Blurry spots in antigen-specific wells

[Guest Mason]

Hi there,

I did a B cell ELISpot (3825-2H) where I coated half of the plate with my antigen (10ug/mL) and half with the kit Anti-IgG antibody (15ug/mL). In the picture I have attached, the top row is antigen-specific where I plated 1 million BM cells/well and the bottom is total IgG where I plated 100,000 BM cells/well.

As you can see, the antigen-specific spots are quite blurry compared to the total IgG ones. Do you think increasing the concentration of antigen help to make these more distinct? And if so, should the concentration be equivalent to the IgG concentration? I had some trouble with getting accurate counts on the ELISpot plate reader as they were a lot smaller and lighter.

Thanks!

[admin]

Hi there Mason!

I don’t think increasing the concentration of the antigen will have much impact. But that is a speculation, could be worth trying.

What I do think would be beneficial is allowing the substrate development process to go on longer in the antigen wells. You have clearly followed my recommendations and NOT overdeveloped which is great. The total igG spots look perfect.

However when coating antigen the epitopes can get smudged against the PVDF membrane and result in less than optimal IgG binding. Spots often come out blurry like this and require much longer incubation times. 
 

Another alternative is to use reverse B cell elispot were you biotinylate your antigen and use it only in the detection phase after capturing total IgG. This often results in sharper and more plentiful spots and we like to explain why with that epitopes are better preserved by the biotinylation.

But first action is increasing substrate development in the antigen wells only. Keep what you are doing in total igG.

[Renata]

Hi Mason, 

Nice to see that you are getting spots in the antigen-coated wells, you seem to be on the right track even if they are now a bit too blurry! You could also definitely try increasing Ag coating concentration like you first suggested. I had a customer recently who got more defined spots after they increased the coating concentration.

What is your current antigen coating concentration? Typically 5 to 20 ug/mL works wells, but it would be best to titrate the antigen and see at which concentration you reach saturation, i.e., spot numbers don't increase with increasing coating concentration. For example, you could try 5, 10, 15, 20 ug/mL. 

Good luck and let us know how it goes!