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Guest oliver

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I recently did a sandwich ELISA that failed. We observed positive detection in our negative controls. I was wondering if someone knew where we messed up the ELISA set up. 

For the ELISA we had a serial dilution of S. aureus culture supernatent as we were trying to detect Protein A given off by S. aureus. We also had a purified Protein A sample that we serially diluted as a positive control. We coated each well with polyclonal rabbit anti-mouse IgG to begin. We then blocked with Bovine Serum Albumin. We then incubated with either S. aureus or Protein A in all by the negative control wells. We then incubated with our secondary antibody which was a monoclonal anti-protein-A antibody conjugated with biotin. We then incubated with streptavadin and peroxidase followed by color development with TMB. 

This procedure resulted in positive detection in all wells including then negative controls, which had no antigen present. 

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Hi Oliver,
Welcome to our Forum!
We have discussed your ELISA problem internally here at Mabtech. Generally, it is hard to analyze Protein A as it binds to several antibodies thereby increasing the risk for high background levels. But as you also can detect positive signals in the negative controls, something else must be wrong. We guess that what has happened in your ELISA is a linkage between the coating and the detection antibody. You use an anti-mouse antibody as coating, do you know the origin of the detection antibody? If it is made in mouse the linkage is obvious, but it is also possible that the polyclonal antibody binds the detection antibody due to cross-linkage.
There are commercial available ELISAs for the detection of Protein A, unfortunately, we don’t provide such a kit.
Let us know if you have any further comments or thoughts.

Good Luck!


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