title: Transferring Luminex® cytokine assays to a wall-less plate technology: Validation and comparison study with plasma and cell culture supernatants.
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® TH1/TH2 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® TH17 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.
authors.affiliation: Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, Baylor College of Medicine, 1102 Bates St., Ste. 550, Houston, TX 77030, United States.
authors.forename: Leroy
authors.initials: L
authors.lastname: Versteeg
authors.affiliation: Curiox Biosystems, 2 Woodlands Spectrum #03-10, Woodlands Sector I, 738068, Singapore.
authors.forename: Xavier
authors.initials: X
authors.lastname: Le Guezennec
authors.affiliation: Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, Baylor College of Medicine, 1102 Bates St., Ste. 550, Houston, TX 77030, United States.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhan
authors.affiliation: Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, Baylor College of Medicine, 1102 Bates St., Ste. 550, Houston, TX 77030, United States.
authors.forename: Zhuyun
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Merck Research Laboratories, Merck & Co., Inc., 33 Avenue Louis Pasteur, Boston, MA 02115, United States.
authors.forename: Minilik
authors.initials: M
authors.lastname: Angagaw
authors.affiliation: Merck Research Laboratories, Merck & Co., Inc., 33 Avenue Louis Pasteur, Boston, MA 02115, United States.
authors.forename: Janice D
authors.initials: JD
authors.lastname: Woodhouse
authors.affiliation: Merck Research Laboratories, Merck & Co., Inc., 33 Avenue Louis Pasteur, Boston, MA 02115, United States. Electronic address: subhabrata.biswas@merck.com.
authors.forename: Subhabrata
authors.initials: S
authors.lastname: Biswas
authors.affiliation: Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, Baylor College of Medicine, 1102 Bates St., Ste. 550, Houston, TX 77030, United States. Electronic address: beaumier@bcm.edu.
authors.forename: Coreen M
authors.initials: CM
authors.lastname: Beaumier
mesh_terms: D000818:Animals; D000947:Antigens, Helminth / Q000008:administration & dosage / Q000276:immunology*; D017165:Ascaris suum / Q000276:immunology*; D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000378:metabolism; D016207:Cytokines / Q000097:blood / Q000378:metabolism*; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000097:blood / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D005260:Female; D015801:Helminth Proteins / Q000008:administration & dosage / Q000276:immunology*; D007114:Immunization; D008807:Mice, Inbred BALB C; D015203:Reproducibility of Results; D013154:Spleen / Q000276:immunology* / Q000378:metabolism; D013997:Time Factors; D057188:Workflow
chemical_list: D000947:Antigens, Helminth; D015415:Biomarkers; D017077:Culture Media, Conditioned; D016207:Cytokines; D015801:Helminth Proteins