Bioplex 2200 system

Antiphospholipid antibodies, including anti-cardiolipin and
anti-β₂-glycoprotein1 (IgG, IgM, IgA), were analysed from
stored plasma by multiplexed bead technology (Luminex) using BioPlex 2200
system (Bio-Rad, Hercules, CA, USA) according to the specifications of the
manufacturer. Study participants with self-reported type 1 diabetes (n = 5)
were excluded.
The coefficient of variation % was < 8.0 E/mL for all isotypes. The
cut-off for anti-CL and anti-β₂GPI positivity was set at the 99th
percentile of the normal population, according to APS criteria.^{10 (link)}Antibodies to specific nuclear antigens, that is., antinuclear antibodies
(including dsDNA, nucleosomes, Smith antigen, Smith antigen
ribonucleoprotein, ribosomal P protein, ribonucleoproteins 68 and A,
Sjögren-syndrome antigen A Ro-52 and Ro-60, Sjögren’ syndrome antigen B)
were also analysed by multiplexed bead technology (Luminex) using BioPlex
2200 system (Bio-Rad, Hercules, CA, USA), in accordance with the
specifications of the manufacturer.

The immunological profile was determined in all patients by established and standardized techniques at the laboratories of clinical immunology and clinical chemistry at Karolinska University Hospital, as previously described [5 ]: e.g., antibodies to specific nuclear antigens (dsDNA, SSA-Ro52, SSA-Ro60, SSB, Sm) and phospholipids (cardiolipin immunoglobulin (Ig) G/IgM and β₂-glycoprotein1 IgG/IgM) were analyzed by multiplexed bead technology (Luminex) using BioPlex 2200 system (Bio-Rad, Hercules, CA, USA) according to the specifications of the manufacturer. Lupus anticoagulant (LA) was determined using a modified Dilute Russel Viper Venom method (Biopool, Umeå, Sweden) and Bioclot lupus anticoagulant. Complement factors C1q, C4, C3, and C3dg were all measured at Karolinska University Hospital. Complement factors C3a and the fluid-phase terminal complement complex, consisting of the components C5b, C6, C7, C8, and C9 and the S-protein were measured by sandwich ELISA as described earlier [13 (link), 14 (link)], in addition C2 concentrations were measured by electroimmunoassay [15 ]. Rheumatoid factor (RF) IgM/IgA/IgG was analyzed by enzyme immune assay using Phadia 2500 (Elia, Phadia Thermo Fisher Scientific, Uppsala, Sweden). The detection range was 0.4–≥ 214 IU/ml for RF-IgM, 0.4–200 IU/ml for RF-IgA, and 0–600 μg/ml for RF-IgG.

Serology against T. gondii was carried out on the available ethylenediaminetetraacetic acid (EDTA) frozen plasma samples obtained at each woman’s enrollment into the STOPPAM project (Fig 1), either at the first ANC visit or at the consultation following it, usually after one month. ToRC (Toxoplasmosis, Rubella, Cytomegalovirus) IgG on the BioPlex^® 2200 System (Bio-Rad) was assessed according to the manufacturer’s recommendations. It is a multiplex immunoassay for quantitative detection of anti-T. gondii and anti-rubella IgG and for qualitative detection of anti-CMV (cytomegalovirus) IgG in a single reaction from a single serum or plasma sample. Anti-rubella and anti-CMV IgG results are not presented here. A specific software associated with the BioPlex 2200 automated analyzer (Bio-Rad) was used, and T. gondii results were expressed according to the following thresholds: results ≤ 9 IU/mL were negative, those between 10–11 IU/mL were doubtful, and results ≥ 12 IU/mL were positive. In case of positive results at the inclusion, the woman was considered as immunized and no further IgG assay was performed.