Bio plex 200

The instrument used was a Bio-Plex 200 instrument (Luminex corp., Ebersberg, Germany) and data collection was performed using the Luminex xPONENT 3.1 software. The software has pre-defined detection gates for each microsphere set based on the fluorescence of two impregnated fluorescent dyes. The fluorescence emitted from bound SAPE was also detected and for each microsphere set, a median fluorescence intensity (MFI) was calculated within the software based on a minimum of 500 detectable microspheres. At least two negative controls were included for each batch of samples analyzed on the instrument, and the mean MFI of the negative controls was subtracted from the MFI obtained for each sample. Throughout the initial assay development stages, the background fluorescence did not exceed an MFI of 150, therefore, this was chosen as the minimum threshold for which a sample was considered positive.

Serum samples were stored at -80°C prior to analysis. Samples were tested by the Cytokine Reference Laboratory (University of Minnesota) using the magnetic bead set PRCYTOMAG-40K (EMD Millipore). Samples were analyzed for Non-Human Primate (NHP)-specific TNFα, IFNγ, IL-6, IL-1β and IL-2 using the Luminex platform and performed as a multi-plex. Fluorescent color-coded beads coated with a specific capture antibody were added to each sample. After incubation and washing, biotinylated detection antibody was added followed by phycoerythrin-conjugated streptavidin. The beads were read on a Luminex instrument (Bioplex 200). Samples were run in duplicate and values were interpolated from five-parameter fitted standard curves.

A series of 20-mer peptides, with 10-residue overlaps, encompassing the entire CHIKV CP were designed (Table 2) and commercially synthesized (Mocell Biotech, Hong Kong). Each peptide was coupled to carboxylated polystyrene beads using the Bio-Plex amine coupling kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The coupled beads were then multiplexed by diluting in microsphere immunoassay (MIA) buffer (1% BSA, 0.05% ProClin 300 (Supelco, Bellefonte PA, USA) in PBS) and distributed to give approximately 2500 of each beadset (~100 µL) per well of a MultiScreen filter plate (Merck Millipore, Billerica, MA, USA). Each anti-CHIKV CP mAb was then tested against the multiplexed beadsets by incubation for 45 min with shaking at room temperature. The wells were then washed three times with PBS/T wash buffer, prior to the addition of R-Phycoerythrin donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA). Following that, the wells were incubated and washed as previously, after which the beads were resuspended in MIA buffer. The Bio-Plex 200 (Luminex, Bio-Rad, Austin, TX, USA) was then utilized to read the plate, measuring the mean fluorescence intensity for each beadset in their respective wells. mAb recognition of peptides was also assessed by ELISA and dot blot as previously described [29 (link)].

A binding antibody multiplex assay (BAMA) was used to quantify HCMV glycoprotein–specific IgG binding and avidity (77 (link), 78 (link)). HCMV gB ectodomain, pentamer complex, gH/gL/gO, gH/gL, pp28, pp150, and UL44 antigens were covalently coupled to intrinsically fluorescent beads (Bio-Rad). Maternal and cord blood sera were diluted at 1:500 (for gB ectodomain, pentamer complex, gH/gL/gO, and gH/gL) and at 1:25 (for pp28, pp150, and UL44) in assay diluent, plated in duplicate, then coincubated with antigen-coupled beads. Antigen-specific IgG binding was detected with mouse anti-human IgG–PE (SouthernBiotech), and MFI was acquired on a Bio-Plex 200 (Luminex). For determination of avidity, duplicate wells were incubated with sodium citric acid (pH 4.0) or 1× PBS (pH 7.4) between the primary and secondary incubations, and RAI was calculated as (MFI with sodium citric acid)/(MFI with PBS) × 100%. A serial dilution of HCMV-hyperimmuneglobulin (Cytogam) was included as a positive control, and the cutoff for positivity was determined by calculating the mean MFI of seronegative serum binding plus 3 SDs. Blank beads and wells were included to account for background signal. Duplicates with CVs greater than 25% were repeated.