I have been using your kit for human INF-gamma ELISPOT-ALPplus. My experimental settings require stimulating the PBMCs one week before performing the ELISPOT and I am using human AB serum during this experiment. My first three ELISPOT worked very well until I changed the serum. I have tried two stocks after that and both gave me high background, is there any way to solve this other than the obvious answer of trying more stocks of serum.
Thank you very much
Dear Rafael! Welcome to our forum. So I have some things to tell you but unfortunately no direct solutions to your problem. I would advise moving away from AB serum:
Mabtech recommends that our 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.
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!
Thanks a lot, Christian,
In my particular settings in which I have to pre-expand the population during a week, do you think FCS can be worst than human serum because this is the only reason why I am still attached to human AB serum? Do you think it could be an interspecific response to the FCS for the PBMCs? Do you think there are differents between inactivated serum vs non-inactivated serum? (with my previous batch of serum I did not inactivate the serum and everything was working just fine, but now that I am having troubles, I am wondering if this could make a difference).
PS. Right now I am still using your pre-coated plates, therefore I do not have all the problems you were mentioning before about coating.
Rafa
Dear Rafa,
I have no reason to think that your pre-expansion will not work fine using FCS instead of human AB serum. We do pretty much all our cell cultures in FCS, it is a common practice. I think you should atleast give it a shot. I have never heard of a interspecific response to FCS from the PBMC.
At the same time, it is concievable that the 1 week expansion could be skewed slightly differently due the lack of AB serum, but impossible to say before trying it out yourself.
When it comes to heat-inactivated versus non-heat inactivated we believe it is important to do it. You do not want complement components to be still active in either your pre-expanded culture or inside of your ELISpot assay.
best,
Christian
Ok. Thanks for your reply. As I am discussing with your colleague by email. I have an experiment running right now. And I now this human serum is providing me background (maybe for the reasons you are telling me or maybe by unspecific activation). So I think washing the cells with PBS and changing the media with FCS, 2-3 days before the ELISPOT running could stop this non-specific activation. What do you think about?
I have never been a big fan of scrambling to change something half way through. It would in my humble opinion be better to finish this experiment using AB serum, learn from the results, and then setup a new culture but this time with FCS.
By keeping the two components separate you can draw some real conclusions.
Unfortunately, I learn last Sunday that this batch of serum provides background, so it is the only way to try to save the experiment.
Anyway thank you for your opinion and help