Cy5 oligonucleotide as antigen

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Cy5 oligonucleotide as antigen

Postby kristaaaaa » Nov 27 2012 8:49 am

Hi. Is it possible to use small oligonucleotide (18nt, labled with Cy5, cannot modified it more) as antigen and attach it onto microplate for ELISA analysis.
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Re: Cy5 oligonucleotide as antigen

Postby CrowSan » Nov 28 2012 11:11 am

Not sure you would get an antibody response to a DNA fragment that small. Also in humans there is a fair amount of cell free DNA in the blood (cfDNA) which does not elicit an immune response (it's around 30 ng/ml in humans which can increase to 180 ng/ml when cancer is present).

Those DNA elements that do generate an immune response (here it is IgG they mention) seem to be bacterial
"anti-DNA in the serum of normal human subjects (NHS) bind DNA in a sequence-specific manner [8,9]. Thus, these antibodies target sites that are present on some, but not all, DNA from bacterial sources. These antibodies can react with both ss as well as dsDNA, although they are highly specific for particular DNA. This specificity reflects binding to non-conserved base sequences that occur variably in DNA depending on species origin."

NHS = Normal human subject. The above is from: "The influence of DNA size on the binding of antibodies to DNA in the sera of normal human subjects and patients with systemic lupus erythematosus (SLE)"
D S Pisetsky and T C Gonzalez
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1905279/

I have not conducted an extensive literature search so you may want to check if anything more applicable has been published.

Perhaps you could do some kind of modification of the "proximity ligation assay" using this oligo?
(Google the above in quotes).
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Re: Cy5 oligonucleotide as antigen

Postby relaxin » Nov 28 2012 12:17 pm

Anti-Cy5 antibody is available from a number of companies. You may try this before generating your own.

As for attaching oligo on solid surface, please read the following:
http://www.idtdna.com/pages/docs/techni ... f?sfvrsn=4
Not affiliated with any company. Mention of a specifc product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
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Re: Cy5 oligonucleotide as antigen

Postby CrowSan » Nov 29 2012 8:17 am

Doh! Of course the ab is against the Cy5 group - not the DNA.... I must have advanced brain rot.
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Re: Cy5 oligonucleotide as antigen

Postby kristaaaaa » Nov 29 2012 9:07 am

Thank you! :D
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