between 3 and 4

Dear Dorotea,

Thank you for your answer, however I think that this is the RZ of the initial pure peroxidase used for carring out the conjugation, the obtained product has usually lower RZ (1,3-04?), but I am not sure about this particular value. May you have any biblio reference?

For QC, can use just use an enzyme assay which would measure the amount of active HRP, which is what is measured by people using the antibody- HRP conjugate anyways?

Dear Ana,

I'm so sorry, but I haven't. Nowadays, I'm in the field of food biotechnology and I'm not in enzymes research. Maybe you can ask some of my collegues of the article.

best wishes

I think a RZ value of 1.0 - 1.5 would be acceptable for a HRP-conjugate. However, the final RZ depends on the total length of the construct indirect proportionally. For shorter conjugates values towards 1.5 would be awaited and for longer constucts down to 1.0 If the measured value is far below 1.0 this could indicate that the peroxidase domain of HRP is not fully saturated with the prosthetic heme group!

Dear all, thanks for your answers.

yes Marcia, of course I will perform a capture ELISA in order to detect the activity of the conjugates, but I would like to know a little more about the possibility of this parameter, RZ.

Thank you Dorotea, I´ll do.

Dear Marcel, your answer is very interesting, thank you for the information. Please, let me know if you have knowledge about an interesting publication regarding this parameter.

I do not have any publication on this topic at the moment but I am working with phylogenetically related fungal heme peroxidases that also have some fusions and they have a RZ value below 0.9 only when they are not fully saturated with heme.

Thank you Marcel, in any case this is the information I need. Thank you very much.

I am not familiar with RZ values.  However since it deals with absorbtion values it probably measures the amount of protein which is related to the amino acid that causes the absorption.  So any test or knowledge of how much of the amino acid that is necessary to act as a reactant in the conjugation reaction will give you the information that you want.

The RZ give you some hibt about the purity of the HRP or HRP labelled proteins. The pure HRP has an RZ of 3 ... 3.5. The RZ of conjugates depends on the molar ratio of the conjugation (mol HRP per mol protein to label), the size of ypor protein you are conugating. 

At al, it's not a good quality parameter to describe the quality of your conjugate. The RZ of a conjugated protein can be helpful to compare different coupling procedures, different lots, ...

Why? In immune reactions detected by labelled antigens / antibodies the affinity determines of the antigen/antibody interaction the detection limit. The labeled partner hat to be in excess present in this reaction. Therefore, also the substrate should never be the limiting part in an ELISA. 

(just returned from vacations) To Camille Solbrig: for heme peroxidases in general and HRP in particular NO amino acid acts as reactant, H2O2 or peroxidic bond in any molecule (also in organic peroxides!) acts as an electron acceptor to activate HRP or other peroxidase to compound I where it can accept electrons from other substances, particularly florescence dyes that are used in ELISA. To Stephan Kiessig, I am not working with HRP conjugates but with other natural peroxidase conjugates and I think that RZ value is an important hint on the purity of the fused protein. Surely for specific ELISA applications there are other parameters that do matter. But at least in complex experiments the obtained RZ value gives the lucky researcher a control value to be sure that (if not working) from the side of peroxidase it is acceptable or not ...