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


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.


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





α-Cyano-4-hydroxycinnamic acid


Colorectal carcinoma




Epitope–antibody reactivities




Immobiline pH gradient


Matrix-assisted laser desorption/ionization


Mut-L-homologue 1






Phenylmethylsulfonyl fluoride


Trifluoroacetic acid


Time of flight



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)


  1. 1.
    Cunningham D, Atkin W, Lenz H-J, Lynch HT, Minsky B, Nordlinger B, Starling N (2010) Colorectal cancer. Lancet 375:1030–1047CrossRefGoogle Scholar
  2. 2.
    Lu H, Goodell V, Disis ML (2007) Targeting serum antibody for cancer diagnosis: a focus on colorectal cancer. Expert Opin Ther Targets 11:235–244CrossRefGoogle Scholar
  3. 3.
    ElKased RF, Koy C, Deierling T, Lorenz P, Qian Z, Li Y, Thiesen H-J, Glocker MO (2009) Mass spectrometric and peptide chip epitope mapping of rheumatoid arthritis autoantigen RA33. Eur J Mass Spectrom 15:747–749CrossRefGoogle Scholar
  4. 4.
    Hecker M, Lorenz P, Steinbeck F, Hong L, Riemekasten G, Li Y, Zettl UK, Thiesen H-J (2011) Computational Analysis of High-Density Peptide Microarray Data with Application from Systemic Sclerosis to Multiple Sclerosis. Autoimmunity Reviews 11:180–190CrossRefGoogle Scholar
  5. 5.
    Lorenz P, Kreutzer M, Zerweck J, Schutkowski M, Thiesen H-J (2009) Book. In: Reineke U, Schutkowski M (eds) Probing the epitope signatures of IgG antibodies in human serum from patients with autoimmune disease. Humana Press, pp 247–258Google Scholar
  6. 6.
    Park J-S, Kim H-S, Park M-Y, Kim C-H, Chung Y-J, Hong Y-K, Kim T-G (2010) Topoisomerase II alpha as a universal tumor antigen: antitumor immunity in murine tumor models and H-2Kb-restricted T cell epitope. Cancer Immunol Immunother 59:747–757CrossRefGoogle Scholar
  7. 7.
    McLeod HL, Douglas F, Oates M, Symonds RP, Prakash D, Van Der Zee AGJ, Kaye SB, Brown R, Keith WN (1994) Topoisomerase I and II activity in human breast, cervix, lung and colon cancer. Int J Cancer 59:607–611CrossRefGoogle Scholar
  8. 8.
    Tsavaris N, Lazaris A, Kosmas C, Gouveris P, Kavantzas N, Kopterides P, Papathomas T, Arapogiannis G, Zorzos H, Kyriakou V, Patsouris E (2009) Topoisomerase I and IIa protein expression in primary colorectal cancer and recurrences following 5-fluorouracil-based adjuvant chemotherapy. Cancer Chemother Pharmacol 64:391–398CrossRefGoogle Scholar
  9. 9.
    Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, Muramatsu S, Steinman RM (1992) Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 176:1693–1702CrossRefGoogle Scholar
  10. 10.
    Spence JM, Phua HH, Mills W, Carpenter AJ, Porter ACG, Farr CJ (2007) Depletion of topoisomerase II a leads to shortening of the metaphase interkinetochore distance and abnormal persistence of PICH-coated anaphase threads. J Cell Sci 120:3952–3964CrossRefGoogle Scholar
  11. 11.
    Cortès F, Pastor N (2003) Induction of endoreduplication by topoisomerase II catalytic inhibitors. Mutagenesis 18:105–112CrossRefGoogle Scholar
  12. 12.
    Schoeffler AJ, Berger JM (2005) Recent advances in understanding structure–function relationships in the type II topoisomerase mechanism. Biochem Soc Trans 33:1465–1470CrossRefGoogle Scholar
  13. 13.
    Wang JC (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 3:430–440CrossRefGoogle Scholar
  14. 14.
