High binding 384 well elisa plates

High-binding 384-well ELISA plates (Corning, MA) were coated overnight at 4°C with 50 µl/well of recombinant protein diluted in carbonate buffer. The next day, plates were washed with 0.1% Tween-20 in PBS and 200 µl of blocking buffer (PBS+ 1% BSA) added to each well for 2 h at room temperature. After blocking, plates are washed five times before serum samples diluted in 0.1% Tween-20+ 0.1% BSA in PBS was added at 50 µl/well and incubated for 30 min at room temperature. After incubation, plates were washed and 50 µl/well of peroxidase labeled-anti-human IgG (Life Technologies CA) in serum diluent was added. Plates were incubated for 30 min at room temperature, and then washed as previously described. To reveal reactions 100 µl of TMB SureBlue Peroxidase Substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added to each well for 15 min at room temperature, after which the reaction was stopped by adding 1 N H₂SO₄. Plates were read within 10 min of reaction stoppage at optical density (OD) 450 nm, using 570 nm as the reference wavelength on a SpectraMax plate reader (Molecular Devices, CA).

High-binding 384-well ELISA plates (Corning, Tewksbury, MA, USA) were coated with recombinant protein, and then serial dilutions of the plasma samples and positive control and a single dilution of negative control were prepared in serum diluent. After incubation, peroxidase-labeled anti-human IgG, IgG1, IgG2, IgG3, IgG4 and IgE (Life Technologies, Carlsbad, CA, USA) in serum diluent was added. End-point titer was determined by Graph Prism Nonlinear regression (curve fit) sigmoidal dose response (variable slope) to interpolate unknowns from the last optical density value greater than a threshold determined by sera from normal human plasma pool.