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. 2017 Apr;443:18–25. doi: 10.1016/j.jim.2017.01.008

Fig. 3.

Fig. 3

Successful depletion of IL-10 from ovarian carcinoma associated ascites using NHS magnetic beads for investigation of paracrine effects.

Schematic display of IL-10 depletion with αIL-10 antibody covalently bound to magnetic beads. (1) Magnetic NHS ester beads are biochemically linked to mouse IgG2b αIL-10 neutralizing antibody or isotype control antibody. The bonds between the NHS ester group on the beads and free amines of the antibody are covalent, permanently immobilizing the antibody on the bead surface. (2) Antibody-coated beads are added to ascites and incubated overnight to allow specific binding of IL-10 to its neutralizing antibody. (3) A magnet is used to remove complexes of magnetic beads, αIL-10 neutralizing antibody and IL-10. (B) Human IgG levels in ascites were measured by sandwich ELISA before (control) and after depletion αIL-10 antibody bound to magnetic NHS beads (IL-10 depleted). n = 4; Wilcoxon matched pairs test, two-tailed; ns = not significant. (C) IL-10 levels in depleted versus non-depleted control or mock-depleted ascites sample were measured by FlowCytomix bead ELISA (n = 1). The IL-10 level in the depleted sample (6.41 pg/ml) falls below assay sensitivity (27 pg/ml). (D) Monocyte-derived DC were stimulated with 1 μg/ml LPS in the presence or absence of 10% ascites from ovarian carcinoma patients which had previously been depleted of IL-10 (IL10-ΔAF) or which had undergone mock depletion (mock-ΔAF). Alternatively, DC cultures were treated with LPS and untreated ascites fluid (AF) in the presence of αIL-10 neutralizing antibody (5 μg/ml); n = 3 (3 independent experiments; monocyte-derived DC from 1 healthy donor, cultured with three ascites samples from three individual ovarian carcinoma patients). IL-6 levels were measured in cell culture supernatants by sandwich ELISA. One-way ANOVA (Friedman test with Bonferroni post-test); ***p < 0.001. (E) As in D; n = 6 (monocyte-derived DC from 3 healthy donors, cultured with two ascites samples from two individual ovarian carcinoma patients). Wilcoxon matched pairs test, two-tailed; **p < 0.01.