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I have yet another enquiry regarding your antibody ab1128 and its problematic cross-reaction with our streptococcal protein. We were trying to find if there was a potential cross-reacting linear epitope by comparison of the amino acid sequences of hBD-3 and our protein. There is a problem with your data sheet which states that the antibody reacts with residues 23-33 of hBD-3 (GIINTLQKYYC). These AAs are in fact residues 1-11 of the protein. Do you know which is the correct reactivity. Also in your reply to my original query you say that the antibody is affinity purified. According to the data sheet it is just an IgG fraction. Could you please clarify these points for me. Many thanks.
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ANSWER: |
Thank you for your enquiry. Please take a look at this website: http://us.expasy.org/cgi-bin/niceprot.pl?P81534 MRIHYLLFAL LFLFLVPVPG HGGIINTLQK YYCRVRGGRC AVLSCLPKEE QIGKCSTRGR According to Swiss Protein, the amino acid sequence listed corresponds to amino acids 23-33 of hBD-3. The antibody is an affinity purified IgG fraction. I hope this helps. |
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We have been trying to use your rabbit antibody to human beta defensin 3 (ab1128) in an ELISA with no success so far due to an extremely strong cross-reaction between the antibody and the protein with which we have coated the plate. The plate was coated with at 1.5 ug/ml with a protein which is secreted by Group A Streptococci and which we have shown, both by functional assay and by ELISA in the opposite orientation, to bind to hBD-3. The plate was blocked with either gelatine or hydrolysed casein (we have tried both), recombinant hBD-3 added at 3 ug/ml, followed by ab1128 diluted 1/1000, and alk. phos. goat anti-rabbit Ig and pNPP. The control wells containing no hBD-3, but merely the coating protein and both antibodies, give the same OD as those containing hBD-3. Control wells with 2nd antibody only, or ligand plus 2nd antibody, are negative as are all non-coated control wells. Alternative blocking reagents tried so far make no difference. Any suggestions/comments? |
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ANSWER: |
We never tried to test this antibody via ELISA Being an affinity purified peptide antibody, the chance of cross-reaction with an unrelated protein seems small, but possible. In all honesty, we have no idea of a helpful suggestion. |
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