Fluorescein isothiocyanate conjugated rabbit anti dog igg

Dog serum samples were tested with a commercial indirect fluorescent antibody test (IFAT) (MegaScreen®FLUO Babesia canis, MegaCor Diagnostic, Horbranz, Austria) to detect the presence of IgG antibodies against B. canis, using canine erythrocytes infected with B. canis as antigens. Positive and negative controls were always included and were provided by the company. Starting, sera were initially diluted 1:64 in phosphate-buffered saline solution (PBS) (pH = 7.2), applied to slide wells and incubated in a moist chamber for 30 min a 37 °C. The slides were washed and reacted with fluorescein isothiocyanate-conjugated rabbit anti-Dog IgG (Sigma-Aldrich, Milan, Italy), incubated at 37 °C in a humid chamber for 30 min. The slides were mounted with buffered glycerin and covered with a coverslip. Finally, the slides were examined with a microscope (Axioscop 2, Zeiss) under a fluorescent light (HBO50). Those samples that had fluorescence at a diluition ≥ 1:64 were considered as seropositive. Samples resulting seropositive were then diluted to determine the end-point titre (i.e. the last dilution in which is observed positive reaction).