Development and evaluation of a flow cytometry microsphere assay to detect anti-histone antibody in dogs

Anti-nuclear antibody (ANA) is one of the diagnostic parameters that support a diagnosis of autoimmune disorders in humans, dogs, and horses, particularly the condition systemic lupus erythematosus (SLE). The most commonly used method for detecting ANA in canine serum is the indirect immunofluorescence antibody assay (IFA) that detects dog IgG with reactivity towards mammalian cell nuclei. Interpretation of the IFA results is very subjective and dependent on the source of tissue/cellular substrate. We have developed a flow cytometry based assay to detect canine serum antibodies specific to histones. Histones were chosen as the target antigen because these nuclear proteins are the most common nuclear substrate for ANA in dogs with SLE. Microsphere beads were coated with histones and incubated with canine sera. Bound anti-histone antibodies were detected by FITC-conjugated rabbit F(ab')2 anti-dog IgG. Sera from four groups of dogs (47 dogs total) were tested for anti-histone antibodies and compared with the traditional IFA assay. The groups included 15 healthy dogs, 15 dogs with noninflammatory diseases, 9 dogs with polyarthritis and positive ANA, and 8 German shepherds with perianal fistulas. The microsphere assay results indicated that only one dog in the noninflammatory group and four out of nine dogs in the polyarthritis group had mean fluorescent intensity values above our established cut-off (defined as 2 S.D. above the mean of healthy controls). There was moderate agreement between the anti-histone assay and the traditional ANA (kappa statistic=0.54). Absorption of ANA positive serum with total histones dramatically diminished the fluorescent signal detected by flow cytometry and the speckled nuclear pattern observed by IFA, whereas preabsorption did not change the diffuse nuclear staining pattern. These findings indicate that the anti-histone assay will not replace the ANA test and that other nuclear proteins, such as ribonucleoproteins may contribute to the diffuse ANA patterns.