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


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.

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Zhu, Y. , Alapati, D. , Costa, J. , Maduskuie, V. , Fawcett, P. and Shaffer, T. (2014) A Comparison of Enzyme-Linked Immunosorbent Assay versus Multiplex Methodology Using an in Vitro Model of Pulmonary Hypertension and Inflammation. Journal of Biomedical Science and Engineering, 7, 419-426. doi: 10.4236/jbise.2014.77044.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Dinarello, C.A. (2000) Proinflammatory Cytokines. Chest, 118, 503-508.
[2] Breen, E.C., Reynolds, S.M., Cox, C., Jacobson, L.P., Magpantay, L., Mulder, C.B., Dibben, O., Margolick, J.B., Bream, J.H., Sambrano, E., Martinez-Maza, O., Sinclair, E., Borrow, P., Landay, A.L., Rinaldo, C.R. and Norris, P.J. (2011) Multisite Comparison of High-Sensitivity Multiplex Cytokine Assays. Clinical and Vaccine Immunology, 18, 1229-1242.
[3] Leng, S.X., McElhaney, J.E., Walston, J.D., Xie, D., Fedarko, N.S. and Kuchel, G.A. (2008) ELISA and Multiplex Technologies for Cytokine Measurement in Inflammation and Aging Research. The Journals of Gerontology: Series A, 63, 879-884.
[4] Elshal, M.F. and McCoy, J.P. (2006) Multiplex Bead Array Assays: Performance Evaluation and Comparison of Sensitivity to ELISA. Methods, 38, 317-323.
[5] Zhu, Y., Chidekel, A. and Shaffer, T.H. (2010) Cultured Human Airway Epithelial Cells (CALU-3): A Model of Human Respiratory Function, Structure, and Inflammatory Responses. Critical Care Research and Practice, 2010, Article ID: 394578.
[6] Zhu, Y., Miller, T.L., Chidekel, A. and Shaffer, T.H. (2008) KL4-Surfactant (Lucinactant) Protects Human Airway Epithelium from Hyperoxia. Pediatric Research, 64, 154-158.
[7] Zhu, Y., Miller, T.L., Singhaus, C.J., Shaffer, T.H. and Chidekel, A. (2008) Effects of Oxygen Concentration and Exposure Time on Cultured Human Airway Epithelial Cells. Pediatric Critical Care Medicine, 9, 224-229.
[8] Kramer, B.W., Jobe, A.H., Bachurski, C.J. and Ikegami, M. (2001) Surfactant Protein A Recruits Neutrophils into the Lungs of Ventilated Preterm Lambs. American Journal of Respiratory and Critical Care Medicine, 163, 158-165.
[9] Allen, G.L., Menendez, I.Y., Ryan, M.A., Mazor, R.L., Wispe, J.R., Fiedler, M.A. and Wong, H.R. (2000) Hyperoxia Synergistically Increases TNF-Alpha-Induced Interleukin-8 Gene Expression in A549 Cells. American Journal of Physiology—Lung Cellular and Molecular Physiology, 278, L253-260.
[10] Horan, P.K. and Wheeless Jr., L.L. (1977) Quantitative Single Cell Analysis and Sorting. Science, 198, 149-157.
[11] Khan, S.S., Smith, M.S., Reda, D., Suffredini, A.F. and McCoy Jr., J.P. (2004) Multiplex Bead Array Assays for Detection of Soluble Cytokines: Comparisons of Sensitivity and Quantitative Values among Kits from Multiple Manufacturers. Cytometry Part B: Clinical Cytometry, 61, 35-39.
[12] Pickering, J.W., Martins, T.B., Schroder, M.C. and Hill, H.R. (2002) Comparison of a Multiplex Flow Cytometric Assay with Enzyme-Linked Immunosorbent Assay for Auantitation of Antibodies to Tetanus, Diphtheria, and Haemophilus Influenzae Type b. Clinical and Diagnostic Laboratory Immunology, 9, 872-876.
[13] de Jager, W., te Velthuis, H., Prakken, B.J., Kuis, W. and Rijkers, G.T. (2003) Simultaneous Detection of 15 Human Cytokines in a Single Sample of Stimulated Peripheral Blood Mononuclear Cells. Clinical and Diagnostic Laboratory Immunology, 10, 133-139.
[14] Price, L.C., Wort, S.J., Perros, F., Dorfmuller, P., Huertas, A., Montani, D., Cohen-Kaminsky, S. and Humbert, M. (2012) Inflammation in pulmonary Arterial Hypertension. Chest, 141, 210-221.
[15] Soon, E., Holmes, A.M., Treacy, C.M., Doughty, N.J., Southgate, L., Machado, R.D., Trembath, R.C., Jennings, S., Barker, L., Nicklin, P., Walker, C., Budd, D.C., Pepke-Zaba, J. and Morrell, N.W. (2010) Elevated Levels of Inflammatory Cytokines Predict Survival in Idiopathic and Familial Pulmonary Arterial Hypertension. Circulation, 122, 920-927.
[16] Farrow, K.N., Lee, K.J., Perez, M., Schriewer, J.M., Wedgwood, S., Lakshminrusimha, S., Smith, C.L., Steinhorn, R.H. and Schumacker, P.T. (2012) Brief Hyperoxia Increases Mitochondrial Oxidation and Increases Phosphodiesterase 5 Activity in Fetal Pulmonary Artery Smooth Muscle Cells. Antioxidants & Redox Signaling, 17, 460-470.
[17] Humbert, M., Monti, G., Brenot, F., Sitbon, O., Portier, A., Grangeot-Keros, L., Duroux, P., Galanaud, P., Simonneau, G. and Emilie, D. (1995) Increased Interleukin-1 and Inter-leukin-6 Serum Concentrations in Severe Primary Pulmonary Hypertension. American Journal of Respiratory and Critical Care Medicine, 151, 1628-1631.
[18] Babu, P.B., Chidekel, A. and Shaffer, T.H. (2005) Hyperoxia-Induced Changes in Human Airway Epithelial Cells: The Protective Effect of Perflubron. Pediatric Critical Care Medicine, 6, 188-194.
[19] Alapati, D., Rong, M., Chen, S., Hehre, D., Hummler, S.C. and Wu, S. (2014) Inhibition of ß-Catenin Signaling Improves Alveolarization and Reduces Pulmonary Hypertension in Experimental BPD. American Journal of Respiratory Cell and Molecular Biology, Epub ahead of print.
[20] Ledur, A., Fitting, C., David, B., Hamberger, C. and Cavaillon, J.M. (1995) Variable Estimates of Cytokine Levels Produced by Commercial ELISA Kits: Results Using International Cytokine Standards. Journal of Immunological Methods, 186, 171-179.

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