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

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 paraformaldeh yde- lysine-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, 11 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 17 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 de- tection 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 im- munostaining systems. Early uses of the ABC system were for detection of immunoglobulins, 12,19 cell mark- ers 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