The continued presence of the 24-kDa antigen on blots after alkaline hydrolysis was demonstrated using a polyclonal antibody prepared to the 24-kDa antigen excised from the gel (Fig. 9). As an additional control, parallel blots were probed with 2B1 McAb, which has been shown to recognize a peptide epitope. Alkali treatment had no detectable effect on the ability of 2B1 McAb to recognize its antigen (Fig. 9). These results suggest that the 24-kDa antigen contains significant amounts of О-linked carbohydrates and that it is these sugar chains that constitute all, or part of, the epitope site recognized by 2D6 McAb.
Species Specificity of the 2D6 Carbohydrate Epitope
Western blots containing reduced NP-40-solubilized proteins from mouse, hamster, bull, boar, and ram spermatozoa were probed with 2D6 McAb to determine the species specificity of the О-linked carbohydrate epitope. As shown in Figure 10, a positive reaction was obtained only with rat sperm proteins. antibiotics for sale online
In both species the 2D6-reactive antigens localized to the flagellum and had a patchy appearance similar to that observed on rat cauda spermatozoa. Note that in Figure 12F, a small proportion (20-30%) of mouse caput spermatozoa stained on the head region. This antigen appears to be intra-acrosomal and disappears during epididymal maturation. Hence, there is no reaction on the Western blot shown in Figure 10, which represents cauda sperm.
FIG. 10. The O-linked carbohydrate epitope recognized by 2D6 McAb appears to be species specific, as it is only detectable in rat spermatozoa. Detergents extracts were prepared from washed cauda (rat, mouse, hamster) and ejaculated (bull, boar, ram) spermatozoa and Western blots probed with 2D6 McAb.