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HOME> Products> Miscellaneous> Others> Epitope Mapping Service Using the DECODE Method

Comprehensive epitope mapping at single–amino acid resolution Epitope Mapping Service Using the DECODE Method

Date：August 06 2026Web Page No：520124

This contract service provides high-resolution epitope mapping at the single–amino acid level using the DECODE^*1 method, developed by a research group led by Dr. Hiroki Ueda (RIKEN / The University of Tokyo).
By combining peptide selection using the DECODE method with next-generation sequencing (NGS) and high-performance computational analysis, the service enables precise identification of antibody epitopes at single–amino acid resolution, along with comprehensive prediction of cross-reactivity.
It supports not only the detailed characterization of monoclonal antibodies but also the evaluation of non-specific binding and cross-reactivity in polyclonal antibodies.

*1 DECODE : Decoding Epitope Composition by Optimized-mRNA-display, Data analysis, and Expression sequencing
* Click here for ordering information.
* The service is for research use only.

Epitope mapping service by the DECODE method

About Epitope Analysis and the DECODE Method
Features
Analysis Items
Applications Examples
Compatible Antibody Types and Required Amount
Publications
How to order

About Epitope Analysis and the DECODE Method

Antibody Epitopes

More than 7 million antibodies are currently commercially available worldwide. However, concerns regarding their reproducibility and reliability have persisted for many years. In particular, non-specific binding—where an antibody recognizes proteins other than its intended target—and lot-to-lot variability in the performance and specificity of polyclonal antibodies remain significant challenges for researchers.
Information about the epitope—the region recognized by an antibody—is critical for evaluating specificity and cross-reactivity. In general, when an antibody recognizes a linear epitope (a continuous amino acid sequence), it typically binds to fewer than 10 amino acid residues. Moreover, within antibody–antigen interactions, especially critical amino acid residues (so-called “hot spots”) are often limited to fewer than five residues.
Accurate epitope information is essential for selecting highly specific antibodies, optimizing experimental conditions, improving reproducibility and reliability, and ensuring the correct interpretation of experimental results.

DECODE-Based Epitope Mapping

The DECODE method is a peptide selection technology based on an improved mRNA display protocol.
In this method, a highly diverse random DNA library (1×10¹³ variants) is first transcribed in vitro to generate mRNA. Puromycin is then ligated to the mRNA molecules (mRNA-Puromycin-DNA). During translation, puromycin enters the ribosomal A site and forms a covalent bond with the C-terminus of the newly synthesized peptide. Through this mechanism, each translated peptide becomes covalently linked to its encoding mRNA, resulting in stable mRNA–peptide fusion molecules (mRNA-peptide). These mRNA–peptide fusions are subsequently incubated with the target antibody immobilized on beads. Sequences that bind to the antibody are selectively captured and recovered for downstream analysis.

After three rounds of this selection, the enriched sequences are analyzed by next-generation sequencing (NGS). Based on the most highly enriched and converged peptide sequences, amino acid frequencies are calculated and visualized as a sequence logo. In this representation, each letter corresponds to the one-letter code of an amino acid, and the size of the letter reflects its relative frequency or importance at that position. When multiple amino acids are stacked vertically at the same position, this indicates sequence variation at that site.

Furthermore, the identified peptide sequence information is systematically screened against the Protein Data Bank (PDB) to estimate potential cross-reactivity, enabling genome-wide assessment of antibody specificity.

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Features

Epitope information can be obtained at single amino acid resolution^*2.
Potential cross-reactivity can be predicted across comprehensive protein databases^*3.
Cost-effective epitope mapping service.

*2 Analysis is limited to antibodies recognizing linear epitopes.
Recognition of conformational epitopes or post-translational modifications,
such as glycosylation, cannot be analyzed.
*3 Please contact us regarding sequences not registered in the protein databases.

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Analysis Items

Identification of the epitope region within the target protein
Identification of key amino acid residues strongly recognized by the antibody
Genome-wide prediction of cross-reactivity (one species selected from mouse, rat, or human)^*4

*4 For advanced analyses, additional analysis targets, or provision of complete computational results for all proteins,
please contact us for consultation.

