Dark membranes in IFN-g ELISpot

[John_K]

Hello Mabtech,

 

 

We are long-time customers of your human IFN-g elispot kit and have generally been very happy their performance. This is the product that we buy:

 

https://www.mabtech.com/products/3420-2a_human-ifn-γ-elispot-kit-alp#tabs-min-1

 

 

In our setup, we use elispot plates from Millipore (MSIP model) that we pre-treat with EtOH prior to adding the capture antibody. The following day, cells are seeded into the plate at 250,000 cells per well (w/wo antigen) and incubated overnight for 24h. Spots are detected according to your protocol.  

 

Now to the problem: Although spots are good in terms of numbers and sharpness, our membranes also tend to be kind of "dark" after developing our plates for 20 min. This lowers the overall contrast and makes the spots more troublesome to count in our elispot reader. Looking at your new web page for this product it is obvious that our membranes are much more darker than what they are supposed to be. 

 

Can you give us some advice on how to avoid this issue? What could be wrong? thx.

[Christian@mabtech.com]

Greeting John_K, you have come to the right place for getting advice on this issue.

 

 

In my experience there are a number of reasons as to why ELISpot membranes can become darker then usual post substrate development and I will go through them in detail below. Since I do not know the specifics of your experimental setup, some of these comments will not necessarily apply to you but they are nonetheless good starting points to look into when ironing out this issue. Also, they may be helpful to other readers of this forum experiencing the same kind of problem.

 

Before I list them, I should point out that other factors will definitely exist as well. This is in no way a "complete" listing of everything that can cause dark ELISpot membranes. Here we go.

 

 

1. Mabtech recommends that customers use cell culture medium supplemented with fetal calf serum (FCS) and not autologous AB serum. Why do we say this? Well, one of the reasons pertains to the potential of getting increased membrane backgrounds in your ELISpot assay. The issue is here two-dimensional. First, since an autologous serum is derived from a human source it poses the potential of containing trace amounts of the cytokine that you are investigating (in your case IFN-g). In such a scenario, the capture antibody in the ELISpot well would bind to the IFN-g at the first instance that cell culture medium is introduced into the assay (i.e. already during the blocking of the ELISpot plate). Instead of spots, a general "darkening" of the membrane will occur as the molecules of IFN-g randomly bind to the capture antibody, leading to a darker membrane than usual once spots are developed. 

 

Second, an autologous serum may also contain heterophilic antibodies (Bjerner J et al, Clin Chem. 2005 Jan;51(1):9-11.). These are commonly found in healthy donors and can cause serious problems of unspecific binding and lead to the same type of phenomenon that you have experienced. 

 

Certainly, in many cases, none these two issues will affect your particular batch of AB serum and everything is fine. Nevertheless, it is good to be aware of what factors can be at play in generating these kind of artifacts.

 

 

2. In situations were cells are pre-stimulated and not washed prior to being added into the ELISpot wells, you can also run into the issue of "darkened" ELISpot membranes. The explanation for this is quite simple and analogous to the situation described above in point number 1; If the cells are pre-stimulated in 15ml tubes, left in the hood for 1h and then pipetted into the ELISpot plate, the cell supernatant may contain considerable amounts of IFN-g that has already been released by the cells during this initial hour of activation. Such molecules of secreted IFN-g will in turn of course immediately bind to the capture antibody and result in darker ELISpot membranes once the plate is developed. Spots will be visible, but just as you point out, the darker membrane will lead to a lower contrast of spots and can result in difficulties of setting up your ELISpot count-settings. 

 

 

3. A potential third source of darkened ELISpot membranes is the use of Tween in wash buffers.

 

 

Mabtech does not recommend Tween and the reason for this is simple: in our hands, with our protocol, it leads to darker ELISpot membranes. There is absolutely no benefit. 

 

At the same time, other ELISpot practitioners are adamant on the use of Tween as a critical component in making ELISpot results look better. How can our viewpoints be so different? Well, it all comes back to how the protocol is setup. We always recommend that you should EtOH treat your plates prior to coating. This increases the binding capacity of the PVDF membrane, and combined with a good amount of capture antibody (1-1.5 ug/well), will always produce better results compared to not using EtOH pre-treatment. With our protocol, Tween provides no benefit and will actually "hurt" your results by making membranes noticeably darker after the plate has been developed. 

 

However, in cases where you decide not to perform the EtOH pre-treatment and only coat using a much lower amount of capture antibody (0.5 ug/ml), results will actually benefit from the use of Tween. Spots will appear more clear and the "dark-membrane effect" seises to exist, or atleast becomes much less noticeable. It would therefore appear that the use of EtOH fundamentally changes the effects on the PVDF membrane introduced by Tween exposure.    

 

So in essence, since you state that your laboratory is using EtOH activated plates, please make sure that Tween is not in any of your wash buffers used for ELISpot plate washing.

 

 

 

I hope this helps! 

 

 

Please come back if you have more questions or comments!   

[John_K]

Thank you for this comprehensive response.

 

Some of the points I had never considered, especially the issue surrounding the use of Tween. We have used this detergent in our elispot-washbuffers from the beginning so this might very well be the reason. 

 

I guess we should do a trial experiment and compare the two protocols (w/wo Tween). thx  

[Christian@mabtech.com]

Thank you for this comprehensive response.

 

Some of the points I had never considered, especially the issue surrounding the use of Tween. We have used this detergent in our elispot-washbuffers from the beginning so this might very well be the reason. 

 

I guess we should do a trial experiment and compare the two protocols (w/wo Tween). thx  

 

Doing this experiment sounds like an excellent idea!

 

 

Please comeback and tell us whether excluding the Tween solved your problems or not.

 

 

Best regards,

Christian