    Coss A, Tosetto M, Fox EJ, Sapetto-Rebow B, Gorman S, Kennedy BN, Lloyd AT, Hyland JM, O’Donoghue DP, Sheahan K, Leahy DT, Mulcahy HE, O’Sullivan JN (2009) Increased topoisomerase IIa expression in colorectal cancer is associated with advanced disease and chemotherapeutic resistance via inhibition of apoptosis. Cancer Lett 276:228–238CrossRefGoogle Scholar
  15. 15.
    Stone B, Schummer M, Paley PJ, Thompson L, Stewart J, Ford M, Crawford M, Urban N, O’Briant K, Nelson BH (2003) Serologic analysis of ovarian tumor antigens reveals a bias toward antigens encoded on 17q. Int J Cancer 104:73–84CrossRefGoogle Scholar
  16. 16.
    Ostwald C, Linnebacher M, Weirich V, Prall F (2009) Chromosomally and microsatellite stable colorectal carcinomas without the CpG island methylator phenotype in a molecular classification. Int J Oncol 35:321–327Google Scholar
  17. 17.
    Roewer C, Koy C, Hecker M, Reimer T, Gerber B, H T Jr, Glocker M (2011) Mass spectrometric characterization of protein structure details refines the proteome signature for invasive ductal breast carcinoma. J Am Soc Mass Spectrom 22:440–456CrossRefGoogle Scholar
  18. 18.
    Sinz A, Bantscheff M, Mikkat S, Ringel B, Drynda S, Kekow J, Thiesen H-J, Glocker MO (2002) Mass spectrometric proteome analyses of synovial fluids and plasmas from patients suffering from rheumatoid arthritis and comparison to reactive arthritis or osteoarthritis. Electrophoresis 23:3445–3456CrossRefGoogle Scholar
  19. 19.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  20. 20.
    Blum H, Beier H, Gross HJ (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93–99CrossRefGoogle Scholar
  21. 21.
    Just T, Gafumbegete E, Gramberg J, Prüfer I, Mikkat S, Ringel B, Pau HW, Glocker MO (2006) Differential proteome analysis of tonsils from children with chronic tonsillitis or with hyperplasia reveals disease-associated protein expression differences. Anal Bioanal Chem 384:1134–1144CrossRefGoogle Scholar
  22. 22.
    Kienbaum M, Koy C, Montgomery HV, Drynda S, Lorenz P, Illges H, Tanaka K, Kekow J, Guthke R, Thiesen H-J, Glocker MO (2009) Mass spectrometric characterization of apheresis samples from rheumatoid arthritis patients for the improvement of immunoadsorption therapy—a pilot study. Proteomics Clin Appl 3:797–809CrossRefGoogle Scholar
  23. 23.
    Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5:725–738CrossRefGoogle Scholar
  24. 24.
    Python script by Bosco H University of California, San FranciscoGoogle Scholar
  25. 25.
    Shrake A, Rupley JA (1973) Environment and exposure to solvent of protein atoms. lysozyme and insulin. J Mol Biol 79:351–364CrossRefGoogle Scholar
  26. 26.
    Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132CrossRefGoogle Scholar
  27. 27.
    Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW, Meltzer SJ, Rodriguez-Bigas MA, Fodde R, Ranzani GN, Srivastava S (1998) A National Cancer Institute Workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 58:5248–5257Google Scholar
  28. 28.
    Bantscheff M, Glocker MO, Weiss V (1998) Probing the tertiary structure of multidomain poteins by limited proteolysis and mass spectrometry. Eur J Mass Spectrom 4:265–271CrossRefGoogle Scholar
  29. 29.
    Bantscheff M, Weiss V, Glocker MO (1999) Identification of linker regions and domain borders of the transcription activator protein NtrC from Escherichia coli by limited proteolysis, in-gel digestion, and mass spectrometry. Biochemistry 38:11012–11020CrossRefGoogle Scholar
  30. 30.
