Detection of Ehrlichia risticii using an avidin-biotin-immunoperoxidase staining system.
An indirect immunoperoxidase procedure using a specific anti-Ehrlichia risticii monoclonal antibody and an avidin-biotin-peroxidase staining method was used to detect E. risticii antigen in infected P388D1 murine monocytes. Several different methods of cytological fixation were used, including acetone (15 min), 95% ethanol (15 min), Bouin's fixative (5 hr), and 10% buffered neutral formalin (24 hr). The E. risticii organisms were labeled effectively and identified in cells fixed with acetone and ethanol. However, infected P388D1 cells fixed in 10% formalin or Bouin's fixative required enzymatic digestion with 1.0% trypsin for 15 min at 37 C before positive results were evident. This indirect immunoperoxidase avidin-biotin staining procedure proved to be a sensitive assay for the detection of intracellular E. risticii and may be an effective diagnostic procedure for formalin-fixed paraffin-embedded tissue.
Breider
M A
MA
Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville 37901.
Callahan
G
G
Corstvet
R E
RE
D000818:Animals; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000032:analysis; D001360:Avidin; D001710:Biotin; D002460:Cell Line; D004536:Ehrlichia / Q000276:immunology / Q000302:isolation & purification*; D006734:Horse Diseases / Q000175:diagnosis; D006736:Horses; D007124:Immunoenzyme Techniques; D008264:Macrophages / Q000382:microbiology; D009000:Monocytes / Q000382:microbiology; D012288:Rickettsiaceae Infections / Q000175:diagnosis / Q000662:veterinary; D013194:Staining and Labeling; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001360:Avidin; D001710:Biotin; D014357:Trypsin