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Applications Examples

Anti-FLAG Monoclonal Antibody (clone: M2)

An anti-FLAG monoclonal antibody, for which the epitope motif (DYKXXD) has been previously reported, was analyzed using the DECODE method.
NGS analysis of the sequences enriched by this antibody showed that peptides containing the reported epitope motif DYKXXD were enriched through the DECODE process, consistent with previous findings.

Evaluation of Monoclonal Antibody Specificity

Three different clones of anti–c-Fos antibodies (Clone I, Clone II, and Clone III) were analyzed using the DECODE method. Antibody-binding regions and hotspot residues were identified, and the top 20% of converged amino acids were visualized as sequence logos (see sequence logo figure).
Peptides (12 amino acids) with high DECODE scores were synthesized and tested for binding to each antibody by ELISA.
The antibodies recognized the DECODE-identified sequences, whereas peptides containing mutations in two predicted hotspot residues did not bind (see ELISA evaluation figure). These results indicate that the DECODE method identifies epitopes at single–amino acid resolution.
Genome-wide cross-reactivity was further assessed by calculating DECODE scores for all mouse proteins obtained from UniProt. Clones I and II showed high specificity, while Clone III exhibited predicted cross-reactivity with other proteins (see genome-wide cross-reactivity figure).

* ELISA validation is not included in the standard analysis items of this service.

	Clone I	Clone II	Clone III
Sequence Logo
ELISA
Genome-wide cross-reactivity

Click the images to enlarge（🔍）

Analysis of Monoclonal Antibodies with Different Clones

Monoclonal anti-p53 antibodies derived from different clones were analyzed using the DECODE method.
The results showed that, despite being distinct clones, certain antibodies recognized identical epitope motifs at the motif level. Specifically, clones A, B, and C (red box in the figure) shared the same recognition motif, while clones D and E (blue box in the figure) recognized a different but common motif.

Analysis of Polyclonal Antibodies (Lot-to-Lot Variation)

Polyclonal antibodies of the same catalog number but from different production lots (Lot 1 and Lot 2) were analyzed using the DECODE method. As expected for polyclonal antibodies, multiple recognized sequences were identified.
Although the catalog number was identical, the two lots were found to recognize different epitope profiles. These results suggest that lot-to-lot variation in polyclonal antibodies may affect epitope recognition and could potentially impact experimental reproducibility.

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Compatible Antibody Types and Required Amount

Compatible antibody type : IgG antibodies^*5 (Purified^*6)
Required antibody amount : 10 μg^*7

*5 Only antibodies recognizing linear (primary structure) epitopes can be analyzed.
Recognition of conformational epitopes or post-translational modifications, such as glycosylation, is not supported.
*6 Unpurified antibodies will not be accepted. For antibodies purified using Protein A/G,
background signals may increase and potentially affect the results.
*7 Please contact us if less than 10 μg of antibody is available.

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Publications

Matsumoto, K., et al., "DECODE enables high-throughput mapping of antibody epitopes at single amino acid resolution", PLoS Biol., 23(1), e3002707 (2025). [PMID:39847587]

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How to order

Please download Quotation Request Sheet and Letter of Assurance, fill out both forms including your antibody information, and send the files to your local distributor. Please note that we only accept Excel and Word files; we cannot accept PDF or FAX. Once reviewed, we will issue a quotation through your local distributor.
Approximate lead time of the service is for 3 months from receiving your antibodies.

* This analysis is available for IgG only.
* Unpurified antibodies are not accepted. Protein A/G-purified antibodies may result in higher background signals.
* Antibodies containing sodium azide or thimerosal are not accepted. Samples containing ProClin can be accepted.
* Please note that this service is limited to the analysis of linear epitopes.
Conformational epitopes and epitopes that depend on post-translational modifications or other structural features cannot be analyzed.
* Any remaining submitted antibody will be discarded after the analysis is completed.
* Antibodies in borate buffer cannot be accepted.

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CONTACT

export@funakoshi.co.jp

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