Antigenic analysis of testicular cytochromes c using monoclonal antibodies

Testicular cytochrome c (cyt ct) was isolated from testes of sexually mature, rat, mouse, rabbit, and bull, among which rat testis is highly rich in cyt ct. By fusion of NS-1 myeloma cells and spleen cells of mice immunized with rat cyt ct, 11 stable mouse hybridoma cell lines were established. Using an enzyme-linked immunosorbent assay, it was determined that 4 of the 11 anti-rat cyt ct monoclonal antibodies (McAb) did not bind to somatic cyt c (cyt cs) of vertebrates nor to cyt ct of mouse, rabbit, and bull. Four other McAb showed no binding to cyt cs but showed different patterns of cross-reactivity with these four cyt ct. Therefore, these McAb appear to be very sensitive and useful probes for the discrimination or identification of extremely similar isocytochromes c. Although the primary amino acid sequences between cyt cs of rat and mouse are identical, the antigenic structure of cyt ct of rat and mouse are clearly distinct with regard to cross-reactivity with some anti-rat cyt ct McAb. Furthermore, these McAb also reveal that the primary amino acid sequences of cyt ct, which reflect differences in the surface conformation of the molecule, are probably species specific.