Biogenic amine ELISA - Non-peptide ELISA (Aug/02/2006 )

I am using a kit to do ELISA to quantifiy serotonin in platelet lysates and I got inconsistent results. I did not follow the instructions provided by the kit's manufacturer's to prepare the lysate (dilute platelet rich plasma, spin down to get a pellet, remove supernatant, add PBS to resuspend, freeze-thaw to lyse, spin down to separate membrane from supernatant).

No one in the lab has done an ELISA with platelet extracts. I followed my PI's instructions for preparing the lysate (add an equal volume of 95% EtOH to platelet rich plasma plus some EDTA).

I don't think that I had a problem with pipetting because my standard curve was good (99% correlation with regression line). The results for the duplicates of my samples were off. The larger value was 3 times the smaller value.

I'm thinking that my PI's protocol for the lysate doesn't work for ELISA. What do you think?

Thanks for any advice.

I have no experience with this kit.
Is it worth to follow the provider's recommendation?
I would at least try that, and if it's still not working, contact the provider of the kit. They can help you.