Hi All,
I am working on conjugating mAbs to the surface of PLGA-PEG-MAL nanoparticles. Free thiols are added to the mAbs using Traut's reagent, which will then be added to the NP suspension.
I am wondering if anyone has had experience with this conjugation and would advise me on which concentrations and ratios of mAbs and NPs would be best.
Any help would be wonderful.
Thank you!
EF
Hi Eduard,
I have had some experience in the past conjugating proteins bearing a thiol to maleimide functionalised nanoparticles (that case gold: https://www.researchgate.net/publication/265733952_Monovalent_maleimide_functionalization_of_gold_nanoparticles_via_copper-free_click_chemistry/stats).
You reaction is slightly different, in that your Ab will have quite a few thiols (how many you have you could estimate if you know the mAb structure). My concern would be that if there are a lot of thiols on your antibodies is that you could possibly cross-link particles if the concentration of thiol-mAb is low, which I guess would be bad for you. My advice would be to have the antibodies in a huge excess (conc. you will have to determine for yourself) over the nanoparticles, to minimize this problem.
Then you can purify your particles from the remaining unreacted Ab's.
Good luck!
Article Monovalent maleimide functionalization of gold nanoparticles...
Hi Daniel,
Thanks for the response. If I understand correctly you are saying a low concentration of high-thiol mAbs could cross-link nanoparticles? Why would a higher concentration be a better option, would that not just lead to increased mAb-mAb coupling instead?
I am planning on trying the conjugation once without using traut's anyway, in this case there should not be a huge number of thiol groups on the mAb. I am hesitant to use a huge excess of mAb only because the one I will be using is very expensive.
Thanks for the help!
The reason to use an excess would be to try to saturate the maleimide binding sites on your particles. This would reduce the chances of the situation where a highly valent thiol antibody binds to the particle and due to incomplete coverage or saturation of the maleimides on your particles, e.g., at low concentrations, or better put, low molar ratio of thiol-mAb to nanoparticles, you are likely to get binding between particles and cross-linking.
Yes, that could be a good idea if the antibody is precious. Do you know whether your antibody has any free thiols? This is important as for the reaction to proceed you require the thiol to be in its reduced state, i.e., not in a disulphide bond.
I had a thought that you could also maybe forgo the Trauts step altogether, if you reduce the disulphides in your antibodies. You could try treating them with some TCEP, a non-thiol containing reducing agent, which would liberate the thiols in disulphides for reaction.
Hope this helps
In papers I have read, that cross-linking of NPs has not been an issue but it makes sense that it is possible. Thioglycerol quenching is performed after conjugation to stop that from happening in storage post conjugation though.
The mAb definitely has cysteine residues, I am already using TCEP*HCl to reverse the mAb accumulation occurring while in storage. I am thinking of adding EDTA to the reduced mAb to keep the reduction from reversing and just trying the conjugation without Traut's.
Incidentally, have you had experience with applying TCEP*HCl to mAb solutions? I am having concerns that the low pH (~4) caused by 5 mM TCEP*HCl in 1x PBS will harm the mAb structure/activity. What do you think?
Thanks,
EF
The thioglycerol step is definitely a good thing to do after conjugation.
I have not had experience with mAbs specifically while using TCEP, but I have with proteins. You are rightly cautious. The way I did this previously was to make a concentrated stock of TCEP in PBS, adjust the pH, and then add the amount I required to the protein in buffer for my desired concentration.
I have considered making a stock and just adjusting pH before use, however, as the attached paper implies ("scheme 2" page 2) free protons are needed for the reduction reaction to proceed. What pH did you raise the stock to when you did your work?
Thanks,
EF
https://gmwgroup.harvard.edu/pubs/pdf/310.pdf
There will still be free protons in your buffer, just less :)
I raised the pH to 7.4, as that's were my protein was happy.
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