Luminex 200 instrument system

Magplex microspheres (region 45) were conjugated to mouse anti-human IgG1 (ThermoFisher) using xMAP Ab Coupling kit, as per the manufacturer’s instructions, and blocked with 1% BSA in PBS overnight. Then, 50 µL microspheres and 50 µL diluted recombinant IgG, purified patient IgG, or patient serum were shaken at 500 rpm in a 96-well plate for 1 h. Microspheres were washed 3 times with 1% BSA in PBS and shaken with nanobody-streptavidin-PE conjugates for 30 min. Microspheres were washed, and median fluorescent intensities were calculated using Luminex 200 Instrument System (ThermoFisher).
For other experiments, Magplex microspheres (region 45) were conjugated to S2G2F-specific clone H9 (10 µg/10⁶ beads), as per the manufacturer’s instructions, and blocked with 1% BSA overnight. Then, 50 µL microspheres and 50 µL diluted recombinant IgG were shaken at 500 rpm in a 96-well plate for 1 h. Microspheres were washed 3 times with 1% BSA in PBS and shaken with R-PE-conjugated Fab₂ goat antihuman IgG Fc (Jackson Immunoresearch) for 30 min. Microspheres were washed, and median fluorescent intensities were calculated using Luminex 200 Instrument System (ThermoFisher).

To characterize the cytokine profiles of maternal plasma, placenta, neonatal umbilical cord, cord blood and amniotic fluid specimens, the Luminex assay was performed using the Bio‐Plex Pro Human Cytokine Screening Panel (Bio‐Rad Laboratories, Hercules, CA, USA), which can detect 48 cytokines and chemokines. The data were collected on a Luminex 200 Instrument System and analysed using Luminex xPONENT (Thermo Fisher Scientific, Waltham, MA, USA). The mean cytokine level in uninfected controls was considered the baseline and the cytokine levels in the specimens were expressed relative to the baseline level and termed "relative secretion levels". For negative controls, we used maternal plasma, placenta, umbilical cord, cord blood and amniotic fluid donated by three, four, two, two and three uninfected pregnant women at delivery respectively.

Cytokine levels were measured using the ProcartaPlex 12-Plex Assay for Mouse (ThermoFisher) (GROα, IL-1^®, IL-6, IL-10, IL-12p70, IP-10, MCP-1, MIP-1α, MIP-1^®, MIP-2α, RANTES, and TNF-α) with the Luminex^™ 200^™ Instrument System (ThermoFisher) located at the University of Washington Histology and Imaging Core.