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Transferring Luminex® cytokine assays to a wall-less plate technology: Validation and comparison study with plasma and cell culture supernatants.
- Source :
-
Journal of immunological methods [J Immunol Methods] 2017 Jan; Vol. 440, pp. 74-82. Date of Electronic Publication: 2016 Nov 11. - Publication Year :
- 2017
-
Abstract
- Luminex® technology provides a powerful methodology for multiplex cytokine detection but remains constrained by high costs and a minimum of 25-50μL sample volume requirement per assay-well often hindering analysis of limited biological samples. Here we compare the results of Luminex-based cytokine multiplexing assay performed using conventional 96-well microtiter plates and a particular 96-well wall-less plate based on Droparray® technology ("DA-Bead"). The application of the DA-Bead plate allows 80% reduction of sample and reagent volume, thus an opportunity for significant cost savings in Luminex reagents with no change to the workflow. To compare the DA-Bead method to the conventional method, two different types of samples were tested with two different commercially available Luminex kits and the results for each method were compared. The first type was splenocyte culture supernatants from murine spleens which were harvested from mice immunized with Ascaris suum protein As24 and followed by cell stimulation ex vivo at various time points with this same antigen. Cytokine levels in these supernatants were evaluated using a Bio-Plex® T <subscript>H</subscript> 1/T <subscript>H</subscript> 2 8-plex kit. The second sample type was plasma from mice from an experimental autoimmune encephalomyelitis (EAE) study, and these samples were evaluated using a Milliplex® T <subscript>H</subscript> 17 25-plex kit. The data showed that the DA-Bead method for analysis was comparable to, if not superior to, the conventional method in terms of consistency/precision, accuracy, sensitivity and dynamic range and these results are not specific to sample type, reagents, or commercial vendor.<br /> (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Subjects :
- Animals
Antigens, Helminth administration & dosage
Biomarkers metabolism
Cells, Cultured
Culture Media, Conditioned metabolism
Cytokines blood
Encephalomyelitis, Autoimmune, Experimental blood
Enzyme-Linked Immunosorbent Assay methods
Equipment Design
Female
Helminth Proteins administration & dosage
Immunization
Mice, Inbred BALB C
Reproducibility of Results
Spleen metabolism
Time Factors
Workflow
Antigens, Helminth immunology
Ascaris suum immunology
Cytokines metabolism
Encephalomyelitis, Autoimmune, Experimental immunology
Enzyme-Linked Immunosorbent Assay instrumentation
Helminth Proteins immunology
Spleen immunology
Subjects
Details
- Language :
- English
- ISSN :
- 1872-7905
- Volume :
- 440
- Database :
- MEDLINE
- Journal :
- Journal of immunological methods
- Publication Type :
- Academic Journal
- Accession number :
- 27840065
- Full Text :
- https://doi.org/10.1016/j.jim.2016.11.003