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Simultaneous detection and quantification of six equine cytokines in plasma using a fluorescent microsphere immunoassay (FMIA).

Hall SA, Stucke D, Morrone B, Lebelt D, Zanella AJ - MethodsX (2015)

Bottom Line: Cytokines are cell signalling proteins that mediate a number of different physiological responses.They are also biomarkers for inflammatory conditions and potential diagnostic references for diseases.The following pro-inflammatory and anti-inflammatory cytokines were quantified in equine plasma and serum samples: interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α. •The objective of this study was to quantify six equine cytokines simultaneously using the BioPlex(®) 200 system in equine EDTA-plasma and serum.•It demonstrates an increased number of detectable cytokines over published studies.•This technology has the advantage of reduced sample volume and assay time compared to traditional sandwich ELISAs.

View Article: PubMed Central - PubMed

Affiliation: Scotland's Rural College, Edinburgh, Scotland, United Kingdom.

ABSTRACT
Cytokines are cell signalling proteins that mediate a number of different physiological responses. They are also biomarkers for inflammatory conditions and potential diagnostic references for diseases. Until recently, simultaneous quantification of cytokine profiles had not been possible. Now however, fluorescent microsphere immunoassays (FMIA) are able to measure multiple cytokines in a single sample. The following pro-inflammatory and anti-inflammatory cytokines were quantified in equine plasma and serum samples: interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α. •The objective of this study was to quantify six equine cytokines simultaneously using the BioPlex(®) 200 system in equine EDTA-plasma and serum.•It demonstrates an increased number of detectable cytokines over published studies.•This technology has the advantage of reduced sample volume and assay time compared to traditional sandwich ELISAs.

No MeSH data available.


Related in: MedlinePlus

Bead map output from BioPlex Manager showing the classification of the individual beads coupled to the six different cytokines.
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fig0010: Bead map output from BioPlex Manager showing the classification of the individual beads coupled to the six different cytokines.

Mentions: Aliquoted plasma samples were first centrifuged at 14,000 rpm for 15 min then diluted 1:4 in assay buffer. The plate was then incubated in the dark with shaking (700 rpm) at room temperature (RT) for 110 min. The plate was washed three times as before, then 100 μl of the detection antibody (in assay buffer) was added to the wells and the plate was incubated as before for 50 min. The plate was washed three times as previously, then 50 μl of 1× Streptavidin-phycoerythrin SAPE (BioRad) was added and the plate was incubated for 30 min as before. The plate was washed for a final three times, and then 125 μl of assay buffer was added. The plate was incubated with shaking for 5 min then the reaction was measured using a Bio-Plex 200® instrument and analysed with the Bio-Plex Manager software version 6.1. During a run, the BioPlex Manager software produces a histogram and a bead map (Fig. 2), which shows graphically the identity of each bead by its classification region based on its individual fluorescence. Additionally during a run the system monitors the flow of beads, bead count, bead regions and platform temperature and will trigger a warning if any problems are detected. The system can detect the percentage of bead aggregation from the doublet discriminator gates which measures light scatter from particles which directly proportional to particle size. The mean fluorescent intensity (MFI) for 100 microspheres corresponding to each individual cytokine analyte was recorded for each well. All reported MFI measurements were background corrected (normalised, means fluorescent F-Fo), where Fo was the background signal determined from the fluorescence measurement of the blank. The standard curve was produced using a logistic 5PL regression where recovery was in the 70–130% range.


Simultaneous detection and quantification of six equine cytokines in plasma using a fluorescent microsphere immunoassay (FMIA).

Hall SA, Stucke D, Morrone B, Lebelt D, Zanella AJ - MethodsX (2015)

Bead map output from BioPlex Manager showing the classification of the individual beads coupled to the six different cytokines.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487921&req=5

fig0010: Bead map output from BioPlex Manager showing the classification of the individual beads coupled to the six different cytokines.
Mentions: Aliquoted plasma samples were first centrifuged at 14,000 rpm for 15 min then diluted 1:4 in assay buffer. The plate was then incubated in the dark with shaking (700 rpm) at room temperature (RT) for 110 min. The plate was washed three times as before, then 100 μl of the detection antibody (in assay buffer) was added to the wells and the plate was incubated as before for 50 min. The plate was washed three times as previously, then 50 μl of 1× Streptavidin-phycoerythrin SAPE (BioRad) was added and the plate was incubated for 30 min as before. The plate was washed for a final three times, and then 125 μl of assay buffer was added. The plate was incubated with shaking for 5 min then the reaction was measured using a Bio-Plex 200® instrument and analysed with the Bio-Plex Manager software version 6.1. During a run, the BioPlex Manager software produces a histogram and a bead map (Fig. 2), which shows graphically the identity of each bead by its classification region based on its individual fluorescence. Additionally during a run the system monitors the flow of beads, bead count, bead regions and platform temperature and will trigger a warning if any problems are detected. The system can detect the percentage of bead aggregation from the doublet discriminator gates which measures light scatter from particles which directly proportional to particle size. The mean fluorescent intensity (MFI) for 100 microspheres corresponding to each individual cytokine analyte was recorded for each well. All reported MFI measurements were background corrected (normalised, means fluorescent F-Fo), where Fo was the background signal determined from the fluorescence measurement of the blank. The standard curve was produced using a logistic 5PL regression where recovery was in the 70–130% range.

Bottom Line: Cytokines are cell signalling proteins that mediate a number of different physiological responses.They are also biomarkers for inflammatory conditions and potential diagnostic references for diseases.The following pro-inflammatory and anti-inflammatory cytokines were quantified in equine plasma and serum samples: interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α. •The objective of this study was to quantify six equine cytokines simultaneously using the BioPlex(®) 200 system in equine EDTA-plasma and serum.•It demonstrates an increased number of detectable cytokines over published studies.•This technology has the advantage of reduced sample volume and assay time compared to traditional sandwich ELISAs.

View Article: PubMed Central - PubMed

Affiliation: Scotland's Rural College, Edinburgh, Scotland, United Kingdom.

ABSTRACT
Cytokines are cell signalling proteins that mediate a number of different physiological responses. They are also biomarkers for inflammatory conditions and potential diagnostic references for diseases. Until recently, simultaneous quantification of cytokine profiles had not been possible. Now however, fluorescent microsphere immunoassays (FMIA) are able to measure multiple cytokines in a single sample. The following pro-inflammatory and anti-inflammatory cytokines were quantified in equine plasma and serum samples: interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α. •The objective of this study was to quantify six equine cytokines simultaneously using the BioPlex(®) 200 system in equine EDTA-plasma and serum.•It demonstrates an increased number of detectable cytokines over published studies.•This technology has the advantage of reduced sample volume and assay time compared to traditional sandwich ELISAs.

No MeSH data available.


Related in: MedlinePlus