I am trying to develop and optimize my own indirect sandwhich ELISA by performing cherkerboard titration. I am currently checking different dilutions of the capture antibody against the antigen while also testing wells with no antigen ( row 0A) and wells with no capture antibody (row 0C) but everything else still present. I have included a picture of the OD values I got back. For some reason the wells with no capture antibody have higher values. What does this mean? Is this a bad thing?
Hi Lauren, I am trying to understand what could be the reason...It is interesting to see that the signal from the no capture wells decreases when the antigen decreases too. How do you detect your capture Ab? Is it biotinylated and you add Strep-HRP?
Hello! I am blocking the plates using 3% BSA and 0.05% Tween-20 and I have tried it both in TBS and PBS.
Also, it is not biotinylated. I use a primary antibody sourced in rat and reactive in mouse to bind to the mouss FV and then I use a HRP goat anti-rat detecting antibody.
Harald Kühnel
I was thinking about looking into the kinetics too. That is a good idea. I have not performed either of these assays, but I think we do have SPR.
Falk Fish We just got some samples that express different levels of the Factor V protein, so I plan on using those next to see if the antibodies will be able to qunatify the different amounts. But yes, my diluent buffer for my antibodies is the same thing as my blocking buffer: 3% BSA and 0.05% Tween-20 in TBS and I also tried PBS.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA