I'd like to perform a competitive ELISA from scratch. Being a first-timer, I would appreciate if someone could help me with the basic concetrations of Ab and antigen in oder to start with and titrate (like 1 ug/ml for capture Ab?). I am aware of the principle of the method and the protocol. Thank you in advance!
The nice thing about working in 96- or 384-well ELISA plates is that there are a lot of wells to work with when you want to optimize conditions. Since every antibody-antigen interaction is different, you will have to identify optimal conditions experimentally. Take advantage of the microplate format to perform serial dilutions of capture antibody and antigen as a matrix, with antibody varying by row (for example) and antigen varying by column. That will allow you to quickly zero in on the optimal pairing of concentrations.
I do direct ELISA which is quite similar to a competitive ELISA. My ELISA system might be of great help to you.
antibodies concentration may go up to around 1, 5 or even 15 ug/ml. however I recommend to vary the coating and primary antibodies by yourself to see which concentrations of Antibodies give the lowest background. Some I use 10 ug/ml (coat) and 15 ug/ml (primary).
For blocking reagent, I use 3% BSA works great for me.
I use primary antibodies labeled with biotin (Thermoscientific), which is an extremely sensitive method.
Next, I use streptavidin-polyHRP(Immunotools) as a secondary antibody 1:500
For washing, I wash 4 times with PBS+0.5% Tween20.
The step are as follows:
Coat antigen in carbonate buffer. Incubate overnight at 4 deg C. 100ul/well.
Wash 4x with PBST
add 200ul/well blocking buffer. Incubate 37 deg C for an hour.
Remove blocking buffer and add 100ul/well of primary Ab, incubate for another hour at 37 deg C.
Wash 4x with PBST
Add strep-polyHRP 1:500 and incubate for another hour at 37.
Wash 4x with PBST
Add TMB for 5 min and stop reaction with 2N Sulfuric acid.
Read OD at 450 and ref wavelength at 620 .
VOILA!
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA