Anti-Human AQP4 Recombinant Antibody (D15107) (CAT#: TAB-0977CLV)

Figure 1 Binding of the mAbs to hAQP4 M23 expressed on the envelope of budded baculovirus.

Time course of binding of 300 ng · mL−1 of C9401, D12092 and D15107 at room temperature. Bound antibodies were measured by ELISA and estimated as percent of maximal binding of D15107 at each temperature.

Miyazaki‐Komine, K., Takai, Y., Huang, P., Kusano‐Arai, O., Iwanari, H., Misu, T., ... & Fujihara, K. (2016). High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin‐4 competing with the neuromyelitis optica autoantibody, NMO‐IgG. British journal of pharmacology, 173(1), 103-114.

Figure 2 Binding of the mAbs to hAQP4 M23 expressed on the envelope of budded baculovirus.

Time course of binding of 300 ng · mL−1 of C9401, D12092 and D15107(blue) at 4°C. Bound antibodies were measured by ELISA and estimated as percent of maximal binding of D15107 at each temperature.

Miyazaki‐Komine, K., Takai, Y., Huang, P., Kusano‐Arai, O., Iwanari, H., Misu, T., ... & Fujihara, K. (2016). High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin‐4 competing with the neuromyelitis optica autoantibody, NMO‐IgG. British journal of pharmacology, 173(1), 103-114.

Figure 3 Binding of the mAbs to hAQP4 M23 expressed on the envelope of budded baculovirus.

Concentration–response binding of C9401, D12092 and D15107(blue) at 4°C overnight. Bound antibodies were measured by ELISA and estimated as percent of maximal binding of D15107.

Miyazaki‐Komine, K., Takai, Y., Huang, P., Kusano‐Arai, O., Iwanari, H., Misu, T., ... & Fujihara, K. (2016). High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin‐4 competing with the neuromyelitis optica autoantibody, NMO‐IgG. British journal of pharmacology, 173(1), 103-114.

Figure 4 Binding of the mAbs to AQP4 expressed in stable CHO cell lines.

Concentration-dependent binding of D15107 to fixed CHO cells expressing M23 alone (green) or M1 alone (blue) at 4°C overnight, or live hAQP4(red) M1 37°C for 1 h.

Miyazaki‐Komine, K., Takai, Y., Huang, P., Kusano‐Arai, O., Iwanari, H., Misu, T., ... & Fujihara, K. (2016). High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin‐4 competing with the neuromyelitis optica autoantibody, NMO‐IgG. British journal of pharmacology, 173(1), 103-114.

Figure 5 Live imaging of binding of fluorescent-labelled mAbs to hAQP4‐M1 cells.

Cells were treated with 10µg/ml Alexa-Fluor-555-labelled C9401, D12092 or D15107, and fluorescence of eGFP expressed in hAQP4-M1 cells and mAbs was observed 24 h after incubation.

Miyazaki‐Komine, K., Takai, Y., Huang, P., Kusano‐Arai, O., Iwanari, H., Misu, T., ... & Fujihara, K. (2016). High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin‐4 competing with the neuromyelitis optica autoantibody, NMO‐IgG. British journal of pharmacology, 173(1), 103-114.

Figure 6 Effect of mAbs on levels of AQP4 in hAQP4-M1 cells.

A typical blot is shown.

Miyazaki‐Komine, K., Takai, Y., Huang, P., Kusano‐Arai, O., Iwanari, H., Misu, T., ... & Fujihara, K. (2016). High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin‐4 competing with the neuromyelitis optica autoantibody, NMO‐IgG. British journal of pharmacology, 173(1), 103-114.

Figure 7 Effect of mAbs on levels of AQP4 in hAQP4-M1 cells.

hAQP4-M1 cells were treated with 5 µg/mL of C9401 (lanes 3 and 4), D15107 (lanes 5 and 6) or mAQP4‐specific E5415B (lane 2) in the absence (lanes 2, 3 and 5) or presence (lanes 4 and 6) of 100 nM bafilomycin A1 at 37°C for 24 h. Amounts of eGFP and AQP4 were determined by Western blotting and estimated as fold relative to non‐treated cells (lane 1).

Miyazaki‐Komine, K., Takai, Y., Huang, P., Kusano‐Arai, O., Iwanari, H., Misu, T., ... & Fujihara, K. (2016). High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin‐4 competing with the neuromyelitis optica autoantibody, NMO‐IgG. British journal of pharmacology, 173(1), 103-114.

Figure 8 Binding of chimeric mAbs to hAQP4 M23 expressed on the envelope of budded baculovirus.

Concentration-dependent binding of original and chimeric D15107 to hAQP4 M23 expressed on the envelope of BV were determined by ELISA at 4°C overnight and shown as percent of maximal binding of each antibody.

Miyazaki‐Komine, K., Takai, Y., Huang, P., Kusano‐Arai, O., Iwanari, H., Misu, T., ... & Fujihara, K. (2016). High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin‐4 competing with the neuromyelitis optica autoantibody, NMO‐IgG. British journal of pharmacology, 173(1), 103-114.

Figure 9 Cytoprotective effect of chimeric D15107 against CDC induced by NMO-IgG.

hAQP4-M1/M23 cells were treated with either rabbit complement (RC), 2 or 10 µg/mL of chimeric D15107 (C-D15107), or 200–500 µg/mL of the purified IgG fractions from different patients and their combinations as indicated at 37°C for 2 h. Cell mortalities were determined by LDH assay and estimated as fold relative to non-treated cells (control).

Miyazaki‐Komine, K., Takai, Y., Huang, P., Kusano‐Arai, O., Iwanari, H., Misu, T., ... & Fujihara, K. (2016). High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin‐4 competing with the neuromyelitis optica autoantibody, NMO‐IgG. British journal of pharmacology, 173(1), 103-114.

Figure 10 Injection (i.p. or intra-cerebral) of chimeric D15107 into mice.

Serum levels of chimeric D15107 in mice after i.p. injection (n = 3 for each time point) determined by ELISA. Concentrations of the antibody detected with secondary anti‐human IgG antibody were below the detectable level in mice injected with PBS (n = 4).

Miyazaki‐Komine, K., Takai, Y., Huang, P., Kusano‐Arai, O., Iwanari, H., Misu, T., ... & Fujihara, K. (2016). High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin‐4 competing with the neuromyelitis optica autoantibody, NMO‐IgG. British journal of pharmacology, 173(1), 103-114.

Figure 11 Binding properties of four established monoclonal antibodies using flow cytometry.

Established anti-AQP4 extracellular domain antibodies, including C9401 (a–d), D12092 (e–h), D15107 (i–l), and D15129 (m–p), were added to CHO cells transiently transfected with either hAQP4 M1 (a, e, i, and m), hAQP4 M23 (b, f, j and n), mAQP4 M1 (c, g, k, and o), or mAQP4 M23 (d, h, l, and p) cDNA connected to EGFP cDNA with IRES, and bound antibodies were estimated with flow cytometry.

Miyazaki, K., Abe, Y., Iwanari, H., Suzuki, Y., Kikuchi, T., Ito, T., ... & Ikeshima-Kataoka, H. (2013). Establishment of monoclonal antibodies against the extracellular domain that block binding of NMO-IgG to AQP4. Journal of neuroimmunology, 260(1-2), 107-116.

Figure 12 Effect of amino acid substitutions in the extracellular loops of hAQP4 on binding of monoclonal antibodies.

C9401 (a–d), D12092 (e–h), D15107 (i–l), and D15129 (m–p) were added to CHO cells transiently transfected with either wild-type hAQP4 M23 (a, e, i, and m), T62S/K64N-hAQP4 M23 (b, f, j and n), M149T-hAQP4 M23 (c, g, k, and o), or E228A-hAQP4 M23 (d, h, l, and p) cDNA connected to EGFP cDNA with IRES, and bound antibodies were estimated with flow cytometry.

