Preparation and biological activity of the monoclonal antibody against the second extracellular loop of the angiotensin II type 1 receptor

Abstract

The current study was to prepare a mouse-derived antibody against the angiotensin II type 1 receptor (AT1-mAb) based on monoclonal antibody technology, to provide a foundation for research on AT1-AA-positive diseases. Balb/C mice were actively immunized with the second extracellular loop of the angiotensin II type 1 receptor (AT-ECII). Then, mouse spleen lymphocytes were fused with myeloma cells and monoclonal hybridomas that secreted AT1-mAb were generated and cultured, after which those in logarithmic-phase were injected into the abdominal cavity of mice to retrieve the ascites. Highly purified AT1-mAb was isolated from mouse ascites after injection with 1 × 107 hybridomas. A greater amount of AT1-mAb was purified from mouse ascites compared to the cell supernatant of hybridomas. AT1-mAb purified from mouse ascites constricted the thoracic aorta of mice and increased the beat frequency of neonatal rat myocardial cells via the AT, identical to the effects of AT1-AA extracted from patients' sera. Murine blood pressure increased after intravenous injection of AT1-mAb via the tail vein. High purity and good biological activity of AT1-mAb can be obtained from mouse ascites after intraperitoneal injection of monoclonal hybridomas that secrete AT1-mAb. These data provide a simple tool for studying AT1-AA-positive diseases.