I am trying to optimize an ELISA for ApoE3 protein. I have paired antibodies and am trying to find the best concentration for the ELISA. I have experienced that with high concentration of detection antibody I have strong positive signal even in the absence of the capture antibody. I have used 1xPBS+1%BSA as a blocking step. Any ideas why is this happening?
This is the kind of problem that calls for performing a matrix-type experiment to simultaneously test several variables at one time and quickly find your solution. Is your antibody directly conjugated with HRP or biotin or are you using a secondary against the detection antibody? If your plate is insufficiently blocked at high concentrations of detection antibody you can just have non-specific binding. Does your wash include a detergent such as Tween20? I would test a range of the following variables:
1. concentration of coating Ab
2. % of blocking reagent
3. washing +/- detergent (start with 0.05% Tween)
4. concentration of detection Ab
You can easily run this on one or two plates with a concentration of ApoE that is towards the middle of your standard curve. And of course run each condition without antigen as your background control. Assay development is a pain but with high rewards once you have everything worked out. Good luck!
Hi Elwira !
It can be a problem of washing, you must wash very well your plate after each step...But you can try also a blocking of 2% BSA (it's the concentration I usually used for ELISA).
Another suggestion : I know that some plates have differences in the adsorption regarding to the material. So it can be also a problem of the type of plates that you use for your experiment.
Best of luck.
This is the kind of problem that calls for performing a matrix-type experiment to simultaneously test several variables at one time and quickly find your solution. Is your antibody directly conjugated with HRP or biotin or are you using a secondary against the detection antibody? If your plate is insufficiently blocked at high concentrations of detection antibody you can just have non-specific binding. Does your wash include a detergent such as Tween20? I would test a range of the following variables:
1. concentration of coating Ab
2. % of blocking reagent
3. washing +/- detergent (start with 0.05% Tween)
4. concentration of detection Ab
You can easily run this on one or two plates with a concentration of ApoE that is towards the middle of your standard curve. And of course run each condition without antigen as your background control. Assay development is a pain but with high rewards once you have everything worked out. Good luck!
I agree with what has been said by others. In my experience, high detection antibody concentrations will produce background. Checkboard your ELISA reagents, as Thomas suggested. Once that it is optimized, run dose response curves with several different blockers (i.e. blotto, 1% casein, bsa/FCS) to determine the best blocker. I've left out a bit of detail, so feel free to clarify with me. Setting up an ELISA doesn't need to take more than 1 week.
Many factors can increase the background in ELISA techniques (high concentration of detection antibody, insufficient washes, blocking problems) but, as you have an strong positive signal even in the absence of the capture antibody, my oppinion is that the main cause is due to the blocking step. As others collegues have recommended, try to increase the BSA percent in the blocking buffer. I have had to increase it even to 3% for some antigens.
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