Miyazaki, K., Abe, Y., Iwanari, H., Suzuki, Y., Kikuchi, T., Ito, T., ... & Ikeshima-Kataoka, H. (2013). Establishment of monoclonal antibodies against the extracellular domain that block binding of NMO-IgG to AQP4. Journal of neuroimmunology, 260(1-2), 107-116.

Figure 13 The monoclonal antibodies have no effect on water permeability of AQP4.

The functional assay of hAQP4 incorporated into liposomes. Water permeability was estimated by the functional assay using liposomes incorporating His-tagged hAQP4 M23 in the presence or absence of the monoclonal antibodies D12092 and D15107.

Miyazaki, K., Abe, Y., Iwanari, H., Suzuki, Y., Kikuchi, T., Ito, T., ... & Ikeshima-Kataoka, H. (2013). Establishment of monoclonal antibodies against the extracellular domain that block binding of NMO-IgG to AQP4. Journal of neuroimmunology, 260(1-2), 107-116.

Figure 14 The monoclonal antibodies have no effect on water permeability of AQP4.

Western blotting of monoclonal antibodies bound to liposomes. Lysate from liposomes with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) hAQP4 treated with vehicle (lanes 1, 2), D12092 (lanes 3, 4), or D15107 (lanes 5, 6) was subjected to Western blotting using an HRP-labeled anti-mouse IgG or anti-AQP4 C-terminal antibody followed by HRP-labeled anti-rabbit IgG. His-tagged hAQP4-M23 is indicated with an arrow.

Miyazaki, K., Abe, Y., Iwanari, H., Suzuki, Y., Kikuchi, T., Ito, T., ... & Ikeshima-Kataoka, H. (2013). Establishment of monoclonal antibodies against the extracellular domain that block binding of NMO-IgG to AQP4. Journal of neuroimmunology, 260(1-2), 107-116.

Figure 15 Effect of treatment with monoclonal antibodies on the binding of NMO-IgG to cells expressing hAQP4.

NMO-IgGs (Pt1-5) were added to CHO cells stably transfected with hAQP4 M23-IRES-EGFP cDNA in the presence (black line histogram) or absence (gray zone histogram) of pretreatment with the monoclonal antibody D12092 and D15107, and bound NMO-IgGs were estimated with flow cytometry. The horizontal axis shows the antibodies binding and the vertical axis shows the cell counts.

Miyazaki, K., Abe, Y., Iwanari, H., Suzuki, Y., Kikuchi, T., Ito, T., ... & Ikeshima-Kataoka, H. (2013). Establishment of monoclonal antibodies against the extracellular domain that block binding of NMO-IgG to AQP4. Journal of neuroimmunology, 260(1-2), 107-116.

Figure 16 Monoclonal antibodies replace the binding of NMO-IgG on the cell expressing AQP4.

CHO cells stably transfected with AQP4 M23-IRES-EGFP were incubated with NMO-IgG from patient 2 for 2 h (a), then they were treated with D12092 (c and f) or D15107 (d and g) in the presence of NMO-IgG and further incubated for 24 h. Cells incubated with NMO-IgG for 26 h were also examined (b and e). Bound NMO-IgG and the monoclonal antibodies were detected by Alexa Fluor 568-cinjugated anti-human IgG (a–d) and Alexa Fluor 555-conjugated anti-mouse IgG (e–g), respectively.

Miyazaki, K., Abe, Y., Iwanari, H., Suzuki, Y., Kikuchi, T., Ito, T., ... & Ikeshima-Kataoka, H. (2013). Establishment of monoclonal antibodies against the extracellular domain that block binding of NMO-IgG to AQP4. Journal of neuroimmunology, 260(1-2), 107-116.