1.In what species was the antibody raised? This can be very important in direct plasma/serum assays

2.What protein was melatonin conjugated to for immunisation?

3.What protein is used in the assay buffer?

4.What position of the melatonin molecule was used to link it to the protein? This is important for specificity.

5.What compounds were tested for cross reactivity? Were 5-methoxytryptamine, serotonin, N-acetyl serotonin, N-acetyl tryptamine tested?

6.How was the sensitivity determined?

7.What was the source of the melatonin standard? Is it an identifiable reference standard?

8.What daytime plasma melatonin levels does the assay report?

Replied:

1. Mouse

2. BSA coupling

3. Skim milk powder

4. By chemical modification, a carboxyl group was introduced at the 6th position of the melatonin molecule, forming 6-carboxyl melatonin

5. Includes 5-methoxytryptamine, serotonin (5-hydroxytryptamine), N-acetylserotonin, N-acetylcysteine, none of which showed significant cross-reaction

6. Sensitivity is usually expressed by the detection limit, defined as the average signal of the blank sample (zero standard) plus 2 or 3 times the standard deviation corresponding to the concentration value

7. Chemically synthesized pure substance (CAS number: 73-31-4)

8. Daytime (0 - 80). Nighttime (40 - 180)