Hrp conjugated goat anti human igg secondary antibody

PBS pre-wetted 96 well plates (Millipore) were coated overnight at 4 °C with goat anti-human IgG (H +  L) capture antibody (Jackson Immunoresearch). About 100 l IMDM was used to block each well for 2 h at 37 °C. A designated number of cells were washed with PBS and resuspended in a 2X cytokine cocktail in IMDM. We added 100 l of cells to the elispot plate directly and incubated at 37 °C in an incubator overnight. Cells were then washed away and plates were washed six times. Secreted IgG was detected by binding to HRP-conjugated goat anti-human IgG secondary antibody (Southern Biotech). Spots were developed with AEC Substrate Kit and Peroxidase (HRP) reagents (Vector Laboratories). The spots were quantified using the Cellular Technology Limited; CTL ImmunoSpot software.

A total 100 μl per well of HIV Env proteins (0.5 μg ml⁻¹ in 0.1 M NaHCO₃, pH 9.5) were adsorbed onto 96-well ELISA plates (Immulon 2HB) overnight at room temperature. Wells were washed four times in PBS+0.02% Tween 20 (ELISA wash buffer) and then blocked with 300 μl per well of PBS containing 10% milk 0.03% Tween 20 for 2 h at 37 °C followed by four washes with ELISA wash buffer. Antibodies were diluted in PBS containing 10% milk; 0.03% Tween 20 (incubation buffer) were added to the wells and incubated for 1 h at 37 °C and then washed four times in ELISA wash buffer. HRP-conjugated goat anti-human IgG secondary antibody (Southern BioTech, Birmingham, AL) was added to the wells (1:3,000 in dilution buffer) and incubated at 37 °C for 1 h and then washed four times in ELISA wash buffer. Then, 50 μl of Ultra TMB substrate (Thermo Scientific) was added to each well. After 3 min at room temperature, an equal volume of 1 N H₂SO₄ was added. The absorbance at 450 nM was measured using a Spectramax M2 plate reader.

384-well plates were coated with 2 μg/mL of antigens in 0.1 M sodium bicarbonate. Plates were stored at 4°C overnight and washed with PBS + 0.05% Tween-20 the following day. Blocking was performed with PBS + 4% (w/v) whey protein, 15% Normal Goat Serum, 0.5% Tween-20, and 0.05% sodium azide for 1 h at 25°C. After blocking plates were washed again with PBS + 0.05% Tween-20, and serial dilutions of antibodies or serum control were added. Antibodies were incubated at 25°C for 90 minutes and then plates were washed again. HRP-conjugated goat anti-human IgG secondary antibody (Southern Biotech) was used to detect binding in conjunction with TMB substrate (Sera Care Life Sciences).