Determination of brush border membrane vesicle orientation using monoclonal antibodies recognizing extracytoplasmic and cytoplasmic domains of neutral endopeptidase-24.11.

A method for determination of the orientation and integrity of brush border membrane vesicles is described. The method takes advantage of the availability of two monoclonal antibodies, 23B11 and 2B12, which recognize a cytoplasmic and an extracytoplasmic domain, respectively, of the neutral endopeptidase-24.11. Specific binding of the antibodies to intact kidney brush border vesicles or to vesicles permeabilized by digitonin is detected by fluorescence using an anti-mouse immunoglobulin G-fluorescein isothiocyanate conjugate. The method allows discrimination between right side out, inside out, and unsealed vesicles. It requires limited amounts of material and can be completed the day of the brush border vesicle preparation. Application to rabbit kidney brush border membranes freshly prepared led to values of 89, 8, and 3% for right side out, inside out, and unsealed vesicles, respectively. Storage at low temperature was associated with a marked increase in the proportion of unsealed vesicles.