Hello, I used different proteins to stimulate porcine PBMCs and compared IFN-γ production levels to screen for proteins that induce high IFN-γ. The PHA-stimulated group showed around 800 spots of IFN-γ, but the unstimulated cell group and the protein-stimulated groups showed no significant difference. Even the whole bacterial protein stimulation group did not induce high IFN-γ production. The cells were from pigs artificially infected with bacteria for 28 days, with protein concentrations of 50/100 μg/mL and an incubation time of 18 hours. What could be the possible reasons for this?
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No high-level spots were produced after protein stimulation
- GMGuest Mary2025-04-13T16:26:46Z
- CChristian@mabtech.com2025-05-28T10:29:48ZAdmin
If the possible control (PHA) has worked well the most logical explanation is that the antigen used does not induce IFNg secretion in your pig T-cells. It is not immunogenic in this regard.
For proteins to work in ELISpot they need to be processed by antigen presenting cells (APC) which in turn present them to memory T-cells. This sort of activation requires the correct cell-to-cell interaction so one needs to try different cell numbers, from 100k cells/well - 500k cells/well.
APC need to be viable otherwise it wont work.Memory T-cells must be present and be viable for it to work.
Can you tell me more about your antigen?
Was it the same protein used for injecting the pigs that was used in the ELISpot assay?