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As described in previous emails, the mouse monoclonal antibody (ab14422) was able be detected by an anti-rabbit secondary antibody. An anti-mouse antibody was used as a control. The experiment was repeated a second time, and still produced the same results. I later informed you that the antibodies that were purchased are widely commercialized and we have had no problems with them cross reacting with other species. Due to confidentiality purposes, we still can not release the information about the concentration of the peptide or the concentration of the antibodies used.
Asked on Nov 11 2011
Following up my previous message from Wednesday, I have received the following protocol from the laboratory that valdiated this antibody for ELISA: We tested the antibody in ELISA with the following conditions: Coating of plate with 100 µl of (2 µg/ml) human beta-Defensin 3 (aa 6-22) in bicarbonate buffer, Blocking with BSA Detection with ab14422 in serial dilutions ranging from 1:250 to 1:1000 gave a good signal Substrate: TMB Is this similar to what you tried? We are confident that what you received is mouse anti-BD3, but the third ELISA experiment results that you described earlier suggests that what you received is defective, if your protocol approximated these conditions, and if your secondary antibody was anti-mouse IgG.
Answered on Nov 11 2011