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Prepare all reagents, samples and standards as instructed in the manual.Add 100 μL of standard or sample to each well.Incubate 2.5 h at RT or O/N at 4 °C.Add 100 μL of prepared biotin antibody to each well.Incubate 1 h at RT.Add 100 μL of prepared Streptavidin solution to each well.Incubate 45 min 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.