The Assessment of Cytokines in Dried Blood Spots (DBS) · Dried Blood Spots •Dried blood spots...
Transcript of The Assessment of Cytokines in Dried Blood Spots (DBS) · Dried Blood Spots •Dried blood spots...
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The Assessment of Cytokines in Dried Blood Spots (DBS)
Elizabeth M. Miller and Thomas W. McDade
Biomarker Network meeting May 2, 2012
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Outline
I. Inflammation, Cytokines and DBS
II. Uniplex Assay: R&D High Sensitivity ELISA for IL-6
III. Multiplex Assay: MesoScale Discovery and Quansys
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Inflammation
• Inflammation is implicated in a host of biological functions, including aging
• C-reactive protein is a broad marker of general inflammation commonly used in population studies
• More complex underlying mechanisms are involved in etiology of inflammation
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Cytokines
• Cytokines are molecules that allow immune cells to communicate
• Implicated in up- and down-regulation of inflammation
– IL-6
– IL-10
– TNF-α
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Cytokine Variation
Inflammatory cytokines vary across populations based on nutritional status, infectious and chronic disease burden, and other factors
From McDade et al. (2011). Am J Phys Anth, 146: 373-384.
Serum IL-6 Serum IL-10
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Limitations
• What is the range of inflammatory states across populations and how is it regulated?
• How do we measure the low concentrations of inflammatory cytokines associated with chronic, as opposed to acute, levels of activity?
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Dried Blood Spots
• Dried blood spots (DBS) are a population-friendly alternative to venipuncture blood collection
• A wide range of biomarkers can be assayed in DBS
– Can we add cytokines to the list?
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IL-6 ELISA
• We have completed validation of a high-sensitivity enzyme-linked immunosorbent assay for IL-6
• Uses DBS calibration materials to control for unique sample matrix
• Innovated the use of filter plates to maximize elution of DBS
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IL-6 ELISA
• Sample quantity: 4 x 3.2 mm discs per well
– 8 discs for duplicate measures
• Lower limit of detection: 0.67 pg/mL
• Precision (within-assay): 6.3%-14.6% across detection range
• Reliability (between assay): 9.4%-15.4% across detection range
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Matched Serum-DBS IL-6
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IL-6 ELISA
• This method can be performed by most wet labs with basic enzyme immunoassay equipment
– Requires specialized equipment for filter plates
• This method only quantifies one biomarker of inflammation
• We would like lower detection limits
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Multiplex Assays
• Assay multiple inflammatory cytokines at once
• Requires specialized equipment
• We are currently evaluating multiplex assay platforms for DBS
– MesoScale Discovery (MSD) electrochemiluminescence
– Quansys
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MSD
• We are finalizing a 7-plex pro-inflammatory cytokine assay protocol on MSD platform
• Lower detection limits
• Very good reliability
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LLD (pg/mL):
IL-6: 0.41
IL-10: 1.21
TNF-α: 1.1
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Conclusion
• Cytokines help us understand the regulation of inflammation across the lifespan and in disease
• DBS cytokine assays will facilitate this research across populations
• We are making exciting headway in assay methodology
• We would like to thank the Biomarker Network for supporting this project