Elisa

ELISA laboratory methods

NVN, UTAD, UAB and HUJ each performed an ELISA , either an in-house or a commercial ELISA (NVN and UTAD) . ELISA was performed in accordance with each laboratory’s specifications [ 9, 13, 33]. UTAD ELISA was performed in accordance with the Leiscan Leishmania ELISA Test protocols [33]. NVN ELISA was conducted in accordance with the MegaELISA LEISH (MEGA- COR Veterinary Diagnostics, Hörbranz, Austria) test protocols [ 34]. In summary, Leishmania antigen was prepared using total L. infantum antigen obtained from 3 × freeze-thawed stationary phase L. infantum promastigotes (strains utilized are unique to each laboratory) [ 9, 13]. Microtitre plates were coated with L. infantum antigen; antigen source and concentration varied by laboratory and in-house protocol [ 9, 13]. Plates were incubated overnight at between 2 and 8 ℃ dependent on the laboratory protocol [ 9, 13]. Canine serum was tested at different dilutions: 1:20 for UTAD, 1:800 for UAB, 1:100 for NVN and 1:1000 for HUJ. For HUJ and UAB protocols, plates were resolved via protein A conjugated to horseradish peroxidase for 1 h at 37 ℃ [ 9, 13]. Each plate was read when the positive canine reference serum’s absorbance (405 nm or 492 nm) reached a pre-determined cut-off value [ 9, 13]. Results were then determined based on each laboratory’s cut-off value: 0.4 for NVN, 0.8 for UTAD, 0.28 for UAB and 0.2 for HUJ [ 9, 13, 33].