Horse radish peroxidase conjugated anti mouse igg

Microtiter plates (Immulon 2HB) were coated overnight at 4°C with either the full-length protein (FL, 50 ng/well) or the repeat peptide (NANP, 20 ng/well) and washed 3 times with PBS + 0.05% Tween-20 (PBS/T). Thes-e plates were then blocked with PBS +1% Casein (PBS/C) for 1 h and washed 3 times with PBS/T. Sera were diluted 1:5,000 in (PBS/C) and serially diluted two-fold down each column of the plate in duplicates. Plates were incubated for 2 h at 22°C and washed 3 times with PBS/T. Fifty μl of 1:15000 diluted horse radish peroxidase conjugated anti-mouse IgG (Southern Biotech, Birmingham, AL) in PBS/C were added per well. After 1 h incubation at 22°C, plates were washed 4 times with PBS/T and developed by the addition of 50 μl/well ABTS peroxidase substrate system (KPL, Gaithersburg, MD) for 1 h at 22°C. The reaction was stopped by adding 50 μl of 5% SDS for 5 min, and the absorbance at 415 nm was measured using a microplate reader (Synergy 4, Biotek, Highland Park, VT). The antibody titer was calculated as the serum dilution that produced an absorbance of 1.0 optical density (O.D.) units using Gen5^TM software (Biotek).

ELISA plates (Immunosorp, Nunc, Denmark) were coated overnight with 1 μg/ml gp140 diluted in 0.1 M carbonate buffer at 4°C. Plates were then washed three times with PBS plus 0.05% Tween and blocked with 1% BSA for 1 h at room temperature. Subsequently, serum was serially diluted in PBS plus 0.05% Tween and incubated overnight at 4°C. After washing the plates three times with PBS plus 0.05% Tween, horseradish peroxidase-conjugated anti-mouse-IgG, anti-mouse-IgG1 or anti-mouse-IgG2a (all Southern Biotech, Birmingham, AL) was added and incubated for 1.5 h at room temperature. After five washes with PBS plus 0.05% Tween, the o-phenylenediamine dihydrochloride substrate (Sigma-Aldrich) was added for detection of antibodies. After 20 min, the reaction was stopped with 1 M HCl and the optical density (OD) at 490 nm was read using and ELISA reader. For calculation of endpoint titers, a cutoff value of OD 0.15 was used. Results are expressed as group means+SEM.