Human erythrocyte pyruvate kinase (L'/R-PK): production and characterization of a monoclonal antibody.

Two preparations of L'/R-type pyruvate kinase from human erythrocytes characterized by SDS-PAGE were used for immunization of BALB/c mice. Their spleen cells were fused with mouse myeloma cells by polyethylene glycol according to standard techniques. Supernatants of hybridomas resulting from two separate experiments were assayed by ELISA and further characterized by immunoblotting. Using those monoclonal antibodies reacting with L'/R-PK in immunoblotting, a major band of 62 KDa MW was recognized in both preparations employed for immunization. Additionally, four smaller bands were detected. Furthermore, the monoclonal antibodies also detected a single band of 62 KDa MW in a highly purified L-PK prepared from human liver. In contrast, they showed no reaction with muscle and brain tissues containing M1-type PK. However, they reacted strongly with a single band of approximately 62 KDa in liver and kidney homogenates which is in line with immunocytochemical studies showing immunoreactive material in hepatocytes and proximal tubules of the kidney.