I'm looking for an antibody either for flow cytometry or histology that recognizes the peptide antigen bound to MHCII on mouse dendritic cells. I want to track antigen transport to the skin draining lymph node after sub-cutaneous immunization.
I know there is the SIINFEKL antibody which recognizes OVA257-264 bound by H-2Kb (mouse MHCI), but I can't seem to find a similar thing for MHCII.
Does anybody know?
Hi Dunja,
Maybe you can give the Y-Ae monoclonal antibody a go. It detects I-Ea peptide 52-68 complexed to I-Ab MHCII on C57BL/6 mice (ref: Nature, 353 (1991), pp. 660–662)
It has been used quite successfully to track antigen trafficking and presentation by FACS (eg ref: Immunity 26, 491–502, April 2007).
Good luck.
Hi Dunja,
I have used the combination of CD11c-FITC (BD) and MHC Class II (I-A/I-E)-APC (eBioscience) by FACS to obtain "mature" dendritic cells from the livers of mice.. I hope it can be useful.
Good luck with your experiment.
Hello Dunja,
for the combination Ak and the peptide HEL48-61 there are two anti-peptide in the groove antibodies available. One has been made in Emil Unanue's lab (Dadaglio et al., (1997) Immunity 6, 727-738), the other one is from Ron Germain's lab at the NIH (Zhong et al. (1997) PNAS 94, 13856-13861). I guess these are not the only ones, but they are well characterized and several other groups (e.g. Paul Roche's laboratory) have published work involving these antibodies.
Best regards,
Robert
Hi Dunja, perhaps the Y3P clone (Janeway, 1984) is something for you. It specifically recognizes mouse MHCII starting from the p10-MHCII processing stage and beyond (van Niel et. al , Immunity 2006, figure 2). You will have to check which alleles it recognizes though, I don't know that by heart. However, it does not recognize a specific antigen loaded MHCII.
The Y-Ae antibody also works, I have used that myself in flow cytometry.
Cheers, Toine.
Regarding the Y-Ae antibody: Despite its widespread use, it is not as specific as originally believed. See Henri et al. 2010 JEM. Vol. 207 No. 1, pp 189-206 (http://www.ncbi.nlm.nih.gov/pubmed/20038600). From the abstract: "We further showed that Y-Ae, an antibody that is widely used to monitor the formation of complexes involving I-Ab molecules and a peptide derived from the I-E alpha chain, recognizes mature skin DCs that express I-Ab molecules in the absence of I-E alpha."
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