Identification of distinct antibody epitopes and mimotopes from a peptide array of 5520 randomly generated sequences

Reineke, Ulrich; Ivascu, Claudia; Schlief, Marén; Landgraf, Christiane; Gericke, Seike; Zahn, Grit; Herzel, Hanspeter; Volkmer, Rudolf; Schneider‐Mergener, Jens

doi:10.1016/s0022-1759(02)00139-4

Cited by 83 publications

(46 citation statements)

References 31 publications

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“…It would be very useful if the peptide sequences identified in the immunosignaturing experiments could be used to identify the immunogenic epitopes in a pathogen or an autoantigen. Some preliminary use of random peptide arrays for epitope mapping was done by Reinke et al, but required subsequent rounds of mutational analysis of the peptides to hone in on the epitope sequence (13). However, it may be possible to use a more sophisticated bioinformatics approach to infer the epitope from these loosely related sequences.…”

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confidence: 99%

Exploring Antibody Recognition of Sequence Space through Random-Sequence Peptide Microarrays

Halperin

Stafford

Johnston

2011

Molecular & Cellular Proteomics

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A universal platform for efficiently mapping antibody epitopes would be of great use for many applications, ranging from antibody therapeutic development to vaccine design. Here we tested the feasibility of using a random peptide microarray to map antibody epitopes. Although peptide microarrays are physically constrained to ϳ10 4 peptides per array, compared with 10 8 permitted in library panning approaches such as phage display, they enable a much more high though put and direct measure of binding. Long (20 mer) random sequence peptides were chosen for this study to look at an unbiased sampling of sequence space. This sampling of sequence space is sparse, as an exact epitope sequence is unlikely to appear. Commercial monoclonal antibodies with known linear epitopes or polyclonal antibodies raised against engineered 20-mer peptides were used to evaluate this array as an epitope mapping platform. Remarkably, peptides with the most sequence similarity to known epitopes were only slightly more likely to be recognized by the antibody than other random peptides. We explored the ability of two methods singly and in combination to predict the actual epitope from the random sequence peptides bound. Though the epitopes were not directly evident, subtle motifs were found among the top binding peptides for each antibody. These motifs did have some predictive ability in searching for the known epitopes among a set of decoy sequences. The second approach using a windowing alignment strategy, was able to score known epitopes of monoclonal antibodies well within the test dataset, but did not perform as well on polyclonals. Random peptide microarrays of even limited diversity may serve as a useful tool to prioritize candidates for epitope mapping or antigen identification. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.000786, 1-10, 2011.Antibodies play an important role in protecting against infectious disease and contribute to pathology in autoimmune disease. Understanding antibody-antigen interactions is important for elucidating disease etiology, as well as facilitating vaccine design and diagnostic test development. In addition to their role in the immune system, antibodies are also very useful as affinity reagents for detection and purification as well as clinical diagnostic tools and pharmaceuticals. Epitope mapping is often an important step in determining if an antibody is suitable for a particular application, sorting among antibodies or determining how it performs its function. Many methods exist for identifying the epitope of an antibody, including crystallography, peptide tiling, and phage display (1, 2). In this study, we will examine whether a faster, less expensive array based approach could be applied to the epitope mapping problem A crystal structure of the antibody bound to the target is generally considered the gold standard of epitope mapping because it gives the most detailed information about the binding mechanism and will work for both conformational and linear epitopes. To identify a linear epitop...

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confidence: 99%

Exploring Antibody Recognition of Sequence Space through Random-Sequence Peptide Microarrays

Halperin

Stafford

Johnston

2011

Molecular & Cellular Proteomics

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“…Therefore, we examined the antigenic peptide sequences in SeV-NP. SPOT peptide arrays have been successfully used for the screening of antibody epitopes [13,28,29]. We have also identified B-cell epitopes in the nucleoprotein of mouse hepatitis virus (MHV) using a SPOT peptide array and developed an ELISA using antigenic peptides derived from the nucleoprotein of MHV for serological diagnosis [1].…”

Section: Discussionmentioning

confidence: 99%

Epitope Mapping of the Nucleocapsid Protein of Sendai Virus and Application of Antigenic Epitopes for the ELISA-Based Diagnosis of Sendai Virus Infection

