Melatonin ELISA

Mix 200µL of plasma with an equal volume of cold Ethyl Acetate. Vortex gently.

Allow layers to separate on ice for 0h 3m 0s. Vortex again and incubated On ice for 0h 2m 0s.

Spin samples at 1000g for 0h 10m 0s at 4°C. Transfer the organic layer to a new tube.

Dry samples and resuspend in 220µLof 1X stabilizer.

Add 100µL of standards' working solutions and samples to provided 96-well plate in duplicates.

Immediately after add 50µL of melatonin tracer to each well (except blanks) followed by 50µLof 1X melatonin antibody (except blanks).

Cover plates with the provided plate sealer. Incubate for 1h 0m 0s at 37°C with 500 rpm shaking.

Decant the solution from each well. Add 400µL of wash solution to each well.

Decant the solution from each well and pat dry against clean absorbent paper.

Repeat wash step 3 times.

Add200µLof melatonin conjugate solution to each well (except blanks). Cover plate with the sealer. Incubate for 0h 30m 0s at Room temperature.

Perform the wash step as described above. Add 200µL of substrate reagent to each well. Incubate for 0h 30m 0s at 37°C protected from light.

Add 50µL of stop solution to each well. Measure the optical density using a micro-plate reader with absorbance set to 450 nm.