Anti-beta 2 Defensin antibody [B 235-I] (ab14426)

In response to your enquiry, here is what Autogen Bioclear wrote:

"The beta-defensin genes were directly expressed (no tags or leader sequence). Sequences are available (let me know if you require them). After fermentation, centrifugation, cell breakage; the proteins are folded into its active form during the folding/oxidation stage, this is when the disulfide bridges form. Specific processes are proprietary. The beta-defensins have a characteristic 6 cysteine structural motif containing 3 disulfide-bridges."

I have tested the performance of your antibody against the denatured forms of my standards, HBD-1 (36aa and 47aa), HBD-2 and HBD-3. Unfortunately these results were negative and the only positive results were the native forms of HBD-1 36 and 47 aa, which gave a clear signal on the dot blot.

Best regards

ANSWER:

Thank you for getting back to me with those details, it was very interesting.

Unfortunately given that the source of the antisera have not been able to provide me with further details as to the synthesis of the immunising peptides it is difficult for me to comment on whether they have been raised against a correctly folded immunogen.

If this was indeed the case then one might speculate that these antibodies only recognise the synthetic peptide and not the native form of the protein. However, checking back to past orders and this antibody is relatively popular and we have not received any complaints as such. I can but speculate that the absence of western or dot blot as applications on the datasheets is a reflection that these antibodies do not recognise their epitopes when immobilised on a membrane.

Please can you provide details of how the synthetic immunising peptide was made, with particular attention to the formation of the cysteine bridges prior to immunisation.

ANSWER:

Thank you for your enquiry.

Further to correspondence with the source of this antiserum I have been informed that the antiserum was raised against tricycle human ß-Defensin 2 antigen. Unfortunately they could not provide details of whether the antisera had been tested against the native protein or your protein standards.

I have therefore not been able to find evidence that these sera have been tested against the native form of this protein. I would definitely be interested to know how these sera performed against the denatured forms of your standards.

Are you getting closer to an answer regarding the exact structure of the antigen peptides used to rise these mab?

ANSWER:

Thank you for contacting me.

Unfortunately I have not been able to obtain a satisfactory answer from the source of this antiserum as yet. I will be in touch as soon as I know more details about the synthesis of the immunising peptides.

I appreciate your patience.

Do your anti-defensin antibodies cross-react with other human defensins? For example does antibody to human beta-defensin 2 (B235-I)cross react with human beta-defensin 1 or vice versa? Cross reactivity to other species is not a problem in our application.

ANSWER:

Thank you for your enquiry and your patience.

The members of the defensin family are classified based on the position of the three íntramolecular disulfide bridges between cystein residues. They are classified as alpha- and beta-defensins. We have monoclonal antibodies specific for beta-defensin 1 and beta-defensin 2. They were generated against the respective synthetic peptides (with correct disulfide bridges). A sequence alignment of the two sequences gives no similarity beyond the position of the cystein residues. It is not likely that the antibodies cross react. However, this has not been tested experimentally.

We hope you will find this information useful.