I'm working on an ELISA for HPV-E7 protein (human). I'm using a polyclonal capture and a monoclonal biotinylated detection antibody format. The following is my protocol, I am getting a high background and also high signal for my negative control. I am using HeLa cell lysate to optimize the assay as it contains HPV18 E7. I'm using C33A cells as the negative control, as they're an HPV-free cancer strand. However, the C33A cells still show a relatively high signal (compared to 1% BSA only). Any thoughts on what is wrong with my protocol, or do you think it's the antibodies? Thanks
I'm using the 37515 pierce superblock and I'm getting quite a high background. I am using a sandwich ELISA assay with a NUNC maxisorp plate.
1. polyclonal capture antibody (8 ug/mL) in coating buffer placed incubated overnight at 4 C.
2. Washed 3x with 300 uL 1% BSA in TBST (.05% tween 20).
3. Blocked 4x with 300 uL 37515, no incubation time
4. Washed 3x with 300 uL 1% BSA in TBST (.05% tween 20)
5. Monoclonal biotinylated detection antibody applied (0.5 ug/mL) diluted in 1% TBST-BSA for 1 hour at room temperature with gentle agitation
6. Washed 3X with 300uL 1%BSA-TBST
7. 100 uL poly-HRP (100 ng/mL)applied for 1 hour at room temperature
8. Washed 6x with 300 uL 1%BSA-TBST
9. 100 uL ultra-TMB applied for 30 minutes
10. 100 uL 2M H2S04 Applied
Do you have any suggestions to better this protocol? Thank you