Cerebral Vasospasm Inhibitor

Preparation of an Anti-HMGB1 Monoclonal Antibody

[0030](a) Immunization of a Rat

[0031]A commercially available 1 mg / mL mixture of bovine thymus-derived HMGB1 and HMGB2 (manufactured by Wako Pure Chemical Industries Ltd., code No. 080-070741) was taken into a 2 mL glass syringe; and was gradually mixed with an equal volume of a Freund's complete adjuvant taken into another 2 mL glass syringe via a connecting tube, to give an emulsion. Into the footpads of the hind limbs of a sevoflurane-anesthetized rat, 0.1 mL each, 0.2 mL in total, of the emulsion was injected. Blood was sampled from the cervical vein two weeks later, and the increase of the antibody titer was confirmed. Then, the swollen iliac lymph nodes were aseptically isolated 5 weeks after the injection. About 6×107 cells could be recovered from the two lymph nodes thus obtained.

[0032](b) Cell Fusion and Cloning

[0033]The iliac lymph node cells and mouse myeloma SP2 / O-Ag14(SP2) cells were fused using polyethylene glycol. The re...

[0037]One week before twelve male rabbits weighing each about 3 to 3.5 kg, obtained from Charles River Laboratories Japan Inc., were made into subarachnoid hemorrhage models, the animals were anesthetized with ketamine hydrochloride (50 mg / kg, intramuscular administration) and pentobarbital (20 mg / kg, intravenous administration). Then, a contrast medium was infused via a catheter inserted from the right femoral artery to the origin of the left vertebral artery, and the basilar artery was photographed by angiography. On the day of the preparation of a subarachnoid hemorrhage model, each rabbit was anesthetized with ketamine hydrochloride (50 mg / kg, intramuscular administration) and pentobarbital (20 mg / kg, intravenous administration), and 1 mL of arterial blood was taken. The arterial blood thus taken was injected into the cisterna magna, i.e. the cerebellomedullary cistern, of the same animal. Then, the animal was held with the head downward for 30 minutes to obtain a subarachnoid h...