Generally i use the manufacturer's protocol for the ELiSA kit from biolegend. If you are still not getting color then,

Could be issues with the dilution of your serum. Try adding whole serum first. Don't use all of them in one go. Set up small experiments to reduce loss of ur samples. Store the samples in minus80 and while using always aliquote the serum.

I used the AssayGenie protocol and worked perfectly for me. There are 2 codes for mouse IL6. You should be able to find the protocols in the websites.

https://www.assaygenie.com/mouse-interleukin-6-il6-elisa-kit/

https://www.assaygenie.com/mouse-il6-elisa-kit-mofi00066/

If you still have trouble, I can send you pictures of the protocol

I'm using life span kit, but this time I did my incubations at 37 degrees.

Thank you.

Demos Kynigopoulos

Could you send me pictures of the protocol?