I have a positive control worked out for my ELISA now, so I know it's working.
Now my issue is: human serum.
I've tried switching polyclonal and monoclonal as capture/detection - each time the detection is biotinylated and streptavidin-HRP is added later.
My basic assay is:
1. coat wells with capture Ab (10ug/mL) in PBS overnight in 4 degrees C
2. aspirate/wash three times with wash buffer
3. Block non-specific binding via 1% BSA solution, at least 1-2 hours
5. Add samples - positive control conditional media and serum samples in 1% BSA reagent diluent, incubate 2 hours
7. Add 0.5ug/mL biotinylated detection (mAb if capture is polyclonal; polyclonal if mAb is capture), 2 hours
9. Add streptavidin-HRP, 20 minutes
11. add 1:1 solution A:solution B (R&D systems) substrate reagent
12. once desired blue intensity is reached (aprrox 10 minutes), stop with 2N sulfuric acid (turns yellow)
13. measure the optical density at 450nm wavelength
any good ideas on how to optimize this system to detect (if it's there) an antigen of small size and possibly low concentration.
should I pretreat the human serum in some way? Any new methods you all know about?