Immunological characterization of cytochrome b9 in crude extracts from rat tissues

Rabbit antiserum against rat liver-soluble cytochrome b9 was produced. The antiserum was specific to cytochrome b9 and did not react with hemoglobin, cytochrome c, cytochrome H-450, serum, hemolysate, and tissue extracts from lung, brain, testis, and skeletal muscle. From Ouchterlony double immunodiffusion and tandem crossed immunoelectrophoresis, the antigen was found in liver, kidney, and heart tissue extracts of rat with complete identity. From crossed immunoelectrophoresis, the antiserum distinguished monomeric and dimeric forms of the antigen. Rocket immunoelectrophoresis for quantitating the antigen was developed with a minimum sensitivity of 5 ng. By using rocket immunoelectrophoresis, the concentration of the antigen in crude tissue extracts from liver, heart, and kidney was determined. The antigen was not detectable in crude extracts from lung, brain, testis, and skeletal muscle by immunological methods. Therefore, the immunological assay method was very useful for identifying and quantitating cytochrome b9 in crude extracts with high sensitivity.