No spots with human perforin ELISpot kit

[Questions from customers]

Hi Mabtech,

I have tried to set up this ELISPot assay for human perforin, but have been unsuccessful. I have been using PMA/Ionomycin stimulated PBMCs (stimulated for 24 hrs which I have done in the past) but have not been able to detect any spots. We are using Multiscreen HA plates (mixed cellulose Ester Membrane plates) which is commonly used in our lab for other ELISpot assays. Otherwise we follow the protocol precisely. Can you please some ideas of what might be the reason for lack of spots?

Regards,

E

[Jens@mabtech.com]

Hi E,

Would you please provide us with some more information of your protocol to allow us to trouble shoot this problem? I understand that you followed our protocol, but there are details in it that we leave for you as a researcher to decide. For example, what substrate did you use? How long did you develop spots for? How many cells did you add to each well? What was the viability of the cells? What medium did you use? What did you wash the plates with?

Kind regards,

Jens

[Questions from customers]

Hi Jens,

We have now tried this ELISPOT three times with slight changes from each, but still has not been able to see any spots. I’m attaching a photo for your reference.

This is the protocol we used for each round:

 

First test:

·      Surfactant-free mixed Cellulose Ester Membrane (Millipore MAHAS4510) plate.

·      pre-wet with PBS 200ul.

·      blocking with RPMI1640 supplemented with 10% FCS.

·      Stimulation PMA (50ng/ml)/Ionomycin (1ug/ml) 24 hrs and media control.

·      Cell concentrations tested: 50 000, 10 000 and 5000.

·      All washing and antibody solutions according to the protocol.

·      Substrate: KPL BCIP/NBT Phosphatase substrate system (used in the lab previously) (Sera Care Cat # 5420-0030(50-81-00).

·      Development 30 min.

 

Second test:

·      Surfactant-free mixed Cellulose Ester Membrane (Millipore MAHAS4510) plate.

·      pre-wet with PBS 200ul.

·      blocking with RPMI1640 supplemented with 10% FCS.

·      Stimulation with new batch of PMA (50ng/ml)/Ionomycin (1ug/ml) 24 hrs and media control.

·      Cell concentrations tested: 50 000, 10 000 and 5000.

·      All washing and antibody solutions according to the protocol.

·      Substrate: KPL BCIP/NBT Phosphatase substrate system (used in the lab previously) AND a different batch of KPL BCIP/NBT Phosphatase substrate system (which had worked in another ELISPOT that week) (Sera Care Cat # 5420-0030(50-81-00).

·      Development 60 min.

 

Third test: 

·      Hydrophobic high protein binding immobile-P membrane (Millipore MSIPS4510) plates.

·      pre-wet with 35% EtOH 15ul.

·      blocking with RPMI1640 supplemented with 10% FCS.

·      Stimulation with new batch of PMA (50ng/ml)/Ionomycin (1ug/ml) 24 hrs and media control.

·      Cell concentrations tested: 100 000, 50 000 and 10 000.

·      All washing and antibody solutions according to the protocol.

·      Substrate: KPL BCIP/NBT Phosphatase substrate system (used in the lab previously) AND a different batch of KPL BCIP/NBT Phosphatase substrate system (which had worked in another ELISPOT that week).

·      Development 60 min.

 

Hope you can give us some pointers of how we can make this ELISPOT work.

[Jens@mabtech.com]

Hi again E,

Thank you for all the details. So, Christian and I have discussed your issue and come up with the following comments:

•    Perforin is a rather tricky cytokine to detect with good results. If you look in the ELISpot-well photo in this page, you see a visual example of how this system looks after 72h stimulation with a strong CEF peptide responder: https://www.mabtech.com/products/human-perforin-elispot-basic-kit-alp-3465-2a-0. You do get spots after 24h with a good positive control, but for antigen specific responses it is recommended to use 48h or 72h incubation.  

•    You are using our ELISpotBASIC kit and thus coat the ELISpot plate with capture antibody yourself; what amount of capture antibody (Pf-80/164) are you adding to each well? Since perforin is a quite weak system, you really have to use the recommended concentration 30 ug/well. This is double our normal concentration and it is really necessary. If you dilute the capture antibody and use e.g. 10 ug/ml, you will get blanks. 

