Peptide phage display using random peptide genes has been applied with limited success over almost 25 years without any substantial changes in the way epitopes are identified. Epitope mapping usually results in a set of a few sequences sharing similar or identical residues with the target. New methods in gene synthesis based on trinucleotide synthesis, high throughput sequencing by next generation sequencing (NGS), availability of computational methods and increased computing power have led to substantial improvements. The general limitations of phage display approaches with respect to the clonal stability, library complexity and reproducibility have, however, remained. The high error rate of NGS creates additional problems in the reliability of counting individual sequences.
- Quality from knowledge
- Amino acid resolved epitopes
- New ways to understand epitope variability
- Superior to mapping with synthetic peptides
- EPITOPIC’s Service requires µg quantities
To overcome these individual limitations, EPITOPIC generated an optimized workflow for the application of all steps from selection to data evaluation.In our hands NGS data sets can be extracted to deliver detailed information about epitopes and their variations accepted by antibodies derived from hundreds and more different sequences, often after a single round of selection. The results of EPITOPIC’s epitope mapping are best visualized as alignment of sequences sharing similarity with the antigen. It is not surprising that most epitopes consist of sets of two or three amino acids interrupted by more or less conserved amino acids. A typical example is the epitope of the well known c-myc antibody. At a central position the antibody cannot differ between Ile and Val, probably because the tip of the side chain is pointing away from the antibody. (see example in Statistical Peptide Phage Display).
Using EPITOPIC’s technologies these variations can be predicted from statistical data alone. (Data with permission from Fraunhofer IZI, Leipzig)
Alignments for CD227 mAb recognizing the Mucin-1 PDTRP motif. Variance of the central amino acid in all sequences containing PDxRP. Already the first selection round (left) generates enough data to generate a complete fingerprint. Second selection round on the right.
A complete report for this antibody is available here.
Header – mouse anti-human CD227 antibody (BD Biosciences, Clone HMPV) in complex with its highlighted epitope sequence PD(T)RP