Question (45981) | Recombinant Dengue Virus 2 NS1 glycoprotein (ab64456)

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Question

Dear Sir/Madam,
We are trying to develop an ELISA for Dengue NS1 detection and since we have been been working for a long time with your products, we have decided to use your dengue products. We are in the process of validating our immunocapture system. The capture antibody we are using is a monoclonal antibody against DEN1-4 NS1 (ab41616) that we purchased together with theDengue NS1 glycoprotein (ab64456). We have already validated the presence of the mouse monoclonalantibody against DEN1-4 NS1 (ab41616) in our ELISA support using a Goat polyclonal Anti-mouse (ab20043). After looking at the citation for themouse monoclonalantibody against DEN1-4 NS1 we are expecting a good immunocapture. However on our initial tries, we cannot detect the presence of the dengue NS1 antigen. So we checked some details in the technical support and we found out that the NS1 antigen has a histidine tag which have been missing in the initial data sheet we had. And so we decided to purchase lasttime a unit of Anti-histidine tag HRP (ab1187) to check on theDengue NS1 glycoprotein (HisTag) (ab64456) but unfortunately even at 2 ug/ml concentration, we are not able to detect its presence. We are thinking of the possibility that the antigen we received may not have histidine tag as initially stated in the product sheet? but as you have guaranteed on our previous inquiry, the antigen is containing histidine tag. As loyal costumers of your company, we would like to ask you for the possibility of requesting replacement of the dengue NS1 glycoprotein (ab64456) that we have initially purchased with a different batch preferably to proceed with our experiments.
We are looking forward in working further with your dengue products.
Thank you for your usual costumer support.
Regards,

Answer

Thank you for contacting us.
I am sorry to hear you are having problems with one of our products. I appreciate the details provided. I am afraid that product ab41616 is not guaranteed to work in ELISA. We have tested this antibody in WB and ICC, but we are not aware of its suitability for ELISA assays.
As far as I understand you are performing an indirect ELISA, coating your plate with the antigen ab64456 and then capturing it with the monoclonal antibody, using ab20043 (secondary) to detect your primary, is that correct? It could well be the primary antibody is not recognising ab64456 as it may not work for this application.
I can also confirm the peptide ab64456 is tagged with 6 histidines. It is also possible the antibody is not detecting this protein if the epitope it recognizes is masked with the histidine tags.
If you wish to I can try to find out the epitope targeted by ab41616 and check whether it is comprised within the tag region.
Regarding the failure of ab1187 (Anti-6X His tag® antibody (HRP)) to detect the glycoprotein, it could be due to the presence of sodium azide to preserve the protein. The antibody is linked to HRP and this enzyme is very sensitive to sodium azide.
In any case, as all of our products are covered by our Abpromise guarantee, if you wish to receive a replacement for ab64456, I will be more than happy to send it to you.
However, if you want to receive the same protein, you may still experience the same problems. In order to target this protein, I can try to find within our catalogue a primary against it that does not detect the epitope within the tagged sequence, or any other solution you may prefer.
I look forward to receiving your reply.

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