Liquichip m100 reader

The multiplex assay was performed as previously described^{41 (link)}. Briefly, 50 μl of maternal or cord plasma (diluted 1:500) was mixed with 50 µl pooled microspheres (3000 microspheres/antigen), incubated 1 h, washed, and then incubated with 100 µl of 2 µg/ml of R-phycoerythrin-conjugated, F(ab')₂ fragment, goat anti-human IgG (Fcγ fragment specific) (Jackson ImmunoResearch). The microspheres were washed and read in a Liquichip M100 reader (QIAGEN). Results were expressed as median fluorescence intensity (MFI). The positive control consisted of 36 plasma samples pooled from multigravidae used at 3 dilutions. Negative controls was pooled plasma from 90 individuals in the USA not exposed to malaria.

For IgG subclass assay, each plasma sample was tested in quadruplicates. After initial incubation of FV2-coupled microspheres with plasma and washing as described above, microspheres were incubated with 100 µL mouse anti-human Ab to detect specific IgG subclasses: (a) mouse anti-human IgG1 (Molecular Probes, Ref. A10630) 1:1000 dilution, (b) mouse anti-human IgG2 (Sigma, ascites HP6014 I5635) 1:1000 dilution, (c) mouse anti-human IgG3 (Sigma, clone HP6050 I7260) 1:2000 dilution, and, (d) mouse anti-human IgG4 (Sigma HP6023 I9888) 1:1000 dilution. Plates were incubated for 1 h, at 25 °C on the shaker. After washing as described above, microspheres were resuspended in 100 µl of R-phycoerythrin-conjugated donkey anti-mouse IgG H + L (Jackson 715-116-150) at 1:250 dilution. Plates were incubated for 1 h, at 25 °C on a shaker, washed as before and resuspended in 100 µl PBS-1 % BSA; 85 µl of the microsphere suspension was analysed using a Liquichip M100 reader (Qiagen, Valencia, CA, USA). To test the quality of mouse-anti-human Ab, beads coupled to human monoclonal IgG1k, human monoclonal IgG2k, human monoclonal IgG3λ and human monoclonal IgG4λ were used. These beads were assayed as described above. Each sub-class was highly specific with no significant cross-reactivity with other IgG sub-classes (Additional file 2).