Luminex flexmap 3d

Peripheral blood samples were collected from 60 COMET patients at three time points (week 0/baseline, week 48, and week 80). Slow off-rate modified aptamers (SOMAmer^©) technology was used to measure 1,129 proteins present in blood samples at each collection time point. A small number of blood proteins in 15 of these samples were later also measured by ELISA; the concentrations of the two platforms were correlated using Pearson’s correlation coefficient.
Bronchoscopy was performed at enrollment in patients who were clinically stable and without evidence of active infection. Luminex FlexMAP 3D (Luminex Corporation, Austin, TX) technology was used to measure 29 cytokines/chemokines in the BAL samples. Samples below the lower limit of detection were set to be ½ the lowest minimum detectable concentration across the standard curves of all analytes. Before inclusion in any analyses, all BAL protein concentrations were normalized to total protein concentration as quantified by a Pierce BCA Protein Assay Kit (Pierce Protein Biology, Rockford, IL).
For more details on peripheral blood and BAL sample collection, please see the supplemental materials.

To measure the pro-inflammatory cytokines present in BALF, a Bio-Plex Pro mouse cytokine 23-plex immunoassay kit (Cat#: M60009RDPD, BioRad) was used, according to the manufacturer’s instructions. Briefly, the diluted magnetic beads were placed into the assay plate and rinsed with wash buffer. The BALF samples and standards were then added into the wells, and shaken at 850 rpm at room temperature for 30 min. After sample incubation, the plates were washed with wash buffer 3 times, then the detection antibody was added and the plates incubated for 30 min at room temperature, shaking at 850 rpm. Next, plates were washed with wash buffer 3 times, and SA-PE was added for 10 min at room temperature, shaking at 850 rpm. After this step, the plates were washed 3 times with wash buffer, and the beads were resuspended in assay buffer for reading. Results are determined via the Luminex flexmap 3d (Luminex Corp.)

Proteins were extracted from five 10 micrometers-thick cryostat sections according to the protocol of Allred et al. (38 (link)). We built and validated a customized Multiplex ELISA panel for the Luminex Flexmap 3D at Protavio (Athens, Greece), coupling different magnetic beads from Luminex with the capture antibody of the duoset ELISA from R&D Systems and Standard ABTS ELISA Development Kit from Peprotech against human INFγ, IL6, IL10, TNFα, IL4, CXCL10, IL17, IL13, CCL18, TGFβ, IL23, CXCL13, CXCL12, and CCL19. Initially, we explored the information content of these 14 markers using unsupervised dimensionality reduction (hierarchical clustering and uMap). Next, we trained machine-learning models (linear discriminant analysis, LDA) using panels of 1 to 14 markers at the time. We fitted each model following a leave-one-out cross-validation scheme. Mean Fluorescence Intensities (MFIs) were normalized (z-scores) before training the LDA models. For each panel size, the best panel was selected as the one maximizing accuracy. Models with the same accuracy were prioritized by minimizing the residual probabilities. Following Ockham’s razor, we want the simplest model that explains the data. To that end, we finally selected the optimal model by applying the elbow criterion on the generated Pareto front to select the smallest panel size that provides a good predictive ability.

The plasm molecules were quantitated by using Human Magnetic Luminex Screening Assay (R&D Systems, Minneapolis, MN, USA). The concentrations of YKL-40 in plasm were determined according to the manufacturer's instructions. Acquisition was performed on the Bio-Plex system (Bio-Rad Laboratories) with the Bio-Plex 3D reader (Luminex FlexMAP 3D) in combination with xPONENT software version 4.2 (Luminex).

After completing the study questionnaire, fasting blood samples were collected in BD Vacutainer SST tubes (Becton Dickson, Franklin Lakes, NJ, USA). Levels of circulating pro- and anti-inflammatory cytokines and chemokines were analyzed on a Luminex FlexMap 3D (Luminex Corp., Austin, TX, USA) using a bead-based multiplex panel that included: GM-CSF, IFNγ, IL10, IL12P70, IL13, IL1B, IL2, IL4, IL5, IL6, IL8, MCP1, and TNFα (EMD Millipore Corp., Billerica, MA, USA, cat. #HCYTOMAG-60K).

Antibodies in serum were quantified by custom multiplex assay (70 (link)). Briefly, fluorescently coded magnetic microspheres (Luminex) were covalently conjugated with TRO gp120 (Immune Technology), cytomegalovirus (CMV) gB (Sino Biological), herpes simplex virus 1 (HSV-1) gD (Immune Technology), and goat anti-human IgM (Millipore Sigma). Mouse serum samples were diluted 1:100 in PBS and incubated with 1,000 of each type of microsphere (Luminex) in a 384-well plate (Grenier Bio One) at room temperature for 2 h with constant agitation. The assay plate was washed with assay wash buffer (PBS, 0.05% Tween 20, 0.1% bovine serum albumin [BSA]) using an automated plate washer (Biotek). The microspheres were then resuspended in assay wash buffer containing goat anti-human IgG Fc-phycoerythrin (PE) (SouthernBiotech) or goat anti-human IgM-PE (SouthernBiotech) at 0.65 μg/mL and incubated for 1 h. The assay plate was washed again, and then the samples were analyzed using a Luminex FlexMAP 3D (Luminex), and the median fluorescent intensity (MFI) for microsphere-bound PE signal was measured. Serum antibody concentrations were interpolated from a standard curve in Prism 9 (GraphPad).

To measure the pro-inflammatory cytokines present in BALF, a Bio-Plex Pro mouse cytokine 23-plex immunoassay kit (Cat#: M60009RDPD, BioRad) was used, according to the manufacturer’s instructions. Briefly, the diluted magnetic beads were placed into the assay plate and rinsed with wash buffer. The BALF samples and standards were then added into the wells, and shaken at 850 rpm at room temperature for 30 min. After sample incubation, the plates were washed with wash buffer 3 times, then the detection antibody was added and the plates incubated for 30 min at room temperature, shaking at 850 rpm. Next, plates were washed with wash buffer 3 times, and SA-PE was added for 10 min at room temperature, shaking at 850 rpm. After this step, the plates were washed 3 times with wash buffer, and the beads were resuspended in assay buffer for reading. Results are determined via the Luminex flexmap 3d (Luminex Corp).