Fig. 2.

Anti-HPV-16 and -18 IgA antibodies in tears of rabbits vaccinated with Cervarix (R2941, R2942, R2943), Gardasil (R2932, R2933) or CRPV L1 (R2938). Tears (1 : 50) from rabbits vaccinated with the human dose of Cervarix and Gardasil or 50 µg CRPV L1 VLPs three times. Standard ELISA was conducted using a 96-well plate coated with CRPV L1 VLPs or HPV-16 L1 and -18 L1 VLPs (0.5–1 µg per well) in 1× PBS buffer at room temperature for 30 min. The plate was washed with 1× PBS buffer for at least three times and blocked with 5 % dry milk/1× PBS buffer for >1 h at room temperature. Tear samples (duplicates or triplicates) were incubated with VLPs at 4 °C overnight. In-house mAbs CRPV1A (1 : 100), H16.V5 (1 : 100) and H18.J4 (1 : 100) were used as positive controls. Negative control (Neg) was without primary antibody. Tears from animals not infected with CRPV or vaccinated were also used as negative controls in our studies. After three washes, the secondary antibody (goat anti-rabbit IgA conjugated with alkaline phosphatase, 1 : 2000; Bethyl) was added and incubated for >1 h at room temperature. The plate was then washed three times with 1× PBS, developed with 1 mg ml⁻¹ of 4-nitrophenyl phosphate disodium salt hexahydrate tablet (Sigma) in alkaline phosphatase buffer (pH 9.5) and analysed at 450 nm with an Opsys MR microplate reader (Dynex Technologies). Values are given as mean±se.