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Prepare all reagents and samples as instructed in the manual.Add 100 μL of sample or positive control to each well.Incubate 2.5 h at RT or O/N at 4 °C.Add 100 μL of prepared primary antibody to each well.Incubate 1 h at RT.Add 100 μL of prepared 1X HRP-Streptavidin to each well.Incubate 1 h at RT.Add 100 μL of TMB One-Step Substrate Reagent to each well.Incubate 30 min at RT.Add 50 μL of Stop Solution to each well.Read at 450 nm immediately.