ELISA Assessment of Anti-Sand Fly Antibodies

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).