Detection of Antinuclear Antibodies by Indirect Immunofluorescence in Dog Sera: Comparison of Rat Liver Tissue and Human Epithelial‐2 Cells as Antigenic Substrate

Abstract

Rat liver sections and a human epithelial cell line (HEp-2) were compared as substrates for the detection of antinuclear antibodies (ANA) in the serum of normal dogs and dogs with suspected autoimmune disease, using a standard indirect immunofluorescence (IIF) technique. Antibody reactivity against rat hepatocyte nuclei was frequently found at low serum dilutions in normal dog sera. Using rat liver sections, a minimum significant positive titer, allowing negativity in more than 95% of normal dog sera, was found to be 1/100. With this titer, ANA positivity could be verified in 64 of 112 (57%) reanalysed serum samples from dogs with suspected autoimmune disease, earlier determined as ANA-positive. No reactivity against nuclei of HEp-2 cells was observed in any of the normal dog sera analyzed at a screening dilution of 1/25. Using this dilution as a minimum significant positive titer, 63 of the 112 (56%) re-evaluated serum samples were positive. These 63 samples were from the same dogs as the 64 samples that were positive on rat liver sections. Thus, the 2 methods of ANA-IIF detected a nearly identical population of dogs with suspected autoimmune disease once the level of significance of a positive titer was adjusted to > 95% specificity for each method. HEp-2 cells were found to be superior to rat liver cryostate sections as ANA substrate because of their low reactivity with normal sera, and the ease of discernment of the ANA fluorescence pattern. The recognition and documentation of specific pattern types may give clues to ANA subspecificities, which could prove useful if they are found to correlate with well-defined subgroups of immune mediated clinical diseases in dogs.

Immune-Mediated Fever in the Dog. Occurrence of Antinuclear Antibodies, Rheumatoid Factor, Tumor Necrosis Factor and Interleukin-6 in Serum

“…The serum dilution 1:20 was chosen on the basis of positive reactions in 70/230 sera (31.3%) from dogs mainly with signs of systemic disease and 0/45 sera from healthy controls, both groups comprising different breeds. This corresponds well with what has been published by [11]. …”

Subgroups of canine antinuclear antibodies in relation to laboratory and clinical findings in immune‐mediated disease

“…The IIF‐ANA test is evaluated by fluorescence microscopy, in which different nuclear fluorescence patterns may allow subdivision of the results into different groups 4,5. In dogs, studies have shown that the use of monolayers of human tissue culture cells (eg, human epithelial‐2 [HEp‐2] cells) as a substrate in the assay makes it possible to divide ANA‐positive results into different subgroups 7,8. In humans, further determination of the specificity of IIF‐ANA–positive sera is used frequently to characterize the ANA reactivity.…”

“…A commonly used method for such determination is the Ouchterlony immunodiffusion test 9,10. In ANA‐positive dogs, different immunodiffusion subgroups could be correlated to different ANA subgroups 7,8…”

Serum C-reactive protein concentrations in Nova Scotia Duck Tolling Retrievers with immune-mediated rheumatic disease

“…Serum samples from IMRD dogs had been collected in connection with other studies [2, 17, 18] and previously tested for the presence of ANA with IIF [19]. Samples were considered positive at a titre of ≥1:100.…”