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

• Published in: Veterinary immunology and immunopathology 2017-07, Vol.189, p.11-16
• Main Authors: Cantos-Barreda, Ana, Escribano, Damián, Bernal, Luis J., Pardo-Marín, Luis, Cerón, José J., Martínez-Subiela, Silvia
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Protozoan - blood; Antibodies, Protozoan - immunology; Cross Reactions; Diagnosis; Dog; Dog Diseases - blood; Dog Diseases - diagnosis; Dog Diseases - immunology; Dog Diseases - parasitology; Dogs - blood; Dogs - immunology; Dogs - parasitology; Enzyme-Linked Immunosorbent Assay - veterinary; Fluorescent Antibody Technique - methods; Fluorescent Antibody Technique - veterinary; IgA; IgG2; Immunoglobulin A - blood; Immunoglobulin A - immunology; Immunoglobulin G - blood; Immunoglobulin G - immunology; Leishmania - immunology; Leishmaniasis - blood; Leishmaniasis - diagnosis; Leishmaniasis - immunology; Leishmaniasis - veterinary; Leishmaniosis; Sensitivity and Specificity; TR-IFMA
• ISSN: 0165-2427; 1873-2534
• DOI: 10.1016/j.vetimm.2017.05.007

•TR-IFMAs to detect anti-Leishmania IgG2 and IgA in canine serum were validated.•These assays are precise, sensitive, accurate and without cross-reactivity.•IgG2 TR-IFMA better differentiate seropositive and seronegative dogs that ELISAs.•They better differentiate different clinical stages of leishmaniosis that ELISAs. 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