Standard curve varying OD

[Angelbio]

Here I am again with another problem!

We are developing assays where we coat each time we do the assay with peptide, usually overnight (2-8C in humidity box, same shelf of same fridge each time).

Next day we block and add rabbit anti-peptide and then anti-rabbit HRP and measure signal. All steps are optimised and controls are good (inter-precision 10% or less, intra precision 4%).

But the OD of the standard curve shifts a lot. - %CV of top standard OD is 20% and we have a range of OD 1.3 - 2.3. (read at 450nM with correction at 620nM)

Why?????? Is the coating just a very variable thing and this will always be the case unless we coat a large batch of plates at a time and then set control specs on each batch of coated plates.

Or do we need to go back and look at the coating concentration more closely. Generally we aim for just below saturation of peptide on the well, by increasing coat concentration until the signal increases only slightly more with more coating peptide.

thanks in advance for any advice

[Elisadeveloper]

I am a confused by your ELISA format...you are coating with peptide, and using a rabbit anti-peptide antibody and detecting with anti-rabbit HRP. What is it that you are measuring? Is it a competitive assay and you are detecting peptide in the samples?

You say the CVs of the controls are good, but the CV of the top standard is high. Is this the inter assay response CV at that standard, or is it the response CV between replicates within a single assay?

Monitoring the OD at 650 and stopping at a particular 650 OD (usually half the 450 OD) will allow you to get a more equal response between assays if that is what is concerning you.

What is the pI and size of the peptide, what plates are you using?

Let me know more details and I may have more suggestions