When parallel blots were probed with a polyclonal antibody to rat CD52, a positive reaction was obtained in the supernatant from PI-PLC treated sperm. Preextraction of cauda spermatozoa with 1 M KCl to remove loosely bound glycoproteins from the plasma membrane did not make 2D6 antigen susceptible to cleavage by PI-PLC (results not shown).
Preliminary Identification of the Epitope Recognized by 2D6 McAb: Protein versus Carbohydrate
Previous work demonstrated the presence of significant amounts of carbohydrate associated with the 24-kDa form of 2D6 antigen. Since sugar structures are well known to be highly antigenic, it was necessary to obtain infor mation regarding the nature of the epitope recognized by the McAb. For this purpose, Western blots containing cauda sperm proteins were incubated with ^-glycanase or subjected to alkaline hydrolysis followed by probing with 2D6 McAb. ^-glycanase digestion, which removes ^-linked carbohydrates from glycoproteins, had no significant effect on the binding of 2D6 McAb (Fig. 8). Alkaline hydrolysis, however, eliminated it completely (Fig. 9). buy proventil inhaler
FIG. 9. 2D6 McAb recognizes an O-linked carbohydrate epitope, as shown, by alkaline hydrolysis of Western blots containing proteins from cauda spermatozoa. Following hydrolysis 2D6 McAb no longer binds to the 24-kDa antigen (upper panel). The presence of the antigen on the blot is demonstrated by its reactivity to a PAb (middle panel). As a control, parallel blots were probed with 2B1 McAb, which recognizes a protein epitope and is unaffected by alkaline hydrolysis (bottom panel).