Are you sure your secondary antibody does not crossreact with the coating one? That could be a simple explanation for high background.

Otherwise, I would suggest to include at least 30 min of blocking after coating, using PBS+BSA (or even better PBS+skim powder milk).

Then, dilute everything in the same blocking solution you used in the previous step, including antigen, primary antibody, secondary antibody and HRP conjugate. Blocking and diluting all proteins in the same blocking solution should avoid non-specific background.

All the reagents also need to be titrated, as too high concentrations of every reagent can result in background.

You should definitely try adding a blocking step. The fact that you get a beautiful titration curve indicates that your sandwich Ab pair is probably working well, which is very promising. Most if not all commercial ELISAs have a blocking step, and people who develop ELISAs for a living tend to name blocking as the #1 most important factor to optimize.

Even the quality of the BSA or other proteins you use for blocking can drastically change the outcome of your assays. Commercial ELISA companies often use different blocking reagents for different antibody pairs, so a blocking solution that works well for one antibody pair may not be optimal for another.

I would recommend obtaining as many different potential blocking buffers as you can, and trying each for suitability in your particular assay. Coat a plate with your primary Ab as you describe, and then block different columns of the plate with different blocking solutions (including a plain PBS "no blocking" control). Incubate the blocking solutions for 1 hour, remove + rinse 2x, and then proceed with the rest of your assay adding dilution curves of your antigen, etc. Thus you can accurately compare the blocking capacity of each buffer.

I would try, to start, 5% nonfat dry milk in PBS, a 1 to 5% BSA solution made with your current BSA stock, 1 to 5 % BSA solutions made with any other nearby lab's BSA stocks (BSA quality varies a lot and matters a lot), and at least one or two commercial ELISA blocking buffers. The commercial blocking buffers can be leftover reagents from commercial ELISA kits - they tend not to go bad quickly at all, and you or other nearby labs may even have these lying around.

Good luck!

Thanks Gertrudis, you are right. It might be that the secondary is giving a high signal by itself, because when I titrated it, I obtained high values.I will try with blocking.

Thanks!!

If the secondary antibody really cross-reacts with the coating antibody (I assume primary and secondary come from different species) blocking wont have any effect. You would have to find a non-crossreacting secondary...Cross-reactivities are often declared by the manufacturers, you need to find one that says minimal crossreactivity "to "immunoglobulins of the speciesto which the primary detection antibody belongs to.

Thanks Both Gertrudis and Emily!

Yes, Gertrudis, both antibodies are made in different species and were purified by immunoaffinity. The capture antibody was made in rabbit and the detection in guinea pig. The secondary biotinylated guinea pig antibody from vector laboratories cross react less than 1% with rabbit IgG (according to the datasheet) . Thanks for the suggestion, I will try with the blocking step. Thanks for you helpful advice!

Thanks Emily, I found a good suggestion compare different blocking solutions (in the case that my secondary antibody does not cross react with my capture one, let's see)

I am not sure, but if my antibodies are biotilynated, may I use milk? I read that is not possible...

Thanks for your suggestions and for giving me hope with my assay too :)

All the best!

Milk can be a problem or not depending on the content of biotin. Some companies sell biotin-free milk. Casein-the real blocking agent within milk- is another option. Several years ago, I used to make a lot of phage pannings using milk as the blocking agent and they worked very well. In that case, it was not milk produced for laboratory use, but a normal commercial one found at the spermarkets (called Marvel).

Milk can make the difference in the blocking efficiency, but its true that you have to make sure that its not interfering in your assay.

Thanks Gertrudis!

Dear Gertrudis, thanks for your help.

After being trying different approaches I found out that the secondary biotinylated antibody is giving the high background.

I tried using different blocking agents at different concentrations and even for two hours and I did not observe any changes.

Do you have a good recommendation of a biotinylated guinea pig antibody? or in which company does it sell good antibodies?

I also tried to use HRP antibody and it seems to work, even though the absorbance range is a little bit narrow. In the case of the HRP antibody I begin with a 0.5 absorbance values, that is still high and blocking does not decrease the absorbance values.

Thanks for your help.

Best,

Carolina

Hi Carolina,

Increasing blocking & wash times prior to starting your experiment should really help. Your negative should provide no data, so maybe running a strip from the plate to check that is does not have high background anyway?

Some great tips here from ELISA Genie under their "ELISA Troubleshooting" section:

https://www.elisagenie.com

But are you diluting the problematic molecule (biotinylated antibody) in the same blocking solution? You can try that not only during the blocking step, and always titrate the dilto find the best dilution for every reagent, including the biotinylated antibody

Thanks Seán and Gertrudis for your help.

I proved to incubate all my antibodies with the exception of the capture one with blocking solutions. Different blocking solutions were 5% BSA, 5% Milk or 5% FBS plus or without tween 0,05%. I have already titrated all the components of the ELISA, the most harder component to titrate was the secondary antibody IgG against the detection antibody. This antibody and the streptavidin when they are not present in the titration plate give zero background, therefore they are making all the noise of the system. The using of a HRP antibody helped but not completely, it reduced the highest background, but the dynamic range were not so gut.

my next idea is to biotinylate the detection antibody, try ti improve the ELISA with a HRP antibody or to choose the dilutions of all the titration that give the less absorbance in the zero value and use the iMAPlate system to increase the sensitivity.

Thanks for your suggestions, they are really very welcome!

Best,

Carolina

I forgot to mention that when I put only different dilutions of the biotinylated antibody in the plate, the gave high absorbance values. I believe that the biotinylated antibody is attaching to the plate.