Hello~~Recently, I used expedon's kit to conjugate 5'-aminated oligo with antibody and it failed. I consulted the technician of Expedon and we can't find the reason. Is there any other kit for this application?
Hallo Chenyu Lu,
in our laboratory we also worked on this issue. We found, that for some antibodies, it is rather easy, and for some others it is quite tricky. Bioconjugations usually need some optimizations, although you have kit. Its is because biomolecules, especially antibodies, are so divers.
I assume you use a SMCC Crosslinker (So NHS-chemistry for amino-modified DNA and Maleimide for -SH Groups of the antibodies?) It is rather important that the buffer system does not contain any primary amines, surfactants, is freshly prepared and the pH is according to the protocol. Its very important the the compounds are as pure as possible. For -SH coupling, the Disulfide of the antibodies (-S-S-) need to be reduced (e.g. with TCEP or with MEA) the reduced antibody is however not stable and the reaction with the SMCC crosslinker should be carried out immediately, otherwise the -SH groups will be oxidized by Oxygen to form -S-S- again. There are also kits from abcam:
https://www.abcam.com/oligonucleotide-conjugation-kit-ab218260.html
Here, they also provided some tips:
https://www.idtdna.com/pages/education/decoded/article/improving-immuno-pcr-by-optimizing-antibody-oligo-conjugation
However, you maybe need to switch the coupling chemistry. You could als use Iodacetmide chemistry croslinking for examples. in the link below, they used it for peptide conjugation, however, it can also be employed for NHs-modified Oligos and reduced antibodies. The Thioesters are usually more stable then the maleimide Michael adducts.
Article Predictable Peptide Conjugation Ratios by Activation of Prot...
Here is maybe some helpful literature:
https://pubs.rsc.org/en/content/articlelanding/2017/ob/c7ob02216f#!divAbstract
(Here we used the same chemistry, however with a thiol modifed DNA and coupled it to -NH2 groups from lysins of the antibodies.
Here we used exactly the same chemistry:
Article An Anticaffeine Antibody-Oligonucleotide Conjugate for DNA-D...
Here they used "click chemistry" (coupling an Azide to an Alkine). However, this is a bit more "advanced":
Article Simple Method To Prepare Oligonucleotide-Conjugated Antibodi...
I hope this helps you a bit!
Best regards
Peter
Hallo Chenyu Lu,
in our laboratory we also worked on this issue. We found, that for some antibodies, it is rather easy, and for some others it is quite tricky. Bioconjugations usually need some optimizations, although you have kit. Its is because biomolecules, especially antibodies, are so divers.
I assume you use a SMCC Crosslinker (So NHS-chemistry for amino-modified DNA and Maleimide for -SH Groups of the antibodies?) It is rather important that the buffer system does not contain any primary amines, surfactants, is freshly prepared and the pH is according to the protocol. Its very important the the compounds are as pure as possible. For -SH coupling, the Disulfide of the antibodies (-S-S-) need to be reduced (e.g. with TCEP or with MEA) the reduced antibody is however not stable and the reaction with the SMCC crosslinker should be carried out immediately, otherwise the -SH groups will be oxidized by Oxygen to form -S-S- again. There are also kits from abcam:
https://www.abcam.com/oligonucleotide-conjugation-kit-ab218260.html
Here, they also provided some tips:
https://www.idtdna.com/pages/education/decoded/article/improving-immuno-pcr-by-optimizing-antibody-oligo-conjugation
However, you maybe need to switch the coupling chemistry. You could als use Iodacetmide chemistry croslinking for examples. in the link below, they used it for peptide conjugation, however, it can also be employed for NHs-modified Oligos and reduced antibodies. The Thioesters are usually more stable then the maleimide Michael adducts.
Article Predictable Peptide Conjugation Ratios by Activation of Prot...
Here is maybe some helpful literature:
https://pubs.rsc.org/en/content/articlelanding/2017/ob/c7ob02216f#!divAbstract
(Here we used the same chemistry, however with a thiol modifed DNA and coupled it to -NH2 groups from lysins of the antibodies.
Here we used exactly the same chemistry:
Article An Anticaffeine Antibody-Oligonucleotide Conjugate for DNA-D...
Here they used "click chemistry" (coupling an Azide to an Alkine). However, this is a bit more "advanced":
Article Simple Method To Prepare Oligonucleotide-Conjugated Antibodi...
I hope this helps you a bit!
Best regards
Peter
Another possible source for oligo labeling kit is Novus Biologicals. Our listings are here:
https://www.linscottsdirectory.com/products?q=oligo%20conjugation
just follow "more info" to their data pages.
My advise is the following, already mentioned by excellent answer from Peter Carl: "Here we used the same chemistry, however with a thiol modifed DNA and coupled it to -NH2 groups from lysins of the antibodies."
Be aware that the Cysteines in antibody are not free but form disulfide bridges. If you want to employ thiol coupling you need to carefully reduce the disulfides in the hinge region (without reducing e.g. those in the variable domains). Then you get 4 - 6 free thiols per antibody molecule. But if you use an -SH group at your Oligos you can use amine coupling to crosslink this to the antibody, which has a lot of Lysines. The disadvantage in this setup is that you may inactivate the antibody by blocking the paratope (antigen binding site), but this can in most cases be overcome. Also, you can make a control experiment using NHS-biotin to quickly assess the activity of your antibody after coupling.
Good luck, Markus
You can get a visualization of conjugation of Antibody and DNA based Drugs from here.
https://www.youtube.com/watch?v=SJEulFA2m5c
This kit helps link Antibody and DNA based drugs
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