Journal
SHOCK
Volume 21, Issue 1, Pages 26-30Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1097/01.shk.0000101668.49265.19
Keywords
multiplex assay; cytokine inhibitors; endotoxin; proteomics; antibodies; microarrays
Funding
- NHLBI NIH HHS [HL48889] Funding Source: Medline
- NIAID NIH HHS [AI46534] Funding Source: Medline
- NIGMS NIH HHS [GM50401, GM44918] Funding Source: Medline
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R01HL048889] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R01AI046534] Funding Source: NIH RePORTER
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Cytokine and cytokine inhibitors represent important components of the inflammatory response in patients with trauma, shock, and sepsis. Many investigators wish to quantify cytokines and it would be advantageous to measure multiple cytokines in a multiplex manner to obtain an inflammatory profile rather than a single value. Using the well-accepted standard enzyme-linked immunoassay (ELISA) as a basis, a microarray immunoassay (MI) was designed to measure 16 different human cytokines simultaneously. The MI was performed by spotting antibodies on nitrocellulose pads affixed to glass slides. Detection of the mediators was performed with biotin-conjugated antibodies followed by fluorescently labeled streptavidin. All antibodies and other reagents were purchased commercially. The MI achieved a lower limit of detection that was generally similar to traditional ELISAs (approximately 4-12 pg/mL) and also had a similar coefficient of variation. In the multiplexed MI, there was no cross reactivity between mediators. To verify the utility of the MI, cytokines and cytokine inhibitors were measured in endotoxin stimulated human blood by both ELISA and MI. Virtually identical cytokine concentrations were measured by both methods. These results describe the development of a sensitive, specific and cost-effective multiplexed microarray immunoassay that produces values similar to traditional ELISAs.
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