Asano

Torigoe

Sasaki

et al. 2013

The Journal of Veterinary Medical Science

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ABSTRACT. Sendai virus (SeV) is one of the most prevalent viral pathogens infecting laboratory mice and rats. To date, mature SeV virions have been used as antigens for serological diagnosis. To develop antigens that are more specific and easier to prepare for diagnosis, we examined the antigenic sites in the nucleocapsid protein (NP) of SeV with antisera from experimentally SeV-infected mice and a peptide array membrane containing overlapping 10-mer peptides covering the entire NP. We found antigenic linear sequences in two regions, amino acids 120-160 and 420-500, of the SeV-NP. From these antigenic sequences, we applied two synthesized peptides, IVKTRDMEYERTTEWL and FVTLHGAERLEEETNDE, which correspond to positions 119-134 and 458-474 of the SeV-NP, respectively, as antigens in an enzymelinked immunosorbent assay (ELISA). Evaluation of the ELISAs using these peptides revealed that they were specific to anti-SeV antisera. Furthermore, the ELISAs using these peptides were able to distinguish between SeV-positive and SeV-negative mouse sera to the same extent as a commercial ELISA kit. These results indicate that these peptides are useful for the serological diagnosis of SeV infection.

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“…Although well located in the antibody paratope, one SCR (Phe 32 from the heavy chain) belonging to group 3 points in a direction opposite to the main orientation of the CD4-binding pocket and was not confirmed by site-directed mutagenesis. Finally, group 4 SCR (Trp 36 and Cys 88 from the light chain and Cys 92 and Tyr 103 from the heavy chain) are clearly not involved in CD4 interaction because an alanine mutation in their Fab fragments did not affect CD4 binding, and they showed a side chain inaccessible to the solvent (Cys 92 , Trp 36 , and Cys 88 ) and/or were at a distance from the CD4-binding pocket, as for Tyr 102 (Fig. 7).…”

Section: Positioning Critical Residues On a Computer Model Of The Varmentioning

confidence: 99%

“…The complete paratope binds the antigen with high affinity, but individual binding sites from each CDR, corresponding to a peptide sequence, are probably characterized by a "degenerated" weak antigen interaction, which is much more difficult to identify. The Spot method can overcome such difficulty because high peptide density at each spot, in the range of 200 -500 nmol/cm 2 (35), coupled with a very sensitive electrochemiluminescence detection system facilitates identification of weak binding peptides due to increased avidity for the probed antigen (36). In addition, several studies have demonstrated that some local flexibility contributes to antigen/antibody recognition, leading to conformational adaptation (37)(38)(39)(40).…”

Section: Positioning Critical Residues On a Computer Model Of The Varmentioning

confidence: 99%

Mapping the Paratope of Anti-CD4 Recombinant Fab 13B8.2 by Combining Parallel Peptide Synthesis and Site-directed Mutagenesis

Bès¹,

Briant-Longuet²,

Cérutti³

et al. 2003

Journal of Biological Chemistry

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We analyzed antigen-binding residues from the variable domains of anti-CD4 antibody 13B8.2 using the Spot method of parallel peptide synthesis. Sixteen amino acids, defined as Spot critical residues (SCR), were identified on the basis of a 50% decrease in CD4 binding to alanine analogs of reactive peptides. Recombinant Fab 13B8.2 mutants were constructed with alanine residues in place of each of the 16 SCR, expressed in the baculovirus cell system, and purified. CD measurements indicated that the mutated proteins were conformationally intact, with a ␤-sheet secondary structure similar to that of wild-type

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Identification of distinct antibody epitopes and mimotopes from a peptide array of 5520 randomly generated sequences

Cited by 83 publications

References 31 publications

Exploring Antibody Recognition of Sequence Space through Random-Sequence Peptide Microarrays

Exploring Antibody Recognition of Sequence Space through Random-Sequence Peptide Microarrays

Epitope Mapping of the Nucleocapsid Protein of Sendai Virus and Application of Antigenic Epitopes for the ELISA-Based Diagnosis of Sendai Virus Infection

Mapping the Paratope of Anti-CD4 Recombinant Fab 13B8.2 by Combining Parallel Peptide Synthesis and Site-directed Mutagenesis

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