Automated elisa reader

Specific IgG2, IgG4 and IgE antibodies against rPpSP32 were measured using ELISA. The optimal conditions of antigen concentration as well as sample, primary antibody and streptavidine-horseradish peroxidase dilutions were previously determined [17 (link), 24 (link)]. The wells coated with rPpSP32 were incubated with diluted serum samples (1:200) for 2 hours at room temperature. After 6 washes, biotin-conjugated anti-human IgG isotypes (Sigma) or IgE (BD Biosciences) were incubated at 37°C for 1 hour at a dilution of 1:20,000 for IgG2, IgG4 and 1:250 for IgE. After 8 additional washes, streptavidin-horseradish peroxidase diluted at 1:15,000 (Amersham, Little Chalfont Buckinghamshire, UK) was added for 30 minutes at 37°C. Antibody-antigen complexes were visualized using TMB (BD Biosciences). The absorbance was measured by an automated ELISA reader (Awareness Technology Inc.) at 450nm. The cut-off for the assays was the mean OD of negative controls plus three standard deviations.

Specific IgG anti-saliva and anti-rPpSP32 antibodies were measured by ELISA as previously described [17 (link), 24 (link)]. Briefly, the wells were coated overnight with SGE from P. papatasi or P. perniciosus (0.5 glands per well = 0.25μg/well) or rPpSP32 (2μg/ml = 0.1μg/well) in 0.1M carbonate-bicarbonate buffer (pH 9.6) at 4°C. After washing and blocking free binding sites for 1 hour at 37°C with PBS-Tween-0.5% gelatin, sera diluted at 1:200 were incubated for 2 hours at 37°C. After washing, peroxidase-conjugated anti-human IgG antibody (Sigma, St. Louis, MO) was incubated for 1 hour at 37°C. Tetramethylbenzidine (TMB) (BD Biosciences, San Diego, CA) was then used to visualize antibody-antigen complexes. The absorbance was measured at 450 nm wavelength using an automated ELISA reader (Awareness Technology Inc, Palm City, FL).