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.