21 HL112656, U01 HL081332, R21 HL117676, HL112656, HL 090785, HL117676
Authors: Ware LB, Bastarache JA, Koyama T, Wickersham NE
Cite As: Bastarache JA, Koyama T, Wickersham NE, Ware LB. Validation of a multiplex electrochemiluminescent immunoassay platform in human and mouse samples. J Immunol Methods 2014 Jun;408:13-23. Epub 2014 Apr 21.
BACKGROUND: Despite the widespread use of multiplex immunoassays, there are very few scientific reports that test the accuracy and reliability of a platform prior to publication of experimental data. Our laboratory has previously demonstrated the need for new assay platform validation prior to use of biologic samples from large studies in order to optimize sample handling and assay performance. METHODS: In this study, our goal was to test the accuracy and reproducibility of an electrochemiluminescent multiplex immunoassay platform (Meso Scale Discovery, MSD®) and compare this platform to validated, singleplex immunoassays (R&D Systems®) using actual study subject (human plasma and mouse bronchoalveolar lavage fluid (BAL) and plasma) samples. RESULTS: We found that the MSD platform performed well on intra- and inter-assay comparisons, spike and recovery and cross-platform comparisons. The mean intra-assay CV% and range for MSD were 3.49 (0.0-10.4) for IL-6 and 2.04 (0.1-7.9) for IL-8. The correlation between values for identical samples measured on both MSD and R&D was R=0.97 for both analytes. The mouse MSD assay had a broader range of CV% with means ranging from 9.5 to 28.5 depending on the analyte. The range of mean CV% was similar for single plex ELISAs at 4.3-23.7 depending on the analyte. Regardless of species or sample type, CV% was more variable at lower protein concentrations. CONCLUSIONS: In conclusion, we validated a multiplex electrochemiluminescent assay system and found that it has superior test characteristics in human plasma compared to mouse BALF and plasma. Both human and MSD assays compared favorably to well-validated singleplex ELISAs.