I’m running a sandwich ELISA to compare diseased and control serum samples, but I consistently get higher signal in my No Tissue (NT) wells compared to both my sample wells. What could be the cause of this elevated NT signal, and how can I troubleshoot or minimize it?
- Capture antibody was coated on the plate 4 degree celsius overnight.
- Blocking was performed with 1.5% BSA in PBS-T 1 hour room temperature moderate shake.
- Serum samples (diseased and control) are diluted in the same buffer and incubate 4 degree celsius overnight.
- Detection is done with biotinylated antibody 1 hour room temperature moderate shake followed by avidin-HRP for 1 hour room temperature moderate shake.
- NT wells receive all reagents except the serum samples.
Hi,
-It could be some unspecific binding of the second antibody or the streptavidin-HRP
I can't tell you the exact reason, but following helped in some cases in the past in my case.
1. Mix the the second biotinylated antibody with the Streptavidin-HRP together in blocking solution (e.g. 1.5% BSA) and give them together in a single well
2. use a higher dilution of the secondary antibody and Streptavidin-HRP (I use 1:10000 usually, the dilution factor depends on your stock solution)
3. How often and how do you wash? You could increase the washing steps. If you have an ELISA washer available, you should consider to use it, since it gets better results than washing by yourself.
You need to run some control experiments to have an idea what is the reason. (In general when you start or develop a new ELISA, it is better to run some control assays first, to see, if everything works as intended)
Example for controls(duplicate should be enough) to find the reason for high background.
1. Your NT control with all reagents
2. No coating of the first antibody, BSA block + (secondary antibody+streptavidin-HRP)
-shows if the problem is the first antibody
3. coating of the first antibody + BSA block + streptavidin-HRP
-show if the problem is the second antibody
4. if you have other PBS based blocking buffer (e.g. fish gelatine, thermo superblock) or BSA from other sources, you can also try in case something is wrong with your blocking solution
Sebastian Reinig Thank you so much! I was considering incubating both the detection antibody and streptavidin-hrp together to see if that works. I will have the results later this week.
Sebastian Reinig
So I just got the results of the ELISA back today and it looks like my values were far below the signal intensity I usually get. The values now were in the 4 digit range where before it was in the 6 to 7 digit range. I am not entirely sure what happened here. The good news is that my background was reduced appreciably which is a start. I made my preincubation buffer which was my antibody (1:1K) and AVIDIN-HRP (1:10K) mixed together in 10 mL of Binding Buffer (1.5 g BSA + 25 uL of Tween-20 + 50 mL of 1x PBS). I incubated it at room temperature for about 30 minutes then centrifuged it at 10,000G for 10 minutes. Do you know what could have gone wrong?
Hard to say,
I think mixing is enough. I usually just mix both secondary AB and streptavidin-HRP for 5 minutes in BSA 2.5% in PBST.
Be careful, streptavidin-hrp concentration should be in excess higher in comparison to your secondary biotinylated antibody. You may change the dilution of your streptavidin-HRP in accordance to your secondary AB.
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