Summary of the prodecure
1. Prepare the reagents and samples according to the manual; 2. Leave one well empy; 3. Place 50ul of standard or sample in each well; 4. Add 50ul of the antibody to each well except the Blank (empty); 5 Allow 60 minutes for incubation at 37 degrees celsius; 6. Wash the wells 5 times; 7. Add 100ul of the conjugate to each well but the Blank one; 8. Allow 30 more minutes for incubation at 37C; 9. Wash 5 more times; 10. Add 50ul of both Substrate A and Substrate B in each well (incl. the Blank) and incubate for 15 minutes; 11. Put 50 ul of Stop Solution in all wells (incl. the Blank) and read the results within maximum 10 minutes; Notes: *Samples might require dilution, follow the sample dilution instructions of the manual. **During the last incubation with Substrates A and B the setting should be protected from any light.