Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion sperm

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Abstract
Transmission electron microscopy was used to confirm that a monoclonal antibody (F79.3E2; class IgGlK) was specifically localized to an antigen in the acrosomal ground substance of stallion sperm. This antibody was used to develop and validate an indirect immunofluorescent procedure to evaluate integrity of the plasma‐acrosomal membranes of stallion sperm. The concept was that primary monoclonal antibody would be “shielded” from its acrosomal antigen by an intact plasma membrane. Conversely, sperm with damaged plasma‐acrosomal membranes would exhibit green acrosomal fluorescence when viewed with an epifluorescence microscope. A lipophilic counterstain (red fluorescence) was used to insure that all sperm were visualized. Sperm in fresh‐extended or frozen‐thawed semen were incubated with hybridoma supernatant containing monoclonal antibody for 30 min at 37°C, then a second antibody (rabbit anti‐mouse IgG‐FITC) was added for 30 min at 37°C. Unbound antibody was removed by dilution and centrifugation. Sperm were resuspended in phosphate‐buffered saline containing Evan's blue as a counterstain. All sperm fluoresced bright red, regardless of the status of cell membranes, except that in cells with damaged plasma‐acrosomal membranes, the green fluorescence associated with antibody was overriding for the rostral portion. By counting fluorescent and nonflourescent “acrosomes”, the percentage of sperm with intact plasma‐acrosomal membranes was easily determined. Evaluation of five mixtures of undamaged and damaged sperm by this procedure gave a correlation of 0.91 between the percentage of damaged sperm in a mixture and the percentage of sperm with a fluorescent acrosome. Intra‐ and interassay coefficients of variability were < 6%.