Hi Pongpawan,

We were able to (very recently) solve our background issue. What I would suggest is to go through a series of tests to troubleshoot the origin of your problem, starting with typical contributors to high background. This is a rundown of what we did.

You background issue may come from any of the things above that we have tested, but test the simplest or most likely first and work your way through each component. Good luck.

I would consider that you may have biotin contamination of your BSA.  You should check that you have ELISA grade BSA.  You may also be using too much detection antibody and getting non-specific binding.  Poor blocking could also lead to the detection antibody binding the plate directly.  I have attached an ELISA development guide from R&D Systems that may be helpful in optimizing your ELISA.  When we are designing a new ELISA, we test several different types of plates, and use a titration grid plate layout with the various reagents similar to that described in the attached guide.  Good Luck!

Hey Jason,

as you are collecting ideas, I will share with you my thoughts. I hope they will help you. If it isn't an antibody problem, then I would definetly have a closer look at your blocking step. I guess that you are (as you wrote) BSA (1%-3%) for blocking right?! This is normally the write thing to do. But you could also try use another blocking buffer (e.g. gelatin). I warn you of using milk powder instead as it may cause problems because of biotin molecules in the extract.

Otherwise, as you set up your own protocol. You could try to play with the antibody concentrations (Dectection and Capture) and see if you have less non-specific binding.

Those would be some ideas I have. I hope this helps you narrow done your problem and that you resolve it soon.

We have a label-free assay that gives real-time data, and we've found that BSA in many applications is not a good blocker.  Try BSA, followed by cassein or dehydrated milk.  We've also found BSA can grow bacteria, so it's possible that it's contaminated if you are making samples of BSA and letting them sit for a long time.

Remember, your blocker is only if it is attracted onto the surface.  Difference surfaces are often resistant to different blockers due to their net charge.  You also might try blocking in a lower ph buffer.  We've found ph 4.7 MES increases blocking a great deal because most proteins are more negative in this solution, and our surface is net positive.  Again, depends on the charge of your sample, solute and the PH of the buffer.

Really nice discussion and I also agree with above suggestions given by Scientists.

My initial thoughts were blocking as most people above. But we have no details on wash steps between each stage? Are you washing with PBS-Tween (0.1% is standard) after each stage? Also how many times do you wash (1-5)? and do you blot final wash on paper towls?

I have tested many blocking buffers for ELISA. Block & Sample 5X Buffer from Promega (G3311) works best. You can also use the same buffer for diluting your samples. Are you sure you optimized the concentration of your Streptavidin-HRP? You can use a pretty low concentration of it. Scientists tend to use to much of it for ELISA. Wash 5X between steps.  Good luck!

Thank you everyone for the insightful responses! Here are some details about the system:

PBS-T (0.05%) manual wash after each stage for 6 times, blot with paper towels.

Blocking is done with 3% BSA (ELISA grade). I have also tried 5% with the same results

Concentrations of the capture mAb, detector mAb, and Streptavidin-HRP have been optimized. For example, Streptavidin-HRP is at 0.1µg/ml.

I will definitely look into the blocking conditions, in terms of type, concentration, pH, etc. There is also the concern of the older detector antibody: Would there be issues with the conjugation since the mAb is now 2 years old?

To determine of it is the blocking agent giving background or the capture or detection antibodies, I would suggest running a control of coating with only blocking agent (not using capture antibody at all) and then adding the detection antibodies. This way, if you still get signal you know that it is the blocking agent. So basically just a process of elimination that can be done for each reagent that you are using - exclude that reagent from the steps you would normally follow and then you can work out which of the reagents are giving unwanted reactions. If it is not one specific reagent that seems to be the culprit, then perhaps concentrations should be looked at using various dilutions and a positive control if you have one available.

There are very useful suggestions provided above. It's a good idea to test the source of background by elimination. It seems your washing steps are fine. I would also pretty much emphasize on optimization of the concentration of all reagents you're using (both capture and detection Abs, and Streptavidin-HRP).

The method used to label the detection Ab with Biotin matters, once we found that when we used a different method (a kit) we improved our ELISA background (I think part of the reason is that different methods use different ways to calculate or quantify the number of Biotin molecules actually attached to the detection Ab). 

We also use Streptavidin-HRP at a higher dilution (1:5000) than the dilution recommended by the manufacturer (1:2000).

Lastly, the substrate you're using may also matter. We use TMB (KPL) and we found that the modified version of TMB (SureBlue Reserve) which is claimed to have higher sensitivity  is giving us higher background.

Good luck.

@Adam Huges, at low pH most protein would be positively charged not negative.

Jason. did you fix these problems yet b/c I'm also having the same problem as yours.

From my experience (which I'm also doing it right now), reducing the the background by redialysis of the antibodies doesn't really help reduce the background.

Your washing step seems to have no problem.  :)  Please tell me how u fixed the problem! Thx in advance!

Hi Pongpawan,

We were able to (very recently) solve our background issue. What I would suggest is to go through a series of tests to troubleshoot the origin of your problem, starting with typical contributors to high background. This is a rundown of what we did.

You background issue may come from any of the things above that we have tested, but test the simplest or most likely first and work your way through each component. Good luck.

Among all above mentioned possibilities, you can also think about antibody combinations. I have experienced certain antibodies show attraction to each other. Therefore correct combination of capturing antibody and detection antibody has to be selected by trials.

We found that some antibodies were very sensitive to pH. An alternative method is to use lower pH buffer as coating buffer.