Hrp conjugated goat anti human igg fc antibody

RSC3/ΔRSC3 proteins dissolved at a concentration of 1 μg/ml in phosphate-buffered saline (PBS, pH 7.4) were used to coat in 96-well ELISA plate at 100 μl/well overnight at 4 °C. Coated plates were blocked with blocking buffer (PBS, pH 7.4, plus 2% BSA and 5% milk) at 200 μl/well for 1 hr at room temperature, followed by incubation with plasma or IgGs/antibody serially diluted in PBST buffer (0.05% Tween 20 in PBS) at 100 μl/well for 1 hour at room temperature. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at 1:10,000 was added for 1 hr at room temperature at 100 μl/well. Plates were washed between each step with PBST buffer at 100 μl/well. Plates were developed using 100 μl of the substrate (KPL SureBlue TMB 1-Component Microwell Peroxidase Substrate, cat#: 52-00-02) and stopped with 50 μl 1 N H₂SO₄. Absorption at 450 nm was read on an automated plate reader (Multiscan Ascent, Thermo Corporation, Finland).

30 µl of supernatant from each well of the 96-well microplate was mixed with 70 µl of PBS and incubated in each well of a Nickel coated 96-well ELISA plate (Thermo Scientific, IL) for two hours at room temperature (RT) or overnight at 4°C. The well was washed once with wash buffer, and then incubated with 100 µl of respective anti-HIV-1 antibody at a concentration of 10 µg/ml in PBS+0.05% Tween 20+0.2% w/v dry milk for 1 hour at RT. After 5-time wash with wash buffer, the wells were incubated with 100 µl of Horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc antibody (Jackson ImmunoResearch Laboratories Inc., PA) at 1∶10,000 in PBS+0.05% Tween 20+1% w/v dry milk for 30 min at RT. The wells were then washed 5 times with wash buffer and developed using TMB at RT for 10 min before addition of 180 mM HCl. The readout was measured at a wavelength of 450 nm. All samples were performed in duplicate.

MaxiSorp™ 96-well ELISA plates (Nunc, Rochester, NY, USA) were coated with 100 μl per well of carbonate-bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) buffer containing mouse anti-EpCAM mAb (anti-GA733 mAb) (R&D Systems, Minneapolis, MN, USA) (50 ng) and were incubated overnight at 4 °C. The plates were washed four times with 1× PBS-T [1× PBS plus 0.5% (v/v) Tween 20]. After washing, serially diluted GA733-Fc and GA733-FcK protein samples (12.5–0.196 ng) were added to each well, and then the plates were incubated for 2 h at 37 °C. The wells were washed four times with 1× PBS-T. One hundred microliters of HRP-conjugated goat anti-human IgG Fc antibody (Jackson, West Grove, PA, USA; diluted 1:5,000) was added to each well as a secondary antibody. After incubating for 2 h at room temperature, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Seracare, Milford, MA, USA) was added to each well, and the color was allowed to develop for 3 min. TMB Stop Solution (Seracare, Milford, MA, USA) was used to stop the reaction. The absorbance of each well was read at 450 nm on a Gen5 microplate reader with version 2.01 software (Biotek, Winooski, VT, USA).