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coating not saturated
I coat my peptide onto the wells at 100ng/ml 100ul/well,
This gives a good signal with the specific antibody and the conjugated anti-rabbit.
But looking back at earlier experiments I suspect that the well is not saturated with peptide at this concentration. What implications will this have for the assay?
As long as I get a decent signal is it okay - there is no issues with NSB, the background is very low.
Could it lead to a variablity in the assay?
Re: coating not saturated
Sounds like your coating concentration is in the right ball park, I am assuming your peptide is small relative to antibodies. If you have acceptable uniformity (response CVs of 7% or less at low and high concentrations across an entire plate) and you get acceptable precision and accuracy throughout the intended assay range within and between batches, then all should be good.
If the peptide coating concentration is too high, you may end up with steric issues when the larger specific antibody binds and you build up a complex which can result in poor precision at high antibody concentrations.
I am not sure about your assay format, is it a competitive assay to measure free peptide?
If variability becomes a problem, I have found that addition of a small amount of protein to the peptide coating solution (casein, or BSA) at similar mass concentrations can improve uniformity and robustness.
Re: coating not saturated
Thanks for the tip with the casein. The assay is now working really well and is flying through its validation. We did have a problem with frozen controls which we have since dropped, and I panicked a bit about the coating concentration, but left it alone after your reassuring words.
The assay is coat with antigen (peptide)
then add polyclonal anti the peptide - to make a standard curve with known amounts of polyclonal antibody,
then add an HRP conjugate anti-rabbit
this is to make a quantitative assay to measure the amount of polyclonal anti-peptide antibodies produced by different rabbits. Our client's product is the polyclonal antibody which we will affinity purify from the rabbit serum in a GMP facility.
I actually have a basic question about the approach to this which I will put up in another thread.
thanks for your help in this question though
|coating , saturated|
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