Development of a mouse-feline chimeric antibody against feline tumor necrosis factor-alpha

DOKI, Tomoyoshi; TAKANO, Tomomi; HOHDATSU, Tsutomu

dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and a transferred nitrocellulose membrane. The blot was blocked with 5% nonfat dry milk powder in TBST (20 mM Tris-HCl, pH 8.0, 0.88% NaCl and 0.05% Tween-20) for 1 hr at 37°C. Following washing, the membrane was incubated with horseradish peroxidase conjugated goat antimouse IgG (H+L chain specific) (Southern Biotechnology Associates Inc., Birmingham, AL, U.S.A.) or horseradish peroxidase conjugated goat anti-feline IgG (whole molecular) (MP Biomedicals, Santa Ana, CA, U.S.A.) for 1 hr at 37°C and then visualized in the substrate for 10 min. Neutralization test of mouse mAb 2-4 and chimeric mAb 2-4 against feline TNF-alpha using WEHI-164 cells: Neutralization test was performed as described previously [4] . Briefly, WEHI-164 cells were suspended at 1 × 10 6 cells/ml in the dilution medium containing 1 µg/ml of Actinomycin D (Sigma Aldrich, St. Louis, MO, U.S.A.) and pre-incubated at 37°C for 3 hr. Serially diluted mouse mAb 2-4, chimeric mAb 2-4 or anti feline APN mAb (mAb R-G-4, as a control for mAb 2-4) was mixed with 40 ng/ml recombinant fTNF-alpha (R&D systems, Minneapolis, MN, U.S.A., 75% cytotoxic activity against WEHI-164 cells) or ascites of cats with FIP that were used as natural feline TNF-alpha samples (final concentration of 1:8, 80% cytotoxic activity against WEHI-164 cells). The mixture was incubated at 37°C for 1 hr. Pre-incubated cells were seeded in a volume of 50 µl in the wells of a 96-well plate. Fifty microliters of the mixture was then added into each well. After incubation at 37°C for 24 hr, 10 µl of WST-8 solution (WST-8 cell proliferation assay kit; Kishida Chemical Co., Ltd., Osaka, Japan) was added, and the cells were returned to the incubator for 1 hr. The absorbance of formazan produced was measured at 450 nm with a 96-well spectrophotometric plate reader, as described by the manufacturer. The percent neutralization was calculated by the following formula: Neutralization Repeated-dose test in cats: The mAb repeated-dose test was performed referring to the method reported by Umehashi et al. [22] . Purified mouse mAb 2-4, chimeric mAb 2-4 or PBS as a control was administered to 5 specific pathogen free (SPF) cats aged 2 months. After sedation with Medetomidine (Domitor, Orion Corporation, Espoo, Finland), the SPF cats received low-(1 mg/kg) or high-dose (5 mg/kg) mAb injection into the cervical vein 5 times at 2-or 4-week intervals. Serum was collected immediately before administration. Blood pressure and pulse were measured at the forearm or root of the tail before mAb administration and 10 min after administration, using a fully automatic electronic sphygmomanometer (Pettrust, Aster Electric Co., Yokohama, Japan). The measurements were performed in triplicate. This animal experiment was performed in accordance with the Guidelines for Animal Experiments of Kitasato University (the number of approval is 14-045). SPF cats were maintained in a temperature-controlled isolated facility.