Hrp conjugated goat anti mouse igg and iga abs

To determine the number of Ab-secreting cells (ASCs), mononuclear cells were isolated from spleen and lung as previously described (34 (link)). Ag-specific ASCs were measured by ELISPOT. In brief, the 96-well ELISPOT MultiScreen®_HTS filter plate (Millipore) were coated with 5 μg/ml of recombinant outer-membrane protein in 50 mM sodium bicarbonate solution (pH 9.6), and incubated overnight at 4°C. After blocking with RPMI-1640 (Invitrogen) supplemented with 10% FBS, serially diluted mononuclear cells were added into each well and incubated for 4 h at 37°C in a humidified incubator providing 5% CO₂. HRP-conjugated goat-anti-mouse IgG and IgA Abs (Southern Biotechnology Associates) were added into each well and incubated for 2 h at room temperature. For color development, peroxidase substrate (amino ethyl carbazole Peroxidase Chromogen substrate kit; ImmunoBioScience Corp., Mukilteo, WA, USA) was used. The number of ASCs was counted using a stereomicroscope (Olympus).

Serum and bronchoalveolar lavage (BAL) fluid were collected 7 days after the last vaccination and specific Ab titers against OMPOT or O. tsutsugamushi were measured by ELISA. Briefly, 96-well Maxisorp plates (Nunc) were coated with 1 μg/ml of recombinant OMPOT or 1.2×10⁴ ICU/ml of O. tsutsugamushi and incubated overnight at 4°C. After blocking with PBS containing 1% BSA (Invitrogen) for 1 h at 37°C, sera and BAL fluid were serially diluted in blocking buffer to each well and incubated for 2 h at 37°C. HRP-conjugated goat anti-mouse IgG and IgA Abs (Southern Biotechnology Associates, Birmingham, AL, USA), diluted in a ratio of 1:3,000 in 0.1% BSA in PBS plus 0.05% Tween-20, were added and incubated for 1 h at room temperature. 3,3′,5,5′-Tetramethylbenzidine (Millipore, Burlington, MA, USA) were added for the color reaction and stopped by adding 50 μl of 2N H₂SO₄. The absorbance at wavelength 450 nm was measured by a microplate reader (Molecular Devices, San Jose, CA, USA) and endpoint titers were determined by the value of 0.2 and expressed as reciprocal log₂ titer by using Softmax pro version 5.4.1 (Molecular Devices).