see http://www.nature.com/articles/srep33935#f6

Hope that helps. At least the paper itself is really worth reading for EV-related technologies. I would highly recommend it. Philippe

For dissociation of antigen-Ab interactions, two methods are commonly employed: use of low pH buffer (such as Glycine buffer pH 3.0-3.5) and high salt. Both methods work adequately well for relatively stable ligand and antibody (depending on what you are purifying, one may be sacrificed if necessary). 

If you choose salt, rather than NaCl, use 2.5 MgCl2 at neutral pH - it is tolerated better than other options.

If possible, try an ultracentrifugation based method to avoid the problem of dissociating interactions by harsh methods.

1. íf you know the ab's epitiop, you can try elute by excess epitop. however, this method was used for small chemical epitops mostly, i think. (nowadays, it is relatively easy to generate rec proteins. however, you should know the epitope of your ab well, if it is glycosylated, method in E coli will not work. According to CD63 biology (just a brief check), this approach does not look promising. 

2. you can try to release from beads by disrupting the binding abs with a reducing agent (DTT, mercaptoethanol) similarly like stripping a blot. it can affect the vesicles probably..

Hi,

When developing the Dynabeads CD63 beads for exosome isolation and analysis  the main focus was to offer a solid support platform for exosomes which could be recognized by the flow scatter and then be able to do detailed analysis of stained exosomes located on the bead surface . The Ab-Ag interaction in this case is very strong and no release mechanism was therefore included in this product. The low pH or high salt strategy can of course be tested but it is most likely not very effective.

kind regards

ketil

Thank you for your suggestions guys, and sorry for my late answer. We have been trying 2 different home-made buffers:

- 1% formic acid in PBS

- 0.2M glycine, Tris-HCl pH 2.8 in PBS

Any of them worked in our hands. So finally we purchased EXOFLOWBUFR-2 from Cambridge Biosciences and is working fine, according to Nanosight and ELISA results.

Hi Victor, 

Elution of exosomes off of magnetic beads is common practice. I would reccomend against a pH-based elution as it can be difficult to regain neutral pH and exosomes can lose some stability when left at pH < 3 for an extended period of time. 

I would recommend either a salt-based elution or using the elution buffer (ExoFlowBuffer2) found in SBI's exosome capture kit (Exo-Flow/IP). There are a variety of different biotinylated antibodies (CD63, CD9, Rab5b, etc.) provided in the ExoFlow kits that can serve your purpose

Hi everybody,

do you know if it is possible to purchase the exoflowbuffer2 without purchasing the whol exo-flow kit?

Thanks in advance,

Stefano

Stefano, yes, I called SBI and they told me that it is possible to purchase only the exoflowbuffer2. You have to call them, it is not possible to order online.

My question now is if the exoflowbuffer2 would also dissociate the antibody from the exosome, since I need the exosomes alone (without bead or antibody) to treat cells. In my case, I stained the exosomes with an antibody for my protein of interest. I want to sort exosomes -/+ for the protein of interest. Then treat cells with the sorted exosomes, but for that, I need the antibody that is bound to the protein of interest on the surface of the exosomes to be eluted, as well as the bead.

TIA in case anyone knows the answer to that question!

Thank you so much!

Mayra Tuiche , in theory the Abs should be chemically linked to the mag bead and should not dissociate in the same way the Ag-Ab interaction is broken with high salt or low pH (we use 0.1M glycine pH 2.2, and then immediately neutralize with Tris-Cl pH 9.0, for what it's worth). It is still possible that the Ab will "leach off" the bead no matter what you do, but that is an issue to take up with the manufacturer.

Hi Maja Kosanovic

sorry, I didn't run any specific quantification of the EVs that remained attached to the beads when using those elution buffers. The eluate did not contain any EV after using 1% formic acid in PBS or 0.2M glycine, Tris-HCl pH 2.8 in PBS. In contrast, we could recover EVs from the beads when using EXOFLOWBUFR-2.

Trifluoroacetic acid. We use this for biopanning (plate coated with EVs )

Hi Chirantan Debnath ,

could you please specify the trifluoroacetic acid concentration you use?

Thank you in advance,

Marco

Hi Victor Llombart ,

I'm also trying to elute the EVs but using the Exosome - Human CD9 Isolation kit.

I tried the EXOFLOWBFR2 solution like you but I'm not sure that it worked for me.

Could you kindly give me your conditions for the elution (time, temperature, rotation etc ...)?

Thank you in advance,

Marco

Hi Victor Llombart,

We have succesfully eluted EVs from CD9, CD61, CD63, and CD81 monoclonal antibodies immobilized inside monolithic columns with pH 11.3 ammonium hydroxide or pH 11.3 carbonate bicarbonate solution. This elution strategy should work with antibodies on beads as well. The preparation of the solutions you can find in this article:

Article Fast isolation of highly specific population of platelet-der...

The eluate can be neutralized with HCl immediately after the elution to reduce exposure to high pH.

Br,

Evgen

Marco Morani we use 14ul TFA+1000ul nuclease-free water. from that we use 100ul. Hope it will help.