Elx800 reader

These experiments were performed as previously described (25 (link)). Briefly, enzyme-linked immunosorbent assay (ELISA) plates (Nunc-Immuno) were coated overnight with 100 μl of carbonate-bicarbonate buffer (Sigma-Aldrich) per well containing 7.5 μg/ml S. Typhimurium LPS antigen (Alexis Biochemicals). Plates were washed with PBS containing 0.05% Tween 20 and blocked with 200 μl/well blocking buffer (PBS–1% bovine serum albumin [BSA]) for 1 h at 37°C. Test serum prepared at 1:20 in dilution buffer (PBS–0.05% Tween 20–1% BSA) was serially diluted 3-fold and incubated at 37°C for 1 h. After washing, 100 μl of 1:2,000 secondary goat anti-human IgG-AP antibodies (Southern Biotech) was added and incubated for 1 h at 37°C. Finally, after washing, 100 μl of SigmaFAST p-nitrophenyl phosphate substrate was added to each plate and the plate was read after 30 min using a BioTek ELx800 reader (BioTek Instruments, USA) at 405 nm.

The serum of breeder chickens was analyzed for the titers of specific antibodies against avian encaphalomyelitis virus (AEV), avian metapneumoviruses (aMPV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and avian avulavirus (AAvV-1). In turn, serum samples from broiler chickens were analyzed for the titers of antibodies against IBV, and those of broiler turkeys for the titers of specific antibodies against aMPV, hemorrhagic enteritis adenovirus (HEV), AAvV-1, and Bordetella avium (BA).
Antibody titers were determined using commercial aMPV, IBV, IBD, and AAvV-1 (Idexx, Westbrook, ME, USA) and AEV, BA, and HEV ELISA kits (Synbiotics, San Diego, CA, USA) following the producers’ guidelines. Individual stages of the ELISA test were performed using an epMotion automatic pipetting station (Eppendorf, Hamburg, Germany) and an ELx405 automatic deep well microplate washer (BioTek, Winooski, VT, USA). Results were read using an Elx800 reader (BioTek) and xCheck (Idexx) and ProFILE software (Synbiotics).

SIV-Gag and Env-specific IgG were detected in plasma using ELISA as previously described [39 (link)]. In brief, purified SIV Gag or Env antigens (Immunodx, Woburn, MA, USA) were used as coating antigen. After washing, serial four-fold dilutions of plasma in blocking buffer were added in wells. Following washing, plates were further incubated with peroxidase-conjugated goat anti-monkey IgG secondary antibodies (Exalpha Biologicals, Shirley, MA, USA) diluted in blocking buffer. After washing, all wells were further incubated with O-Phenylenediamine Dihydrochloride substrate (Sigma-Aldrich, St. Louis, MO, USA), color was developed, and reaction was stopped by adding 4N sulfuric acid. The plate was read at an optical density (OD) of 490nm using Elx800 reader (Biotek, Winooski, VT, USA). All samples were assayed in duplicates with appropriate positive and negative controls. For quantification of SIVGag or Env specific IgG responses, rhesus IgG (NIH-NHP Reagent Resource) standard was used. Nonlinear regression using a sigmoidal dose-response variable slope model was used to interpolate concentrations from the standard curve as reported earlier [39 (link)].