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Prepare all Standards before starting assay procedure (Please read Reagents Preparation). It is recommended that all Standards and Samples be added in duplicate to the Microtiter Plate. 1. Secure the desired number of coated wells in the holder then add 50 μL of Standards or Samples to the appropriate well of the antibody pre-coated Microtiter Plate. 2. Add 100 μL of Conjugate to each well. Mix well. Complete mixing in this step is important. Cover and incubate for 1 hour at 37 °C. 3. Wash the Microtiter Plate using one of the specified methods indicated below: 4. Manual Washing: Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well completely with wash solution, then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure four more times for a total of FIVE washes. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly hen washing the plate to assure that all strips remain securely in frame. 5. Automated Washing: Aspirate all wells, and then wash plate FIVE times using wash solution. Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350 μL/well/wash (range: 350-400 μL). After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. It is recommended that the washer be set for a soaking time of 10 seconds or shaking time of 5 seconds between washes. 6. Add 50 μL Substrate A to each well. 7.Add 50 μL Substrate B to each well. Cover and incubate for 15 minutes at 20-25 °C. (avoid sunlight) 8. Add 50 μL of Stop Solution to each well. Mix well. 9. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader immediately.