Rhesus Macaque IL-21 ELISpot?

[Questions from customers]

Hi Mabtech!

 

I want to measure IL-21 secreting T cells from Rhesus Macaque-samples in ELISpot, but in your table for human and NHP cross-reactivity you only state that your IL-21 antibodies cross-reacts with Cynomolgus macaques: https://www.mabtech.com/knowledge-center/tutorials-and-guidelines/non-human-primate-cross-reactivity

 

Does that mean that they don’t cross-react with Rhesus or does it only mean that you haven’t tested it?

 

//S

 

[Jens@mabtech.com]

Hi S!

The absence of a dot in that table means that we haven’t tested them in Rhesus (if the antibodies have been tested and found that they don’t cross-react, that will be indicated in the table as a red dot). But if you’re interested in evaluating potential cross-reactivity yourself, we could send you a trial ELISpot kit to test on your Rhesus samples?

//Jens

[Questions from customers]

Hi Jens,

 

That sounds great! We’d like to freshly coat the plate, i.e. use the BASIC kit with ALP if that’s alright? We routinely do ELISpot, so manual coating is not a problem.

Do you have any tips on what kind of positive control to use? We were thinking of PMA+ionomycin but maybe there’s other alternatives?

 

//S

[Jens@mabtech.com]

Hi again S,

Sure thing, coat yourselves! Please note that you need to complement BASIC-kits with plates (https://www.mabtech.com/products/3654-wp-10_elispot-pvdf-plate-white) and that the plates should be pre-treated with ethanol before use. Please refer to this tutorial video for our recommendation of ethanol pre-treatment: https://www.youtube.com/watch?v=c0lC3CiIB5A

In addition, the ALP-enzyme should be used in conjunction with the BCIP/NBT-plus substrate (https://www.mabtech.com/products/3650-10_bcip-nbt-plus-substrate-elispot).

Regarding positive control for IL-21 ELISpot, I would recommend our agonistic anti-CD3 antibody that’s reactive with Cynomolgus as well as Rhesus (https://www.mabtech.com/products/3610-1-50_anti-monkey-cd3-mab-cd3-1-purified). Do a titration from 25 000, 50 000, 100 000 and 200 000 cells/well together with anti-CD3 and see if you get any IL-21 spots. In some other wells, I would also stimulate with PMA+Ionomycin, but starting with lower cell numbers (maybe 10 000, 25 000, 50 000, 100 000 and 200 000) since this stimulus totally circumvent normal activating pathways and thus probably give higher responses.

//Jens

[Questions from customers]

Hi Jens,

 

We have now finished the IL-21 ELISpot assay on Rhesus splenocytes. As seen in the attached photo, the kit works perfectly well with Rhesus. We see a lot of spots with PMA+Ionomycin and correspondingly no spots in negative control wells. However, the anti-CD3 antibody didn’t perform as expected, which is a little bit surprising. 

 

Anyway, good that the kit works.

 

//S

 

Rhesus monkey IL-21 Mabtech_ELISPOT.pdf

[Jens@mabtech.com]

Thank you for this feedback, S!

I am very surprised that the anti-CD3 stimulation didn’t give a response even at 100 000 cells/well. Sometimes certain T cell population just aren’t stimulated well with anti-CD3. An even higher cell number (200 000) perhaps would have resulted in spots. In this paper, authors Skinner et al seeded 250 000 Rheusus-PBMCs in each well before stimulation with anti-CD3: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248778/

 

Their readout was IFN-gamma secretion but there’s no reason to believe IL-21 wouldn’t be affected as well.

But good thing you included the PMA+Ionomycin in your assay! Thank you again for that feedback.

Kind regards,
Jens