ELISA Testing Protocol
Label the individual strips that will be used and aliquot reagents as the following example:
||Volume per Reaction
|Kanamycin Antibody #1
|HRP-Conjugated Ab #2
|1X Wash Solution
- Add 50 uL of each Kanamycin Standards in duplicate into different wells (Add standards to plate only in the order from low concentration to high concentration).
- Add 50 uL of each sample in duplicate into different sample wells.
- Add 50 uL of HRP-Conjugated Ab#2 to each well and 50 uL of Kanamycin Antibody #1 to each well. Mix well by gently rocking the plate manually for 30s.
- Incubate the plate for 30 minutes at room temperature (20 – 25°C / 68 – 77°F). (Avoid direct sunlight and cold bench tops during the incubation. Covering the microtiter plate while incubating is recommended).
- Wash the plate 4 times with 250 uL of 1X Wash Solution. After the last wash, invert the plate and gently tap the plate dry on paper towels (Perform the next step immediately after plate washings. Do not allow the plate to air dry between working steps).
- Add 100 uL of TMB substrate. Time the reaction immediately after adding the substrate. Mix the solution by gently rocking the plate manually for 30s while incubating(Do not put any substrate back to the original container to avoid any potential contamination. Any substrate solution exhibiting coloration is indicative of deterioration and should be discarded. Covering the microtiter plate while incubating is recommended).
- After incubating for 15 minutes at room temperature (20 – 25°C / 68 – 77°F), add 100 uL of Stop Buffer to stop the enzyme reaction.
- Read the plate as soon as possible following the addition of Stop Buffer on a plate reader with 450 nm wavelength (Before reading, use a lint-free wipe on the bottom of the plate to ensure no moisture or fingerprints interfere with the readings).