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
  • Resolution of epitopes at the amino acid level
  • 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.

Several approaches for mapping the epitopes of antibodies are known, but they are usually resulting in approximate information about the location of the epitope on the antigen. Peptide arrays result in the epitope recognized in the protein with a precision of +/-3 amino acids. Similar results are obtained from phage display based on a limited number of sequences sharing similarity with the antigen. Additional studies with antigen mutants or scanning peptide epitopes by stepwise replacement of the amino acids with alanine may reveal essential amino acids but they are laborious and will not result in any information comparable to epitope fingerprinting.

The examples on the left 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: 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 described in Epitope Mapping. The initial epitope data suggested that the antibody prefers other amino acids but Threonine in the central position. Indeed is the Glutamic Acid enriched as well. So the recognized motif should be denominated as PD(E/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.

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