A Comparison of Enzyme-Linked Immunosorbent Assay versus Multiplex Methodology Using an in Vitro Model of Pulmonary Hypertension and Inflammation

Yan Zhu, Deepthi Alapati, Joanna Costa, Victoria L. Maduskuie, Paul T. Fawcett, Thomas H. Shaffer

Center for Clinical Diagnostics, Nemours Alfred I duPont Hospital for Children, Wilmington, USA.
Nemours Research Lung Center, Biomedical Research, Nemours Alfred I duPont Hospital for Children, Wilmington, USA.

DOI: 10.4236/jbise.2014.77044                

Abstract

Enzyme-linked immunosorbent assay (ELISA) is the most widely used method for measuring a single cytokine. Recent developments in cytokine quantification such as multiple arrays measure multiple cytokines simultaneously. Although good correlations between ELISA and multiplex methods have been observed, side by side comparisons are limited. In the present study we hypothesized that ELISA and Luminex techniques are comparable in detecting cytokines in culture medium when pulmonary artery smooth muscle cells (PASMC) are exposed to stress. Primary human PASMC were cultured in modular chambers and exposed to 21% FiO₂ and peak inspiratory and positive end expiratory pressure of 24 and 8 cmH₂O respectively, and 95% FiO₂. At 24 hours, culture medium was collected and assayed for interleukin-6 (IL-6) and IL-8 by quantitative ELISA and by Human Cytokine 25-Plex Panel using a Luminex 200 analyzer. A comparative analysis of agreement between our ELISA and Luminex data was detailed for control and stress conditions using the Bland-Altman plot analysis. Each assay resulted in comparable increased (p < 0.001) levels of IL-6 and IL-8 as compared to control in response to oxidative and biophysical stress. The Bland-Altman analysis demonstrated that 95% of the differences between ELISA and Luminex values were within ±1.96 SD from the mean difference indicated by the 95% limits of agreement for the measurements of IL-6 and IL-8. There was no systematic bias as a function of inflammation level. We conclude that in this cell culture model, ELISA and Luminex are comparable in detecting the levels of IL-6 and IL-8 in the culture medium. If measurements of multiple cytokines are demanded and the amount of sample is limited, Luminex multi-analyte profiling technology is accurate and sensitive.

Keywords

Enzyme-Linked Immunosorbent Assay (ELISA), Luminex, Pulmonary Artery Smooth Muscle Cells (PASMC), Inflammation, Bland-Altman Plot Analysis

Conflicts of Interest

The authors declare no conflicts of interest.

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