Fever Virus Antigen in Paraffin-Embedded Tissues A Comparison of Three Avidin-Biotin Complex Immunoenzyme Systems for Detection of African Swine

The sensitivity and specificity of 3 avidin-biotin complex (ABC) immunostaining systems were compared on paraffin-embedded tissues from African swine fever virus (ASFV)-infected pigs. Results were also compared with immunofluorescent detection on cryosections of the same tissue for optimal detection of ASFV antigen. The ABC-alkaline phosphatase (ABC-AP) and ABC-peroxidase (ABC-PO) systems were at least as sensitive as direct fluorescent antibody (FA) and 10-fold more sensitive than the ABC-glucose oxidase system. Three ABC-AP and 2 ABC-PO chromagens with different counterstains were compared. In addition, 2 fixatives, 2 biotinylation procedures, 7 endogenous peroxidase blocking regimes, 6 tissue adhesives, and 3 mounting media were compared. The ABC-AP system with a red chromagen and hematoxylin counterstaining was preferred and most closely approximated routinely stained pathologic sections. Fixation in paraformaldehydelysine-periodate fixative preserved ASFV antigen for research studies for at least 3 years. Formalin-fixed tissues retained some staining for up to 10 years. The fluorescent antibody (FA) technique for antigen localization in tissue sections has been the mainstay of microbiologic diagnostics and research for decades. An FA procedure for detection of African swine fever virus (ASFV) antigen was developed in 1966, and methods have changed little since then. Using this technique, the pathogenesis of ASFV was elucidated, and cells of the mononuclear phagocytic system were shown to be primarily infected. Recent pathogenesis studies using FA and histopathology have found monocytic cells in the paracortex of the mandibular lymph node to be the earliest cells infected. Further characterization of these cells was hampered by the inherent limitations of FA immunostaining, including poor morphology of cryosections and poor antigen detection by FA on paraffin sections. These problems have limited the usefulness of the FA technique for further characterization of these cells. The avidin-biotin complex (ABC) immunoenzyme system provides higher amplification than other immunostaining systems. Early uses of the ABC system were for detection of immunoglobulins, 12,19 cell markers 8,10,13 and hormones. 2-5,15 These antigens are often abundant and relatively stable in tissue as compared with viral antigens. We have developed an ABC system for the detection From the Foreign Animal Disease Diagnostic Laboratory, National Veterinary Services Laboratories, APHIS, USDA, Greenport, NY 11944 (Gregg, Mebus), and New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 (Schlafer). Received for publication November 27, 1992. of ASFV antigen and have used it extensively for more than 6 years. Numerous protocols and modifications have been tested, including different tissue fixation, biotin conjugation procedures, 3 different enzyme detection systems with different chromagens and counterstains, various tissue adhesives, enzyme digestion treatments, blocking agents, and mounting media. This study critically compares different ABC systems for the detection of ASFV antigen on paraffin-embedded tissues. Methods reported here summarize our extensive testing. Maximum sensitivity and intensity of specific staining has been retained with minimum background staining. Materials and methods Tissue. Spleen and mandibular lymph node tissues were selected from normal and ASFV-infected Yorkshire crossbred pigs inoculated by the oronasal route with an ASFV 1978 isolate from the Dominican Republic (DR-2) or a Portugese isolate from 1960 (Lisbon 60). Pigs were euthanized at 0, 1, 2, 3, 4, 5, and 7 days postinoculation (DPI) using an overdose of sodium barbital. Tissues were immediately fixed in paraformaldehyde-lysine-periodate (PLP) (0.01 M periodate, 0.075 M lysine, and 2% paraformaldehyde) and held at least 1 wk before processing. Frozen tissues were collected, snap frozen in cryoembedding medium, and stored at -70 C for cryostat sectioning. To assess the effects on ASFV antigen during storage in fixative, additional spleen samples were stored in 10% formalin or PLP from pigs euthanized during acute African swine fever (ASF). Samples included material from pigs infected with various isolates of ASFV kept at room temperature for 1-10 yr. Biotin-labeled anti-ASFV antiserum (B-ASFV). Hyperimmune anti-ASFV antiserum was produced in 1 pig. Four