Nitrophenyl phosphate liquid substrate system

Specific IgG antibodies to P. kandelakii saliva were assessed by ELISA. Ninety-six-well high binding microtiter plates (Thermo scientific, Rochester, NY, USA) were coated with 50 µl of SGH diluted to (1 pair/ml) in 0.1 M carbonate-bicarbonate buffer overnight at 4 °C. The wells were then washed in Tris-buffered saline (TBS) added with 0.05%Tween 20 and incubated with TBS–4% bovine serum albumin (BSA) for 1 hour at room temperature (RT) to block free binding sites. After three washes 50 µl of diluted Plasma (1:100) were added and incubated for 1 hour at 37 °C. Antibody-antigen complexes were detected using alkaline phosphatase-conjugated goat anti-human IgG (H + L) antibodies (Sigma, St Louis, MO) diluted at 1:5000 for 1 hour at RT and were visualized using nitrophenyl phosphate liquid substrate system (Sigma). The absorbance was measured at 405 nm using a Versamax microplate reader (Molecular devices, San Jose, CA). The cut-off for the assays was the mean optical density obtained with sera of eight negative controls plus three standard deviations.

Specific anti-P. duboscqi saliva IgG antibodies were assessed by ELISA. A 96-well high binding microtiter plate (Thermo Scientific, Rochester, NY) was coated with 50 μl of SGH diluted to one pair/ml in 0.1 M carbonate-bicarbonate buffer overnight at 4°C. The wells were then washed in Tris-buffered-saline (TBS) with 0.05% Tween 20 and incubated with TBS-4% Bovine Serum Albumin (BSA) for one hour at room temperature (RT) to block free binding sites. After three washes, 50 μl of 1:100 diluted sample plasma was added and incubated for 1 hour at 37°C. Antibody-antigen complexes were detected using alkaline phosphatase-conjugated goat anti-human IgG (H+L) antibodies (Sigma, MO) diluted at 1:5000 for 1 hour at RT and were visualized using nitrophenyl phosphate liquid substrate system (Sigma). The absorbance was measured at 405 nm using a Versamax microplate reader (Molecular devices).