Problems with central membrane white spot and retained blue color

[Guest Saswati Bhattacharya]

I am using the ELISpot Plus: Monkey IFN-γ (HRP) assay and have some assay problems. First, there are white spots in the middle of the membrane. Is this from drying out of the membrane? Is there any other reason that white spots would be there? And why are the internal wells retaining the blue color? The positive control is the anti-CD3 antibody. 

[Christian@mabtech.com]

Dear Saswati,

Since these plates are precoated we know the issue is not in the coating. My suspicion is that something with either the cells or the stimuli explain this dark membrane background (that is even). 

I have to ask: well B1 looks perfect. What is difference in this well compared to well B2?

We know from experience that certain stimuli can induce weird membrane effects. For example, certain peptides are "sticky", bind to the membrane then makes the detection antibody bind to the membrane evenly. Another factor is that too high DMSO concentration can cause weird darkness of the membranes. Is your stimuli a new peptide with very high DMSO concentration in it? What is the final concentration in the ELISpot well?

When you get an artefact introduced into the ELISpot where the detection antibody and/or SA-HRP interact with the membrane then you can get deviations in substrate development that comes out looking white patches in the membrane.