IgG and IgA antibodies against the receptor binding domain (RBD) of the SARS-CoV-2 virus were analyzed with a multiplex-based human serology kit (Bio-rad, CA, USA) according to the manufacturer’s instructions. Briefly, diluted samples were incubated with coupled beads for 30 minutes at room temperature (RT), followed by incubation with secondary antibodies and streptavidin-phycoerythrin. After proper wash and resuspension of the beads, the reactions were read on a BioPlex-200 equipment (Bio-Rad), and the results were expressed as median fluorescence intensity (MFI).
Bioplex 200 equipment
Manufactured by Bio-Rad
Sourced in United States, France, Italy
The BioPlex-200 is a versatile multiplex immunoassay system designed for high-throughput protein and antibody detection. It utilizes proprietary magnetic beads coated with capture reagents to enable simultaneous quantification of multiple analytes from a single sample. The system is capable of precisely measuring a wide range of biomolecules, making it a valuable tool for various applications in research and clinical settings.
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6 protocols using bioplex 200 equipment
1
Multiplex Assay for SARS-CoV-2 Antibodies
2
Cytokine Analysis in Exercise Intervention
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An independent investigator who was blinded to the group assignment of the patients performed the cytokine analysis. At baseline, 5-mL fasting blood samples were collected 1 week before the beginning of the interventions. Post-intervention measurement of cytokines was performed 72 h after the last exercise session in the DEP group and 6 months after the basal measurement in the MD and control groups. All samples were stored in gel serum separator tubes and centrifuged at 3,000 rpm for 15 min.
Cytokine quantification was performed using the Human Cytokine kit (Millipore, Billerica, MA, United States) by means of the Luminex® technique according to the manufacturer’s instructions. Reading of plates was performed using Bio-Plex 200 equipment (Bio–Rad Laboratories, Hercules, CA, United States) with the Bio-Plex Manager Software. Cytokine concentrations were determined by evaluating the average fluorescence intensities in a calibration curve.
Cytokine quantification was performed using the Human Cytokine kit (Millipore, Billerica, MA, United States) by means of the Luminex® technique according to the manufacturer’s instructions. Reading of plates was performed using Bio-Plex 200 equipment (Bio–Rad Laboratories, Hercules, CA, United States) with the Bio-Plex Manager Software. Cytokine concentrations were determined by evaluating the average fluorescence intensities in a calibration curve.
3
Coupling Proteins and Peptides to Luminex Beads
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We described in detail in our previous works the protocol for coupling proteins and peptides to Luminex microsphere beads [12 (link),13 (link)]. In brief, recombinant spike proteins (1 μg/1.25 × 106 beads) and nucleocapsid (2 μg/1.25 × 106 beads) were covalently coupled on carboxyl functionalized fluorescent magnetic beads (Luminex Corp., Austin, TX) with the BioPlex amine coupling kit (Bio-Rad Laboratories, Marnes-la-Coquette, France) according to the manufacturer’s instructions. For each recombinant protein-coupled bead set, we used 2000 beads/μl of assay buffer. Preliminary experiments on different plasma dilutions (1/100−1/1000) showed that the dilution 1/200 gave the best signal to noise ratio. Diluted samples were incubated with coupled beads for 16 h at 4 °C. Reactions were revealed after incubation with a biotin-labeled anti-human IgG and streptavidin-R-phycoerythrin conjugate. Antigen-antibody reactions were read on BioPlex-200 equipment (Bio-Rad, Marnes-la-Coquette. France) and the results were expressed as median fluorescence intensity (MFI) per 100 beads. To determine IgG titers of a subset of samples against the different antigens tested, we performed a 2-fold serial dilution of these samples from 1/100 to 1/12,800 and tested them as described above. The titer was the highest value of reciprocal dilution factor given a signal above the cut-off.
4
Serological Assay for Ebola Antibodies
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Plasma samples were tested for each EVD survivor for Ebola virus antibodies using our previously described serological assay based on Luminex technology [27 (link)]. Recombinant proteins of NP, viral protein-40 (VP40), and GP (from Mayinga and Kissoudougou strains) from Zaire EBOV species were used, and plasma samples were tested at 1:1000 dilution as previously reported. Antigen and antibody reactions were subsequently read on BioPlex-200 equipment (BioRad, Marnes-la-Coquette, France), and the results were expressed as median fluorescence intensity (MFI) per 100 beads.
5
Multiplexed Luminex ADCD Assay
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Antibody-Dependent Complement Deposition (ADCD) was quantified using 96-well based customized multiplexed Luminex assay. Bulk IgG was purified and separated from other serum proteins using Melon Gel resin according to the manufacturer’s instructions (Thermo Scientific #45208). Purified IgG at 1:75 dilution for IgG anti-RBD and at 1:150 dilution for IgG anti-S1, -S2, -S, -NP and -TT, was incubated with antigen-coupled beads for 2 h at 37°C on 700 rpm orbital shaker. After incubation, each sample was incubated with human complement serum (Sigma #S1764) at a concentration of 1:50 at 37°C for 30 min. Samples were then incubated at RT for 30 min with biotinylated monoclonal anti-human C3d (Quidel #207) at 1 µg/ml final concentration. Finally, 1 µg/ml of Streptavidin-RPE (Prozyme #PJ31S) was added to each well and incubated at 37°C in the dark for 1 h. Complement deposition was determined on a BioPlex-200 equipment (Bio-Rad) and measured as MFI. Assays performed without IgG and with heat-inactivated human complement serum were used as negative controls.
6
Bisphenol-induced Cytokine and VEGF Secretion
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Analysis of inflammatory cytokines and cytokine-induced vascular endothelial growth factor (VEGF) was performed in the medium of trophoblast cell culture, where soluble factors can be released after bisphenols exposure. After the experimental treatments, the medium was collected in 2 mL tubes, centrifuged at 1000 × g for 10 min at 4°C, and supernatant was kept at -80°C until analysis. Secretions of IL-6 (interleukin 6), IL-8 (interleukin 8), IL-1β (interleukin 1β), TNF-α (tumor necrosis factor-α), chemokine CCL2 (CC Motif Chemokine Ligand 2), and VEGF were analysed using the immunological kit for Human Magnetic Luminex Screening Assay (Bio-techne s.r.l. Milan, Italy). The analysis was performed using the BioPlex200 equipment (BIO-RAD Laboratories srl, Milan, Italy). Sample concentrations of the different compounds were estimated through a standard curve using a 5-order polynomial curve and expressed pg/mL (Bio-Plex Manager software 5.0). Data were normalized against cell count.
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