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Analytical and Bioanalytical Chemistry

, Volume 403, Issue 1, pp 227–238 | Cite as

Clonality characterization of natural epitope-specific antibodies against the tumor-related antigen topoisomerase IIa by peptide chip and proteome analysis: a pilot study with colorectal carcinoma patient samples

  • Michael Linnebacher
  • Peter Lorenz
  • Cornelia Koy
  • Annika Jahnke
  • Nadine Born
  • Felix Steinbeck
  • Johannes Wollbold
  • Tobias Latzkow
  • Hans-Jürgen Thiesen
  • Michael O. GlockerEmail author
Original Paper

Abstract

Patient-specific sequential epitopes were identified by peptide chip analysis using 15mer peptides immobilized on glass slides that covered the topoisomerase IIa protein with a frameshift of five amino acids. Binding specificities of serum antibodies against sequential epitopes were confirmed as being mono-specific by peptide chip re-analysis of epitope-affinity-purified antibody pools. These results demonstrate that serum samples from colon carcinoma patients contain antibodies against sequential epitopes from the topoisomerase IIa antigen. Interactions of patients’ antibodies with sequential epitopes displayed by peptides on glass surfaces may thus mirror disease-specific immune situations. Consequently, these data suggest epitope–antibody reactivities on peptide chips as potential diagnostic readouts of individual immune response characteristics, especially because monospecific antibodies can be interrogated. Subsequently, the clonality of the antibodies present in the mono-specific antibody pools was characterized by 2D gel electrophoresis. This analysis suggested that the affinity-purified antibodies were oligoclonal. Similarly to large-scale screening approaches for specific antigen–antibody interactions in order to improve disease diagnostic, we suggest that “protein-wide” screening for specific epitope–paratope interactions may help to develop novel assays for monitoring of personalized therapies, since individual properties of antigen–antibody interactions remain distinguishable.

Keywords

Topoisomerase IIa Peptide chip analysis Epitope mapping Epitope–antibody reactivities Antibody profiling Mass spectrometry 

Abbreviations

CHAPS

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

CHCA

α-Cyano-4-hydroxycinnamic acid

CRC

Colorectal carcinoma

DDT

Dithiothreitol

EAR

Epitope–antibody reactivities

IAA

Iodoacetamide

IPG

Immobiline pH gradient

MALDI

Matrix-assisted laser desorption/ionization

MLH-1

Mut-L-homologue 1

NL

Non-linear

n-OGP

n-O-Octyl-β-d-glucopyranoside

PMSF

Phenylmethylsulfonyl fluoride

TFA

Trifluoroacetic acid

ToF

Time of flight

Notes

Acknowledgements

We thank Mrs M. Ruß for excellent technical assistance. We also acknowledge the state Mecklenburg-Western Pomerania for financial support (initiative Exzellenz-MV funding, FKZ: UR 08051). Michael Linnebacher received funding from the “Deutsche Krebshilfe” (grant number: 108919; Norddeutsche Tumorbank für das kolorektale Karzinom).

Supplementary material

216_2012_5781_MOESM1_ESM.pdf (287 kb)
ESM 1 (PDF 287 kb)

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Michael Linnebacher
    • 1
  • Peter Lorenz
    • 2
  • Cornelia Koy
    • 3
  • Annika Jahnke
    • 1
  • Nadine Born
    • 2
  • Felix Steinbeck
    • 2
    • 4
  • Johannes Wollbold
    • 2
    • 4
  • Tobias Latzkow
    • 4
  • Hans-Jürgen Thiesen
    • 2
    • 4
  • Michael O. Glocker
    • 3
    Email author
  1. 1.Department of General Surgery, Molecular Oncology and Immunotherapy, Medical FacultyUniversity of RostockRostockGermany
  2. 2.Institute of Immunology, Medical FacultyUniversity of RostockRostockGermany
  3. 3.Proteome Center Rostock, Medical Faculty and Natural Science FacultyUniversity of RostockRostockGermany
  4. 4.IndyMED GmbHRostockGermany

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