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Add 100L standard or sample to the wells. Incubate for 90 min at 37C 2. Discard the liquid, immediately add 100L Biotinylated Detection Ab working solution to each well. Incubate for 60 min at 37C 3. Aspirate and wash the plate for 3 times 4. Add 100L HRP conjugate working solution. Incubate for 30 min at 37C. Aspirate and wash the plate for 5 times 5. Add 90L Substrate Reagent. Incubate for 15 min at 37C 6. Add 50L Stop Solution 7. Read the plate at 450nm immediately. Calculation of the results

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