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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

Steps of optimised equine fluorescent microsphere immunoassay (FMIA).
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fig0005: Steps of optimised equine fluorescent microsphere immunoassay (FMIA).

Mentions: Polyclonal capture and detection antibodies and recombinant cytokines were purchased as matched pairs if available. Coupling amounts of capture antibody protein were optimised based on manufacturer’s and literature guidelines (see Table 1 for details). Individual cytokine measurements were optimised as a single-plex for interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α before combining them to perform a six-plex, which was then further optimised. Optimisations (summarised in Table 1) included the amount of capture antibody coupled to magnetic microspheres, detection antibody concentration, incubation time and assay buffer matrix. The optimal assay buffer used was 81% distilled water, 10% Reagent Diluent (R&D systems) and 9% heat inactivated fetal calf serum (Gibco, Life Technologies, UK). For the FMIA (Fig.1), 50 μl of magnetic microspheres coupled with 20 μg/ml of capture antibody were first added to a 96 well, black, flat-bottomed plate (BioRad 171-025001) and washed twice with wash buffer (BioRad 171-025001) using a Bio-Plex Pro II wash station. To this, 50 μl of prepared recombinant standards, unknown samples, controls or blanks (assay buffer only) were added.


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)

Steps of optimised equine fluorescent microsphere immunoassay (FMIA).
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig0005: Steps of optimised equine fluorescent microsphere immunoassay (FMIA).
Mentions: Polyclonal capture and detection antibodies and recombinant cytokines were purchased as matched pairs if available. Coupling amounts of capture antibody protein were optimised based on manufacturer’s and literature guidelines (see Table 1 for details). Individual cytokine measurements were optimised as a single-plex for interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α before combining them to perform a six-plex, which was then further optimised. Optimisations (summarised in Table 1) included the amount of capture antibody coupled to magnetic microspheres, detection antibody concentration, incubation time and assay buffer matrix. The optimal assay buffer used was 81% distilled water, 10% Reagent Diluent (R&D systems) and 9% heat inactivated fetal calf serum (Gibco, Life Technologies, UK). For the FMIA (Fig.1), 50 μl of magnetic microspheres coupled with 20 μg/ml of capture antibody were first added to a 96 well, black, flat-bottomed plate (BioRad 171-025001) and washed twice with wash buffer (BioRad 171-025001) using a Bio-Plex Pro II wash station. To this, 50 μl of prepared recombinant standards, unknown samples, controls or blanks (assay buffer only) were added.

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