Confirm Your Target Peptides
The imuPep Class I Peptide Binding Analysis is an innovative, high-throughput service of imusyn to determine the stability of peptide/HLA class I complexes. We offer this service for an unmatched portfolio of eukaryotic recombinant HLA class I alleles, thus allowing a quick and reliable assessment of immunogenic peptides.
Increasing attention is being paid to the response of CD8 positive T cells to new vaccines, whether against infections or against cancer. Often, the peptides of interest are identified or rationally designed in in silico studies. While these studies usually give a good idea of peptide affinities, the stability of the resulting peptide/HLA class I complex remains unknown many times. To fulfil their antigen-presenting function, however, HLA class I molecules must not only bind the peptides generated inside the cell but also remain at the cell surface long enough to elicit a T cell response. Data are accumulating that suggest that this stability of the peptide/HLA class I complex is one of the most important determinants for immunogenicity of a peptide. Therefore, a reliable assessment of complex stability is an important step in the development of new T cell epitopes to identify optimal peptides at a very early stage of a project.
By combining imusyn’s cutting edge technologies, we are able to offer a new and innovative way to determine the stability of HLA Class I/peptide complex.
imusyn has established a method to load recombinant soluble HLA class I proteins with peptides. Following this, we determine peptide binding by using two approaches: first, we measure the change in intrinsic protein fluorescence during a temperature ramp (differential scanning fluorimetry, DSF). A shift in the spectrum of tryptophan and tyrosine fluorescence emission indicates denaturation of the protein/peptide complex. The denaturation temperature (here: inflection point temperature, Ti) is directly correlated with the thermal stability of the complex.
In borderline cases, where DSF alone is not giving a clear result, we confirm peptide binding by running the loaded complex on a native PAGE. Imusyn’s HLA class I are loaded with endogenous peptides of different charges, resulting in a ladder-like appearance of the protein on the gel. If the peptide is loaded successfully, this ladder is usually reduced to one or two bands on the gel, or one of the bands becomes significantly more prominent (a comparable approach can be found in Mancino et al., 2002 ⧉).
The combination of the two assays give a reliable conclusion on peptide binding. The peptide binders for one allotype can be ranked by their Ti.
(1) Choose from more than 60 different HLA class I allotypes (more available on request)
(2) No need to disclose your sequences if you provide the peptides
(3) Quick and reliable
When looking at your data, there are two possible scenarios:
(1) There is a considerable shift (more than 1°C) in the Ti of the HLA class I + peptide sample in comparison to the control measurement without peptide. This is a clear indication of peptide binding.
(2) There is little or no shift in the Ti. If the “steepness” of the curve remains identical to the control, this indicates that the peptide has not bound. If there is a difference in the curve shape, we run a native PAGE as a second method to confirm peptide binding. If the band pattern changes with the peptide added, this is an additional conformation of peptide binding.
Weak peptide binders can have Tis as low as 40°C. In these cases, a second Ti is usually visible, which stems from the re-binding of endogenous peptides (Figure 1). Peptides that bind well usually have a Ti in a range of 65°C to 75°C, whereas extremely stable peptides form complexes with a Ti higher than 75°C.
In addition, the overall stability of the complex depends strongly on the allotype you are looking at. The control measurement can give you a good idea of the allotype’s thermal stability, giving you another parameter on which to judge the stability of your peptide/HLA class I complex.
In our experience, peptides with a dissociation constant greater than 10 µM do not bind in our assay. However, this may vary depending on the allotype you are testing.
Please use our order form for your orders or if you need a quote.
When you order the imuPep Class I Peptide Binding Analysis from imusyn, you can either
(1) have the peptides synthesized by us (lead time of about four weeks), or
(2) provide your own peptides.
If you send us peptides, we prefer them in liquid formulation. Please inform us about the concentration, buffer, and the number of tyrosine or tryptophan residues. We require at least 100 µl of ≥ 1 mg/ml peptide. If you cannot meet these criteria, please contact us beforehand.
We also accept lyophilized peptides, if you provide us with a detailed protocol for reconstitution. Please send at least 100 µg of your peptide.
All information about the peptides can be put into the order form.
Should we not be able to determine if a peptide binds to a specific HLA class I or not, you will not pay for this assay.
Due to the measurement principle with intrinsic fluorescence, it may be that peptides with more than one tyrosine or peptides with tryptophan will not give meaningful results in a DSF measurement. In this case, we can still determine general peptide binding by native PAGE.
Peptides that only reconstitute in denaturing buffers, e.g. buffers with trifluoroacetic acid (TFA) or buffers that have an extreme pH, may not work in the imuPep Class I Peptide Binding Analysis. In addition, reducing agents such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) can influence the measurements. Please get in touch with us if you are unsure whether your buffer conditions fall into this category. We may refuse to measure peptides in unfavorable buffers.
If you have specific questions or want to learn more about imuPep, you can contact us via our contact form.