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the specificity of HRP-conjugated antibody - (Oct/28/2010 )

Hello guys

Recently, I am setting up a sandwich ELISA assay using the monoclonal antibody as a capture and rabbit polyclonal antioby conjugated to HRP as a detector.

The problem comes from the inconsistent result of HRP-conjugated polyclonal antibody. For HRP conjugation, I use sulfo-SMCC (for activation of HRP) and sulfo-SPDP (for polyclonal antibody).

After conjugation to HRP, I tried to titrate this HRP conjugate onto the plate where antigen is directly coated onto. The result was very good (titer was very high), indicating that HRP conjugation worked well. But the problem comes from the sandwich ELISA. When I use this antibody as a detector in my sandwich ELISA system, the standard curve doesn't look good (the background level was very high) and this antibody cannot detect antigen from the sera samples which were positive before. I am using the same batch of rabbit polyclonal antibody for HRP conjugation. My result certainly seems to indicate that the specificity of antibody is changed alittle bit after conjugation although it can still recognize the antigen which is directly coated onto the plate. I guess this can happen because antibody structure could be changed by HRP conjugation (especially when multiple HRPs are conjugated per antibody) and then its activity could be changed too.

My guess is that my conjugation worked too well (too many HRP per antibody: molar ration of HRP and antibody was 10:1). So I am trying to reduce the molar ratio to 1 or 5: 1 and reduce the incubaiton time for coupling.

Is there anybody who has same experience like me or can anybody explaine this?


I have seen this before. Not only the molar ratio used in the procedure but also the buffer and pH can be critical. The specificity of your antibody has not are using the same batch indicated in your posting. You may not have the system completely optimized and be on an edge with respect to one of the variables. As long as you hold the ab and hrp lots constant and vary other aspects you should be able to reproduce your working material. Hold some of the previous "good" batch in reserve so you can do testing in parallel with the new material.



Thank you for your comment. I've read on this matter and found out that this actually happens especially when multiple labels (such as fluorescent dye, HRP, or other enzymes) are conjugated into one antibody molecule. I also heard that the lot number of HRP can affect too. As you suggested, I will try to keep antibody and HRP lots constant and vary other variables.


I am also experiencing some problems with regard to conjugation of HRP to an IgM monoclonal Ab. I am usng the Lightning-Link HRP conjugation kit from Innova Biosciences. I performed the conjugation procedure according to the manufacturer's instructions, however, when performing a direct ELISA I observe colour development in my negative - no coat controls. I have diluted the conjugate Ab down and eventually there is a point in the negative control where there is no colour, however, at this conjugate dilution there is also no colour in the wells where the coat antigen is present.

The antibody that I am conjugating has been used before in an indirect ELISA to detect the same coat antigen and I have not observed any non-specific colour development in these instances.

Any advice in this regard will be greatly appreciated.


The IgMconj could be very sticky. If your blocking and washing steps are typical one idea would be to prereact the plate before you add your conjugate with the same species non specific IgM incubate and wash...then add your specific conjugate. Other options are more rigorous washing (ie higher surfactant concentrations) With the higher surfactant conc you can follow by a wash with plain distilled water. other thoughts: leave the wash in the plate for 1 minute prior to decanting/aspiration.

let us know how it goes...