MAJOR HISTOCOMPABILITY COMPLEX CLASS II

 

Assays and molecules

 

 

 

Our products and services include:

- MHCII affinity and stability assays.

- Customized biotinylated MHCII/peptide tetramers.

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 MHC II affinity and stability assays

Computational methods to identify MHC binding peptides exist, they do however tend to be imprecise and over-predictive.

Affinity assays
Immunitrack offers state of the art peptide MHC affinity measurements by very sensitive assays. The assays use nanomolar concentration of MHC II and measure the direct binding of peptide to MHC II. The assays have been validated in several NIH funded projects and peer reviewed articles and account for more than 100.000 Kd measurements in the Immune Epitope Database (IEDB).

Stability assays

For MHC I it was shown that stability is a better indicator of immunogenicity than affinity, preliminary results from Immunitrack supports this hypothesis for MHC II. Our stability assay measures the decay of MHC II complexes at physiological temperature enabling precise half-life determinations. All assays are amendable for HTS screening and does not require any modifications of peptide.

 

Affinity vs. stability EBV

                       MHCII stability affinity

Figure shows two EBV derived peptides of nearly equal affinity (Kd of 1 and 6 nM). Though both peptides show strong affinity to DRB1*15:01 only the known T cell epitope (orange) have a high stability or long half-life, underlining the importance of stability analysis.


 Affinity vs. stability CMV


            Picture1                                 

Stability vs. Affinity. Peptides with a Kd lower than ~100 nM are normally considered potential T Cell epitopes. This figure underlines the fact that affinity not necessarily correlates with stability and that stability assessment is a better measure for peptide immunogenicity. x axis: Kd measurements of CMV derived peptides, y axis: stability measurements on same peptides, 100% reference peptide is not shown. Red circles represents “true” CD4 T cell epitopes identified by Elispot and MHC II tetramer analysis (PLoS One. 2014; 9(4)).


Biotinylated MHC II complexes

Our biotinylated MHC II complexes have been used as tetramer reagents in several publications. The previous challenges of producing MHC II molecules of a sufficient quality have hampered the disseminated use of MHC II tetramers. By using a tag on the peptide our production method ensures a 100 % peptide loading of complexes.

                            Picture6                                                               

              
 
PBMCs were incubated with PE- and APC-conjugated HLA-DRB5*01:01- IE1211-225(h6) tetramers. Double positive cells were cultured for 14 days with irradiated autologous dendritic cells pulsed with IE1211-225 and subsequently tetramer analyzed again with relevant and irrelevant tetramer. Adapted from PLoS One. 2013 Sep. 2;8(9):e73648. doi: 10.1371/journal.pone.0073648


Alleles available

Immunitrack is continuously expanding its MHC II repertoire and are currently able to deliver assays or complexes on the following alleles:

Human Murine
DR DQ DP
DRA1*0101 DQA1*0101 DPA1*0103 Iab (availble end Q4 2016)
DRB1*0101 DQB1*0301 DPA1*0202
DRB1*0301 DQB1*0501 DPB1*0401
DRB1*0401 DPB1*0402
DRB1*0701
DRB1*1101
DRB1*1501
DRB3*0101
DRB3*0202
DRB4*0101
DRB5*0101

MHCII alleles not present on the list may be synthesized and produced upon request. 


 

Publications where the MHC II molecules were used

1.

A carbon nanotube-polymer composite for T-cell therapy.

Nat Nanotechnol. 2014 Aug;9(8):639-47

PMID: 25086604

2.

Identification and HLA-tetramer-validation of human CD4+ and CD8+ T cell responses against HCMV proteins IE1 and IE2. 

PLoS One. 2014 Apr 23;9(4):e94892.

PMID: 24760079

3.

Adsorption of multimeric T cell antigens on carbon nanotubes: effect on protein structure and antigen-specific T cell stimulation. 

Small. 2013 Mar 11;9(5):666-72.

PMID: 23090793

4.

MHC class II tetramers made from isolated recombinantαandβchains refolded with affinity-tagged peptides. 

PLoS One. 2013 Sep 2;8(9):e73648.

PMID: 24023895

5.

Chaperone-assisted thermostability engineering of a soluble T cell receptor using phage display. 

Sci Rep. 2013;3:1162.

PMID: 23362461

6.

Identification of MHC class II restricted T-cell-mediated reactivity against MHC class I binding Mycobacterium tuberculosis peptides. 

Immunology. 2011 Apr;132(4):482-91.

PMID: 21294723

7.

NetMHCIIpan-2.0 - Improved pan-specific HLA-DR predictions using a novel concurrent alignment and weight optimization training procedure. 

Immunome Res. 2010 Nov 13;6:9

8.

HLA class I binding 9mer peptides from influenza A virus induce CD4 T cell responses. 

PLoS One. 2010 May 7;5(5):e10533.

PMID: 20479886

9.

Functional recombinant MHC class II molecules and high-throughput peptide-binding assays. 

Immunome Res. 2009 May 5;5:2.

PMID: 19416502

10.

Quantitative predictions of peptide binding to any HLA-DR molecule of known sequence: NetMHCIIpan. 

PLoS Comput Biol. 2008 Jul 4;4(7):e1000107.

PMID: 18604266

 

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