New wide dynamic range assays for quantification of anti-Leishmania IgG2 and IgA antibodies in canine serum

The aims of this study were (1) to develop and validate time resolved-immunofluorometric assays for the detection of anti-Leishmania IgG2 and IgA antibodies in canine serum and (2) to evaluate the ability of these assays to quantify different amounts of anti-Leishmania antibodies in Leishmania-seronegative and seropositive dogs, determined by a commercial ELISA assay, and between different clinical stages according to LeishVet guidelines. The analytical validation showed that the assays had a good precision with intra- and inter-assay coefficients of variation lower than 10%. In addition, the assays allowed the quantification of very low concentration of antibodies as well as demonstrated a high level of accuracy, as determined by linearity under dilution (R2 = 0.99) and recovery tests (>85%). Moreover, no cross-reactions with Ehrlichia canis, Canine Parvovirus Type 2, Anaplasma phagocytophilum, Babesia canis, Dirofilaria immitis and pyometra were found. The assays were able to detect higher values of anti-Leishmania IgG2 and IgA antibodies in seropositive dogs compared with seronegative dogs (p < 0.0001), although an overlap between groups existed in the case of IgA. In addition, significantly higher values for both antibodies were detected in LeishVet groups II (p < 0.05) and III (p < 0.01) when compared with LeishVet group I. From our study, it could be concluded that the immunofluorometric assays developed would be suitable for determination of anti-Leishmania IgG2 and IgA antibodies in serum samples with an adequate precision, analytical sensitivity and accuracy. In addition, these assays showed a wider difference in the concentration of both IgG2 and IgA antibodies between seronegative and seropositive dogs and between different clinical stages of CanL than a current commercial ELISA kit. Further studies would be recommended to evaluate the diagnostic sensitivity and specificity of these new assays as well as their application in monitoring CanL.