Peroxidase conjugated goat anti mouse igg antibody

Production of HPV 16-, 18-, and 58L1-specific antibodies was tested by enzyme-linked immunosorbent assay (ELISA) using pseudoviruses as coating antigens. Sixty microliters of pseudovirus (0.001 mg/ml) was added to each well of a 96-well plate and incubated for 16 hours at 4°C. After washing, plates were blocked with 2% (w/v) bovine serum albumin in PBS containing 0.1% Nonidet P-40 (Sigma). Serially diluted mouse sera or vaginal washings (60 ul/well) were added and incubated at room temperature for 2 hours. After washing, peroxidase-conjugated goat anti-mouse IgG antibody (1∶2000; Santa Cruz Biotechnology) or goat anti-mouse IgA antibody (1∶1000; Santa Cruz Biotechnology) was added. For color development, 1-Step Turbo TMB (3,39,5,59-tetramethyl benzidine substrate solution; Pierce, Rockford, IL, USA) was added. Endpoint titers were defined as the highest serum dilutions that resulted in an absorbance value twice that of non-immunized serum (cutoff value, 0.1) and were expressed as group means ± SDs.

AF-594 succinimidyl ester, purchased from Molecular Probes (catalogue number, A-20004), was dissolved in DMSO. The mAb against SV-A capsid protein was kindly provided by Dr M. Dauber (Friedrich Loeffler Institute, Germany). FITC goat anti-mouse IgG antibody and peroxidase-conjugated goat anti-mouse IgG antibody were purchased from Santa Cruz Biotechnology. HRP-conjugated streptavidin was obtained from Jackson Immuno Research Lab. SlowFade Gold Antifade Reagent (Invitrogen) containing DAPI was purchased from Invitrogen.

The levels of antibodies specific for HPV16L1-, -18L1, and -58L1 were measured by enzyme-linked immunosorbent assay (ELISA) as preciously described [12 (link)]. Briefly, ELISA plates were coated with 1 μg/ml of HPV16, -18, or -58 PVs. After incubation for 16 hours at 4°C, the plates were washed and blocked with 2% (w/v) bovine serum albumin. Serially diluted mouse sera or vaginal secretion samples were added and incubated for 2 hours at room temperature. After washing, the plates were incubated with peroxidase-conjugated goat anti-mouse IgG antibody (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat anti-mouse IgA antibody (1:1000; Santa Cruz Biotechnology) for 1 hour at 37°C. For color development, 1-Step Turbo TMB (3,3′,5,5′-tetramethyl benzidine substrate solution, Pierce, USA) was added. The reaction was stopped by adding 1N H₂SO₄, and the absorbance was measured at 450 nm. Endpoint titers were defined as the highest serum dilutions that resulted in an absorbance for non-immunized serum that reached a cutoff value, and were expressed as the group geometric means ± SD.