5 replies to this topic

[vaccinator]

Hi there,

What kind of controls do people use for murine IFN-g ELISPOTs? We usually perform these experiments with mouse splenocytes or lymphocytes and use a media negative control and PMA stimulated positive control. However, the PMA control always gives a strong response. We sometimes get lower responses than expected with our experimental groups and find that we can't really compare plates setup on different days.  We'd like to use an antigen to stimulate on the ELISPOT plate that all mice should have a similar IFN-g response to. Does anyone know of such a thing or have any other controls that we could try?

Thanks very much

[Carlton H]

Why do you say that you can't compare plates from different days?  Since you're doing the positive control, can't you just normalize to that control?  So long as each experiment has something to normalize to, then there's no reason that you shouldn't be able to compare experimental groups from different experiments.

[vaccinator]

We don't always do the same positive control - sometimes the positive control vaccine has slightly different ingredients etc. There's just not enough room on the plate or the mice to spare to include the same group each time.  But I have noticed that for a particular vaccine we have tested several times, some times there are different number of spots, size of spots, intensity of spots etc. It could be the vaccine formulation, the quality of the peptides used for in vitro stimulation, the mouse supply etc. For this reason I'd like to stimulate the cells in vitro with an antigen to which i can expect a standard response.

[Carlton H]

... I'm confusing myself because I'm trying to think of a solution with very limited information.  If you can give as much detail of your experiment as possible, I can probably help.

One thing in particular that I'm getting caught on: You mention a positive control vaccine that's changing, but before you said you were positive controlling with PMA injection.  Which are you using? ... And if it's PMA, can't you just make up the PMA the same each time?

Also, since your negative control is just media, you really should have the positive control every time, specifically because you're never going to be able to compare between experiments without it (unless you can ensure the conditions are near exact, which seems to be part of the problem in the first place).  Your negative control is this instance is zero, and you can never normalize to zero.

As to an antigen... Well, I've never performed this precise experiment, but I'd imagine you could pick a good surface protein from a murine-infectious bacteria, clone it into inert bacteria, grow it up, purify it, and you should be set.

Hope I'm approaching your problem from the correct mindframe.  Feel free to bounce some more thoughts off me / ask questions / whatever.  I'm here to help.

Cheers,
-Carlton

[vaccinator]

Thanks for your help, I really do appreciate it!  Here is some more details about our experiments.  We vaccinate mice with different vaccine formulations in order to test the response they mount against different antigens formulated in the vaccines. We use HLA-A2 transgenic mice to test human antigens. There are about 4 test groups with 4 mice per group.  8 days later, the mice are euthanized and splenocytes or lymph node cells used in ELISPOT. We stimulate the cells on the ELISPOT plate with the antigen they were vaccinated with and also an irrelevant antigen (seperatly). We always include 2 naive mice that did not receive any vaccination. As a positive control, we stimulate cells with a mix of PMA and ionomycin, which as I said, usually gives a strong response. And as a negative control, the cells are not stimulated with anything (media only). Plates are developed the next day as usual.

We have recently started to perform a PMA/Ionomycin dilution in order to establish a calibration curve, but still the response is non-specific so it can't really tell us if each animal can mount an antigen specific response or not.  Does this make sense or am I making it too complicated?

[Carlton H]

Has the PMA always been PMA/ionomycin, or was it just PMA at first?  Regardless, you could try using PMA and ionomycin separately as positive controls and see if one of these is the source of your nonspecific IFN-g response.  If each individually is still giving dose-independent results, then I guess we've just talked ourselves in a circle and we're back the the original problem...

...if that indeed is the case, and you really just need to change antigens, you could always try tiny (picomolar, if not less) concentrations of a "superantigen", such as staphyloccoal enterotoxin B (SEB).  It'll certainly produce an immune response.

I also used to use heat-killed e-coli (easy to grow, easy to use, cheap as dirt) to produce a primary immune response in zebrafish (I was looking at SAA expression levels).  I didn't do anything with secondary immunity in those experiments, but if you can spare the mice it's worth a shot.

-Carlton