Epitope fingerprinting for Antibodies

Epitope Fingerprinting

Antibody binding requires the recognition of several amino acids or at least some of their properties in the epitope either in a continuous sequence or structurally close residues. The adjacent and often intermitting amino acids can vary to some extent. With epitope fingerprinting services EPITOPIC defines the complete variability of the motif recognized by an antibody. Even for antibodies recognizing apparently the same epitope the method will render different allowed variations in those positions not essential for binding and facilitates the determination of the minimal requirements of amino acids essential for binding. This is important in many aspects like determining the specificity of an antibody or to enable patentability of antibodies against the same antigen (biosimilars).

  • Quality from knowledge
  • Amino acid resolved epitopes
  • New ways to understand epitope variability
  • Superior to mapping with synthetic peptides


Figure 1: Alignments of peptides found on anti-FLAG® M1 (left) resp. anti-FLAG® M2 (right). The antibodies were raised against the DYKDDDDK-peptide. Anti-FLAG® M2 is considered to be more specific. Relative abundance of the amino acids surrounding the DYK motif explains the higher specificity of the anti-FLAG® M2 antibody. Data is based on 1,347 resp. 1,940 different DYK-sequences.

The examples on the left (Fig. 1) show fingerprints of the FLAG®-M1 and –M2 antibodies (Sigma) raised against the DYKDDDDK peptide. This analysis of all sequences containing DYK was carried out for the amino acids following this motif and results in the fingerprints of the observed amino acid distribution. The mostly preferred and more specific FLAG®-M2 antibody shows a significantly more specific fingerprint. (Data with permission from Fraunhofer IZI, Leipzig)


Figure 2: Fingerprint for CD227 mAb recognizing the Mucin-1 PDTRP motif. Variance of the central amino acid in all sequences containing PDxRP.

Figure 2 represents a fingerprint for the epitope PDTRP recognized by the Mucin-1 antibody. The statistical epitope fingerprinting data suggests that this antibody prefers other amino acids but Threonine in the central position. Other amino acids are enriched as well. So the recognized motif should be denominated as PD(T)RP instead. This would be in perfect agreement with the structural problem to recognize all five consecutive amino acids with the same precision. It is also indicating a potential contact in the antibody’s structure, which cannot be discovered with standard methods. A complete report for the Mucin-1 antibody is available here.

FLAG® and anti-FLAG® are registered trademarks of Sigma-Aldrich Co. LLC.
Header – mouse anti-human CD227 antibody (BD Biosciences, Clone HMPV) in complex with its highlighted epitope sequence PD(T)RP

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