Hello,
During an ELISPOT experiment using hamster splenocytes after vaccination, my development came out looking a bit weird with some wells showing some crazy looking spots.
Top 3 rows are negative control and should also not show any spots, other 5 are recall antigens.
I was wondering if you could have any advice on how this could occur and how one could prevent it.
Kind regards
Elias
That looks very strange indeed.
I presume you have coated this plate yourself. What protocol did you use for the coating process? Capture mab concentration? Did you use etoh pre-activation?
Also, please describe your detection protocol.
One easy solution would be to buy our precoated plates instead. Good quality spots guaranteed as long as cells are of viable:
https://www.mabtech.com/products/elispot-plus-hamster-ifn-gamma-alp_3102-4apw-2
Plate type MAIPS4510 from Millipore was used. Membranes were prewetted with 50µl 70% ethanol for two minutes (without taking off the underdrain). All coating steps were performed according to the protocol (15 ug/ml of coating antibody was used as described) (https://www.mabtech.com/products/elispot-flex-hamster-ifn-gamma-alp_3102-2a). Plates were stored with coating antibody over the weekend, not overnight, at 4°C. No fluid had leaked from the plates.
All washing steps were performed with sterile PBS, no Tween. Cell suspensions were filtered over 70µm filters prior to plating, 500k cells were plated per well. Plates were put into 37°C and 5% CO2 incubator, no wrapping. After approximately 18h (all wells were still filled, so no leakage), the spot detection protocol was followed exactly as in the protocol. Substrate was filtered over a 0.45µm filter.
Artifacts seem to be present as solids in the well as some are attached and hanging on the sides of the wells that seem to be effected the hardest.
Did you use Mabtech substrate?
If not, that could be the source of the problem. Some substrates are precipiating but not optimal for ELISpot.