I want to know if any of you has an idea about how much does the using of a DTT treated protein (as immunogen in mice) affects the mAb production process?
denaturation with DTT can affect the protein structure, and modify epitopes which are the target is CD4 + T cells, or dendritic cells and finally the B-cell immune response can then be very different between a natural protein, and denatured recombinant molecule.
On the other hand DTT may have adjuvant activity, mainly by targeting certain receptors of dendritic cells unsuitable for a good immune response (eg scavenger receptors).
This type of misuse of immunization is wanted in allergy, for example.
Thanks Jean,
Then It will better to use the PD-10 column or the dialysis to remove the urea/DTT, just before the immunization.
You are aware that even if you remove DTT/urea with PD-10, in most cases, you still have a denatured protein and not a native antigen? Only small proteins spontaneously fold to the right conformation. For which purpose do you want to make your monoclonal antibodies?
Thanks @Michael,
The ultimate target is to establish an ELISA methodology to measure the specific Ab in salmon serum against a particular Ag.
The problem is that we have to use DTT with Tev protease to remove our tag before immunizing the mice with the tag free r-protein. any tips?
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