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Simultaneous measurement of multiple ear proteins with multiplex ELISA assays.
Hear Res. 2011 May; 275(1-2):1-7.HR

Abstract

A recent advancement in enzyme-linked immunosorbent assay (ELISA) technology is the multiplex antibody array that measures multiple proteins simultaneously within a single sample. This allows reduction in sample volume, time, labor, and material costs, while increasing sensitivity over single ELISA. Current multiplex platforms include planar-based systems using microplates or slides, or bead-based suspension assay with microspheres. To determine the applicability of this technology for ear research, we used 3 different multiplex ELISA-based immunoassay arrays from 4 different companies to measure cytokine levels in mouse middle and inner ear tissue lysate extracts 24 h following transtympanic Haemophilus influenzae inoculation. Middle and inner ear tissue lysates were analyzed using testing services from Quansys Biosciences, Aushon Biosystems SearchLight (both microplate-based), MILLIPLEX MAP Sample (bead-based), and a RayBiotech, Inc (slide-based) kit. Samples were assayed in duplicate or triplicate. Results were compared to determine their relative sensitivity and reliability for measures of cytokines related to inflammation. The cytokine pg/ml amounts varied among the multiplex assays, so a comparison also was made of the mean fold increase in cytokines from untreated controls. Several cytokines and chemokines were elevated, the extent dependent upon the assay sensitivity. Those most significantly elevated were IL-1α, IL-1β, IL-6, TNFα, VEGF, and IL-8/MIP-2. The results of the multiplex systems were compared with single ELISA kits (IL-1β, IL-6) to assess sensitivity over the traditional method. Overall, the Quansys Biosciences and SearchLight arrays showed the greatest sensitivity, both employing the same multiplex methodology of a spotted array within a microplate well with chemiluminescent detection. They also were more sensitive than the traditional single ELISA performed with commercial kits and matched gene expression changes determined by quantitative RT-PCR. The Quansys array showed a limit of detection for ear IL-6 down to 2-4 pg/ml, indicating it is sufficiently sensitive to detect ear proteins present in low concentrations. Thus, the multiplex ELISA procedures appear suitable and reliable for the study of hearing related proteins, providing accurate, quantitative, reproducible results with considerable improvement in sensitivity and economy.

Authors+Show Affiliations

Oregon Hearing Research Center, Department of Otolaryngology-Head & Neck Surgery, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Mail Code NRC04, Portland, OR 97239-3098, USA. truned@ohsu.eduNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

21144888

Citation

Trune, Dennis R., et al. "Simultaneous Measurement of Multiple Ear Proteins With Multiplex ELISA Assays." Hearing Research, vol. 275, no. 1-2, 2011, pp. 1-7.
Trune DR, Larrain BE, Hausman FA, et al. Simultaneous measurement of multiple ear proteins with multiplex ELISA assays. Hear Res. 2011;275(1-2):1-7.
Trune, D. R., Larrain, B. E., Hausman, F. A., Kempton, J. B., & MacArthur, C. J. (2011). Simultaneous measurement of multiple ear proteins with multiplex ELISA assays. Hearing Research, 275(1-2), 1-7. https://doi.org/10.1016/j.heares.2010.11.009
Trune DR, et al. Simultaneous Measurement of Multiple Ear Proteins With Multiplex ELISA Assays. Hear Res. 2011;275(1-2):1-7. PubMed PMID: 21144888.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Simultaneous measurement of multiple ear proteins with multiplex ELISA assays. AU - Trune,Dennis R, AU - Larrain,Barbara E, AU - Hausman,Frances A, AU - Kempton,J Beth, AU - MacArthur,Carol J, Y1 - 2010/12/07/ PY - 2010/09/16/received PY - 2010/11/09/revised PY - 2010/11/29/accepted PY - 2010/12/15/entrez PY - 2010/12/15/pubmed PY - 2011/9/15/medline SP - 1 EP - 7 JF - Hearing research JO - Hear. Res. VL - 275 IS - 1-2 N2 - A recent advancement in enzyme-linked immunosorbent assay (ELISA) technology is the multiplex antibody array that measures multiple proteins simultaneously within a single sample. This allows reduction in sample volume, time, labor, and material costs, while increasing sensitivity over single ELISA. Current multiplex platforms include planar-based systems using microplates or slides, or bead-based suspension assay with microspheres. To determine the applicability of this technology for ear research, we used 3 different multiplex ELISA-based immunoassay arrays from 4 different companies to measure cytokine levels in mouse middle and inner ear tissue lysate extracts 24 h following transtympanic Haemophilus influenzae inoculation. Middle and inner ear tissue lysates were analyzed using testing services from Quansys Biosciences, Aushon Biosystems SearchLight (both microplate-based), MILLIPLEX MAP Sample (bead-based), and a RayBiotech, Inc (slide-based) kit. Samples were assayed in duplicate or triplicate. Results were compared to determine their relative sensitivity and reliability for measures of cytokines related to inflammation. The cytokine pg/ml amounts varied among the multiplex assays, so a comparison also was made of the mean fold increase in cytokines from untreated controls. Several cytokines and chemokines were elevated, the extent dependent upon the assay sensitivity. Those most significantly elevated were IL-1α, IL-1β, IL-6, TNFα, VEGF, and IL-8/MIP-2. The results of the multiplex systems were compared with single ELISA kits (IL-1β, IL-6) to assess sensitivity over the traditional method. Overall, the Quansys Biosciences and SearchLight arrays showed the greatest sensitivity, both employing the same multiplex methodology of a spotted array within a microplate well with chemiluminescent detection. They also were more sensitive than the traditional single ELISA performed with commercial kits and matched gene expression changes determined by quantitative RT-PCR. The Quansys array showed a limit of detection for ear IL-6 down to 2-4 pg/ml, indicating it is sufficiently sensitive to detect ear proteins present in low concentrations. Thus, the multiplex ELISA procedures appear suitable and reliable for the study of hearing related proteins, providing accurate, quantitative, reproducible results with considerable improvement in sensitivity and economy. SN - 1878-5891 UR - https://www.unboundmedicine.com/medline/citation/21144888/Simultaneous_measurement_of_multiple_ear_proteins_with_multiplex_ELISA_assays_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0378-5955(10)00431-4 DB - PRIME DP - Unbound Medicine ER -