IMMUNOLOGY question

Hello everyone!

I am trying to optimise a co-culture system of:

1. Hybridoma T cells known as DO11.10 cells, which are OVALBUMIN (OVA) specific

2. Murine Splenocytes

3. Ovalbumin

I have been trying to pulse the 10^6 splenocytes (in a volume of 1ml) with 0.5mg/ml OVA for 6 hours, and then co-culturing 10^5 OVA-pulsed splenocytes (in 100ul) with 10^5 Hybroma T cells - DO11.10 (in 100ul) in a 96-well plate (total volume of 200ul). I have used splenocytes-pulsed with DO11.10 OVA peptide (OVA323-339) as positive control. I have tested the IL-2 production by the DO11.10 using ELISA after 48 hours. Although the positive control gives a good signal, the other samples which are pulsed with OVA do not seem to show anything.

Does anyone have any suggestion of what I could do to make this system work? I have read papers that made this work already, so it must work. I must be missing some important detail. Any help would be massively appreciated.

Many thanks,

Molecule!