Figure 4

Evaluation of commercial antibodies for the intracellular detection of recombinant bovine and ovine IL-17A. The eight commercial antibodies listed in Table 1 were tested against fixed, permeabilised untransfected (UTF) CHO cells and CHO cells transfected with cDNA encoding bovIL-17A or ovIL-17A for their capacity to detect intracellular recombinant IL-17A by flow cytometry. Results are shown for one polyclonal antibody (pab) produced against bovIL-17A (A) and seven monoclonal antibodies (mabs) produced against human or mouse IL-17A (B–D). Profiles of the relevant control antibodies listed in Table 2 are included in the overlapping histograms. Events were acquired on the MacsQuant according to the gating strategy described previously (in brief) and shown in Additional file 2. Line colours representing different antibody treatments are given in parentheses: A Primary rabbit anti-bovine IL-17A pab PB0274B-100 at 1 μg/mL (A.1, red) or negative control primary anti-bovine CD34 pab (in-house) at an estimated 1 μg/mL equivalent (a, black) then detected with a secondary goat anti-rabbit alexafluor 488 at 1 μg/mL; B Directly conjugated mouse anti-human IL-17A eBio64DEC17-phycoerythrin (PE) mab (IgG1) at 2.5 μg/mL (B.1, red) and control IgG1 VPM21 mab (in-house) at an estimated 2.5 μg/mL equivalent (b, black) and detected with goat anti-mouse PE at 1 μg/mL; C Primary mouse anti-human IL-17A mabs MT44.6 (C.1, blue), MT241 (C.2, green), MT2770 (C.3, brown) and MT504 (C.4, red) [all IgG1] at 0.5 μg/mL and control IgG1 VPM21 mab (in-house) at an estimated 0.5 μg/mL equivalent (black), all detected with goat anti-mouse PE at 1 μg/mL; D Primary mouse anti-human IL-17A mabs #41809 (D.1, red) (IgG2b) and #41802 (D.2, blue) (IgG1) at 2.5 μg/mL and a mixture of control mabs VPM21 (IgG1) and VPM22 (IgG2b) at an estimated 2.5 μg/mL equivalent (d, black), all detected with goat anti-mouse PE at 1 μg/mL.