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. Author manuscript; available in PMC: 2021 Jun 11.
Published in final edited form as: Cell Host Microbe. 2019 Jun 18;26(1):100–113.e8. doi: 10.1016/j.chom.2019.05.003

Figure 3. A Pathogenic, APS-Derived β2GPI R39-R43-Specific Autoantibody Cross-Reacts with R. int DNA Methyltransferase.

Figure 3.

APS-derived monoclonal antibodies P1-117 and P2-6 were cloned as full-length human IgG1 and purified. Full-length mature rhβ2GPI, a mutant version containing alanines in positions R39-R43 (rhβ2GPIΔ39-43), R. int DNA methyltransferase (R. int DNMT, WP_118597735.1), and a mutant version containing alanines in positions R122-R126 (R. int DNMTΔ122-126) were expressed and purified.

(A) Representative ELISA titration curve showing P1-117 binds to R39-R43 within domain I (DI) of β2GPI.

(B) Representative R. int ELISA showing P1-117, P2-6 negative control (binding domain I outside R39-R43), R. int-immunized, and sham-immunized mouse sera, respectively. X-axis shows serum dilutions (1:100 serum = 10 μg/mL antibody) in 2-fold dilutions.

(C) Representative ELISA titration curve of P1-117 and P2-6 binding to R. int DNMT or R. int DNMTΔ122-126, respectively.

(D) Human trophoblast cells were treated with media, P1-117 (50 μg/mL) or IgG isotype control (50 μg/mL). After 48 h, cell migration was measured (n = 4, *p < 0.05).

(E and F) Human endometrial endothelial cells (red) were plated on Matrigel overnight to allow tube formation after which trophoblast cells (green) were added with media alone, P1-117 (50 μg/mL) or IgG isotype control (50 μg/mL), and then co-cultured for 48 h.

(E) Representative images from each experiment (n = 3) are shown.

(F) Number of tubes counted per field (n = 3, *p < 0.05). Panels A–C, two-way ANOVA with Bonferroni correction. D, one-way ANOVA with Tukey’s multiple comparison test. E, Friedman test with Dunn’s multiple comparison test. Error bars represent ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001.