title: Methods for detection of immune-mediated neutropenia in horses, using antineutrophil serum of rabbit origin.
abstract: Equine neutrophil antibody was raised in rabbits inoculated with equine neutrophils isolated to purity greater than 99.0%, using Percoll density-gradient sedimentation. Neutrophil antibody was detected by use of agar gel diffusion, leukoagglutination, indirect immunofluorescence, staphylococcal protein A and streptococcal protein G binding, and phagocytic inhibition techniques. Precipitin lines and leukoagglutination were seen in antiserum dilutions of 1:4 and 1:64, respectively. The specific nature of leukoagglutination was characterized by the formation of rosette-like clumps of neutrophils. Specific bright membranous fluorescence was seen in neutrophils treated with the antiserum and exposed to fluorescein-conjugated goat anti-rabbit immunoglobulin, and staphylococcal protein A and streptococcal protein G. Whereas the indirect immunofluorescence and protein G-binding tests were equally sensitive and resulted in titer of 1:256, the protein A-binding test was less sensitive and resulted in titer of only 1:32. Nonspecific binding of protein A and protein G was noticed as uniform or patchy cellular fluorescence in a small number of neutrophils. Treatment of neutrophils with antiserum up to dilution of 1:8 resulted in a significant (P less than 0.05) suppression of phagocytosis of opsonized zymosan particles. Thus, protein G-binding and indirect immunofluorescence tests are highly sensitive to detect neutrophil antibody and may be used to diagnose immune-mediated neutropenias in horses and, possibly, in other animal species.
authors.affiliation: Department of Clinical Pathology, School of Veterinary Medicine, University of California, Davis 95616.
authors.forename: N C
authors.initials: NC
authors.lastname: Jain
authors.forename: J L
authors.initials: JL
authors.lastname: Vegad
authors.forename: C S
authors.initials: CS
authors.lastname: Kono
mesh_terms: D000380:Agranulocytosis / Q000662:veterinary*; D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000276:immunology; D005455:Fluorescent Antibody Technique / Q000662:veterinary; D006734:Horse Diseases / Q000276:immunology*; D006736:Horses; D005779:Immunodiffusion / Q000662:veterinary; D009419:Nerve Tissue Proteins; D009503:Neutropenia / Q000276:immunology / Q000662:veterinary*; D009504:Neutrophils / Q000502:physiology; D010587:Phagocytosis; D013205:Staphylococcal Protein A
chemical_list: D000906:Antibodies; C029152:G-substrate; D009419:Nerve Tissue Proteins; D013205:Staphylococcal Protein A