I want to make an assay of in vitro proliferation of PBMC in animals immunized using the same vaccine antigen. To do this I have to process the vaccine. Among the existing techniques as UV, sonication, heating, etc. Which ones are best??
It depends on the nature of the antigen and the susceptibility of the CD4 and CD8 epitopes to disruption via the various processing methods you've listed. Are you using a whole/native antigen, subunits, or perhaps peptides that have been characterized as the specific CD8 and CD4 epitopes for your pathogen? When we test vaccines against various pathogens, we usually use panels of known immunodominant peptides corresponding to the antigens used in the vaccine. This is employed for cell proliferation assays as well as ICS. It avoids inadvertent alteration of the peptides during chemical/physical processing and also eliminates the need for APCs to process antigen macromolecules into peptides. This is straightforward if the immunodominant peptides have been characterized for your model, such as LCMV or PR8 influenza in B6 mice. In cases where the actual peptide recognized by the CD4 or CD8 T cells is unknown or unavailable, then we have employed various methods of processing, usually using previously published protocols as a starting point. Again it depends on the nature of the antigen. The processing we use for mycobacterial peptides (whole cell lysates) are different from those used for Borrelia (heating is unrewarding) and much different from viruses and fungal pathogens. Perhaps you could clarify the nature of your antigen.
Most vaccines also have additional ingredients (adjuvants, preservatives, etc.) that you probably do not want to use in your assay. If the antigen is a single protein or conjugate, I would try to obtain some of the purified protein commercially. If it is not commercially available, you could try to contact the vaccine manufacturer to see if they would be interested in supplying the material they use as part of a collaboration (they may be interested in the results).
Additional information would be helpful. Are you using an inbred mouse model? Do you know the amino acid sequence(s) of your antigen(s)? Have you considered stimulating your T cells (+ exogenous APC) using overlapping peptide pools to determine the immunodominant peptides?
+1 to Greg's suggestion of overlapping peptide pools to determine immunodominance, it can be super effective when available.
The three proposed techniques are methods commonly used to study the aging of the active ingredient of the vaccine: the heating being the most common.
Regarding the antigen itself, the study focuses instead on the detergents, preferably non-ionic detergents.
Finally, the use of PBMC proliferation assays gradually gave way to tests comparing the actions of the antigen on different populations of DC (mDCs, pDCs, IpC ...), isolated from PBMC.
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