5 replies to this topic

[chicho]

Anyone know of any serum protein that might be reacting with an HRP substrate, such as TMB, to give off signal?

Edited by chicho, 23 September 2011 - 10:33 AM.

[Ben Lomond]

If this relates to a background signal in a regular sandwich ELISA used to detect an analyte within serum, then it is most likely due to some form of interference (either specific or non-specific) of one of the components within the ELISA. If you can let us know the design of the assay, and specifics of the problem, I would be more than happy to help.

[PAO_ahac]

Is your question: Serum added directly to the substrate causes color change in absence of HRP? Or, you have background signal in your assay in which BOTH serum and HRP conjugate are being used?

[chicho]

PAO_ahac, on 26 September 2011 - 02:11 AM, said:

Is your question: Serum added directly to the substrate causes color change in absence of HRP? Or, you have background signal in your assay in which BOTH serum and HRP conjugate are being used?

"Serum added directly to the substrate causes color change in absence of HRP?"

Yep, basically that is my question.  If you add mix serum and the HRP-substrate, will they react to produce a color reaction?

[PAO_ahac]

I have never done that.  Usually the question is how to eliminate background color in the absence of binding.  If you have a panel of samples and hrp you can certainly run a quick experiment.

[Mohamad Zulkeflee Sabri]

Hi chicho. I don't think HRP will react with serum, unless you conjugated HRP with certain antibody (HRP-conjugated Ab) and added TMB then it will change colour. Antibody will react with its antigen in serum, and basically its HRP that reacts with TMB to produce the blue colour.