That can happen if the enzyme sterically blocks the recognition site, but that is not the rule.

Another point: HRP-conjugated antibodies are normally used as secondary, why would you use them as primary? The binding is like this:

antigen - primary - secondary - HRP

Thanks a lot for your reply!

I'm using another antibody to bind to HRP-conjugated antibody because I would like to get a fluorescence signal. So I'm adding one more layer to the binding. Would that work?

Hello Emilia

I agree with Engelbert. Why do you want to use a HRP-conjugated primary antibody ? If you want to obtain a fluorescent signal you can use a primary antibody to your target antigen and then add fluorescent dye conjugated secondary antibody . As mentioned by Engelbert the binding will be as follows:

Antigen - primary antibody - secondary antibody-fluorescent dye.

As I said, it may work unless there is steric hindrance. Alternatively, you could use HRP substrates that form an insoluble, fluorescent product, this would increase the sensitivity of detection. Molecular Probes (now part of Calbiochem) floggs such stuff.

You can try this combination of antibodies (by Western blot) if you have purified protein as positive control.

Dear Emilia,

you can try the following antibody sandwich:

Primary ab (HRP conjugated)

Anti-HRP-Ab (e.g. Rabbit anti-HRP from Sigma)

Secondary ab with fluorophor against the species of the anti-HRP-ab (e.g. anti-Rabbit Ig)

But you must test first if the anti-HRP ab binds to endogenous structures.

Also, you can try if the HRP concentration of your labelled first ab is sufficient to carry out the reaction used for ABC-staining (

ABC Peroxidase Standard Staining Kit ), i.e. that a fluorescent precipitate is formed by the enzyme.

https://www.fishersci.se/shop/products/pierce-abc-staining-kits/10435685

The discribed AG-Secondary -ABs reaction are working but you can use a biotinylated Tyramid enhancment combined with Streptavidin- fluophores. It is discribed in

1. Adams J. C.:

“Biotin Amplification of Biotin and Horseradish Peroxidase Signals in Histochmical Stains”; J. Histochem & Cytochem 40 (10): 1457-1463 (1992)

Thanks a lot for all your replies and suggestions! I'll try them out, or order new antibodies if it doesn't work, thanks!

By using the indirect ELISA technique, we can detect the primary antibody by adding HRP- conjugated secondary antibody. Then adding substrate

Hi Emilia, did it work? We are on it too... The secondary is HRP conjugated and we would like to add another ab for flourescence signal Emilia Betty Au-Yeung