•    Christian doesn’t think one can expect good results with Cellulose plates for this system. A plate with PVDF membrane, which is pre-treated with EtOH, is essential. The sensitivity of MAHAS4510-plates is lower, and there is a strong chance they won’t work with perforin.

•    When you pre-wet the PVDF plate with EtOH in your Test 3, does it go well? Have you seen our tutorial on YouTube?:

•    PMA and ionomycin are very strong stimuli, but they can seize to function for example after light exposure. There is also quite large variance in how well they work, at least in Christian’s experience. As a result, we would recommend to use anti-CD3 and anti-CD28 mAbs as positive control. The anti-CD3 mAb from Mabtech is very reliable, and together with anti-CD28 the stimulus is even more potent

•    For weak cytokines like perforin, we really recommend using the Mabtech ELISpot substrate. We only use BCIP/NBT-plus for ALP. The "plus" is important as it is much stronger than normal BCIP/NBT. You can see the difference in the bar graph here: https://www.mabtech.com/knowledge-center/tutorials-and-guidelines/enzymes-substrates/elispot-substrates

•    As you might have gathered from other threads in this Forum, most problems in ELISpot can be traced back to issues in capture antibody coating. As a result, Mabtech have pushed harder and harder for pre-coated ELISpot plates. They are more expensive, but they are produced under optimal conditions at Mabtech HQ in Stockholm, and in addition, pre-coated plates allow you to be spontaneous in that you can just take the plate out of the wrapper and start doing your assay. When coating yourself, you have to do it the day before, often when you are the most tired in the end of the day. So, please consider trying our pre-coated ELISpot perforin-kit. 

•    Viability of cells is essential for ELISpot, and for perforin it is even more crucial than for other cytokines. For IFNg, you can still get some spots although you have only e.g. 60% viable cells at 100 000 cells/well, but with perforin the plate could be blank at that level of viability. According to Smith et al (2007) (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1865640/), 89% viability is a sort of "threshold" for getting reliable antigen specific responses in ELISpot. Below that, and you see a significant drop in spot counts of IFNg. Had the this study been done for perforin, we are pretty certain the viability demands could have been even higher. So, can you tell us something about your source of your cells? Are you assessing viability using trypan blue exclusion? Fresh isolated or thawed PBMC?
 

Kind regards, 

Christian and Jens

[Questions from customers]

Hi Jens and Christian,

Thanks for your very detailed response with lots of info and some good points.
Here are answers to your questions:

•    I have in the past had very good results with the perforin ELISPot testing PMA/Ionomycin stim of sorted subset of cells. I only had 5000 cells per well then and stimulated 24 hrs which worked really well. That was when I was located in different lab though and I cannot remember which plates we used, but pretty sure they were PVDF.

•    We know that the PMA+Ionomycin is working. We only purchased it a few weeks ago (actually just before the ELISPOT reagents arrived) and IFNg comes up positive in ELISAs and we detected IFNg, TNF and CD107 by FACS after PMA/Ion stimulation. We have some anti-CD3 and anti-CD28 here that we could try but I don’t think that would make a difference since I know our stock of PMA/Ion is new and working well.

•    We used the coating antibody at the recommended concentration (30ug/ml).

•    We are coating the plates ourselves because we wanted to use this on sorted cells again and would not use a whole plate at a time, thus it would be wasted with pre-coated plates.

•    When we pre-wetted the plate with EtOH (test 3) the wells went gray and we left it for no more than 1 min as the protocol suggested.

•    Potentially the substrate is not ideal although would you not expect to see something (faint spots maybe?). Anyway, I will try the BCIP/NBT-plus. 

•    We use frozen PBMCs and we do cell counts with trypan blue and we consistently have very good cell viability around 90% and above. Later on for sorted cells these will be sorted in presence of a live/dead stain so that only viable cells are sorted.

•    I agree that I think the issues lie in the coating (and the plates we are using may not be ideal) and also in the substrate. I think it would be worthwhile testing a pre-coated plate side by side with one of our plates. 

Regards,

E

[Jens@mabtech.com]

Hi E, 

Great to hear that you have good viability of the cells, and that you are willing try the BCIP/NBT-plus substrate as well as pre-coated plates. Please share your results if you’re up for it!

Kind regards,
Jens