Hrp conjugated igm antibodies

After incubation, plates were removed from the refrigerator and left to warm to room temperature. Once warmed, plates were washed five times with 1X Wash Buffer A. HRP conjugated rec-Protein G antibodies (Invitrogen, 101223) were diluted (diluent mixture described above) by a factor of 4000, HRP conjugated IgM antibodies (ThermoFisher, A18835) were diluted by a factor of 2000, HRP conjugated IgA antibodies (ThermoFisher, PAI-74395) were diluted by a factor of 8000, HRP conjugated IgG1 antibodies (Invitrogen, MH1715) were diluted by a factor of 1000, HRP conjugated IgG3 antibodies (Invitrogen, 05–3620) were diluted by a factor of 1000, and HRP conjugated IgG4 antibodies (ThermoFisher, MH1742) were diluted by a factor of 2000 (Table 1). Plates were incubated for one hour in the dark at room temperature, then washed five times with 1X Wash Buffer A followed by one wash with PBS. Plates then received 100 μL of Tetramethylbenzidine (TMB) (SeraCare, Milford, MA) per well. After 4–10 minutes; (Table 1) depending on secondary antibody, 25 μL of 1N H₂SO₄ (Sigma, St. Louis, MO) was added to each well to halt the TMB reaction. Plates were read at a wavelength of 450 nm with a reference filter set at 570 nm using a SpectraMax i3x plate reader (Molecular Devices, San Jose, CA) and SoftMax Pro 6.4.2 analysis software.