ELISA pH problem

A H Finn A.Finn at sheffield.ac.uk
Mon Aug 5 08:30:02 EST 1996

This is one for all you immunochemists out there-
We have a cytokine ELISA which uses a monoclonal capture antibody
We found the signal was influenced by the pH of the unknown - with a 
higher signal at pH 5-6 than at pH 7.5
We found that dialysing samples to remove small molecular weight solutes 
- ions, urea etc (we were measuring the cytokine in urine) - restored the 
signal and we could reduce it again by replacing them.
So we decided to use an indicator (BDH universal) to assess the pH of 
samples, so we could quickly acidify them as necessary prior to assay, 
as dialysing them all wasn't feasible and might interfere iwth our 
cytokine (IL8).
Bizarrely, the indicator seems to abolish the pH dependent change in 
signal (without altering the pH)
If we could only understand this, we could legitimise this approach to 
fixing our assay. But we don't.
BDH says there is isopropanol in the indicator, so we're checking that 
out. The rest of the magic chemicals are secret!
Has anyone any idea what is going on here?
Many thanks

Adam Finn
Dept Paediatrics
University of Sheffield
A.Finn at sheffield.ac.uk

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