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

DOKI, Tomoyoshi; TAKANO, Tomomi; HOHDATSU, Tsutomu

Changes in anti-mouse antibody in mouse mAb 2-4-or chimeric mAb 2-4-injected cat serum: Immulon 4HBX ELISA plates (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) were coated overnight at 4°C with purified mouse mAb 2-4 (500 ng/100 µl/well) diluted with carbonate buffer (0.05 M, pH 9.6). After washing with phosphate buffered saline (PBS) containing 0.02% Tween-20, the plates were blocked with a blocking buffer containing 0.5% skim milk in PBS at 37°C for 60 min. Each well of the plates then received 100 µl of 200-fold diluted serum collected from mAb treated cats. After 60 min incubation at 37°C, the plates were washed, and horseradish peroxidase conjugated goat anti-feline IgG (whole molecular) was diluted to the optimal concentrations, and then, 100 µl of the dilution was added to each well of the plates. After incubation at 37°C for 30 min, the plates were washed, and each well received 100 µl of substrate solution and was incubated at 25°C for 10 min in the dark. The substrate solution was prepared by dissolving o-phenylenediamine dihydrochloride at a concentration of 0.4 mg/ml in 0.1 M citric acid and 0.2 M Na 2 HPO 4 buffer (pH 4.8) and adding 0.2 µl/ml of 30% H 2 O 2 . The reaction was stopped with 3 N H 2 SO 4 solution, and the optical density (OD) at 492 nm was determined.