Detection of anti-equine neutrophil antibody by use of flow cytometry

Flow cytometric and conventional fluorescence microscopic methods were compared to detect heterologous (rabbit) neutrophil antibody bound to equine neutrophils. Unfixed and paraformaldehyde-fixed neutrophils were treated with normal rabbit serum or various dilutions of an antineutrophil serum. The cells were then reacted with fluorescein conjugates of goat anti-rabbit IgG, staphylococcal protein A, and streptococcal protein G. Antibody binding was evaluated by use of fluorescence microscopy and flow cytometry. Unfixed neutrophils treated with normal rabbit serum did not fluoresce, whereas many of the fixed neutrophils had distinct cytoplasmic and some membranous (nonspecific) fluorescence. Unfixed cells treated with the antiserum had localized areas (capping) of intense membrane fluorescence, whereas fixed cells had bright uniform membranous fluorescence. The intensity of specific fluorescence varied with the antiserum dilution and the conjugate. On flow cytometry, over 80% of unfixed cells treated with antiserum dilutions up to 1:1,024, 1:2,048, and 1:256 fluoresced, respectively, with anti-IgG, protein-G, and protein-A conjugates. Fixed cells generally had similar percentages of fluorescent cells, but at a higher (1-step) antiserum dilution. It was concluded that flow cytometry is more sensitive than conventional fluorescence microscopy to detect antibodies associated with equine neutrophils.