    Dickey JS, Osheroff N (2005) Impact of the C-terminal domain of topoisomerase II a on the DNA cleavage activity of the human enzyme. Biochemistry 44:11546–11554CrossRefGoogle Scholar
  31. 31.
    Jensen S, Andersen AH, Kjeldsen E, Biersack H, Olsen EHN, Andersen TB, Westergaard O, Jakobsen BK (1996) Analysis of functional domain organization in DNA topoisomerase II from humans and Saccharomyces cerevisiae. Mol Cell Biol 16:3866–3877Google Scholar
  32. 32.
    Nitiss JL (2009) DNA topoisomerase II and its growing repertoire of biological functions. Nat Rev Cancer 9:327–337CrossRefGoogle Scholar
  33. 33.
    El-Kased RF, Koy C, Lorenz P, Drynda S, Guthke R, Qian Z, Koczan D, Li Y, Kekow J, Thiesen H-J, Glocker MO (2010) Mass spectrometric and peptide chip epitope analysis on the RA33 autoantigen with sera from rheumatoid arthritis patients. Eur J Mass Spectrom 16:443–451CrossRefGoogle Scholar
  34. 34.
    Mahler M, Blüthner M, Pollard KM (2003) Advances in B-cell epitope analysis of autoantigens in connective tissue diseases. Clin Immunol 107:65–79CrossRefGoogle Scholar
  35. 35.
    Manea M, Kalászi A, Mezo G, Horváti K, Bodor A, Horváth A, Farkas O, Perczel A, Przybylski M, Hudecz F (2008) Antibody recognition and conformational flexibility of a plaque-specific .ß-amyloid epitope modulated by non-native peptide flanking regions. J Med Chem 51:1150–1161CrossRefGoogle Scholar
  36. 36.
    Al-Kuraya K, Novotny H, Bavi P, Siraj AK, Uddin S, Ezzat A, Sanea NA, Al-Dayel F, Al-Mana H, Sheikh SS, Mirlacher M, Tapia C, Simon R, Sauter G, Terracciano L, Tornillo L (2007) HER2, TOP2A, CCND1, EGFR and C-MYC oncogene amplification in colorectal cancer. J Clin Pathol 60:768–772CrossRefGoogle Scholar
  37. 37.
    Willman JH, Holden JA (2000) Immunohistochemical staining for DNA topoisomerase II-alpha in benign, premalignant, and malignant lesions of the prostate. Prostate 42:280–286CrossRefGoogle Scholar
  38. 38.
    Zhao H, Yu H, Liu Y, Wang Y, Cai W (2008) DNA topoisomerase II-α as a proliferation marker in human gliomas: correlation with PCNA expression and patient survival. Clin Neuropathol 27:83–90Google Scholar
  39. 39.
    Kanta SY, Yamane T, Dobashi Y, Mitsui F, Kono K, Ooi A (2006) Topoisomerase IIa gene amplification in gastric carcinomas: correlation with the HER2 Gene. An immunohistochemical, immunoblotting, and multicolor fluorescence in situ hybridization study. Hum Pathol 37:1333–1343CrossRefGoogle Scholar
  40. 40.
    Mano MS, Awada A, Di Leo A, Durbecq V, Paesmans M, Cardoso F, Larsimont D, Piccart M (2004) Rates of topoisomerase II-alpha and HER-2 gene amplification and expression in epithelial ovarian carcinoma. Gynecol Oncol 92:887–895CrossRefGoogle Scholar
  41. 41.
    Kosari F, Munz JMA, Savci-Heijink CD, Spiro C, Klee EW, Kube DM, Tillmans L, Slezak J, Karnes RJ, Cheville JC, Vasmatzis G (2008) Identification of prognostic biomarkers for prostate cancer. Clin Cancer Res 14:1734–1743CrossRefGoogle Scholar
  42. 42.
    Wong N, Yeo W, Wong W-L, Wong NLY, Chan KYY, Mo FKF, Koh J, Chan SL, Chan ATC, Lai PBS, Ching AKK, Tong JHM, Ng H-K, Johnson PJ, To K-F (2009) TOP2A overexpression in hepatocellular carcinoma correlates with early age onset, shorter patients survival and chemoresistance. Int J Cancer 124:644–652CrossRefGoogle Scholar
  43. 43.
    Pellequer JL, Westhof E, van Regenmortel MH (1991) Predicting location of continuous epitopes in proteins from their primary structures. Methods Enzymol 203:176–201CrossRefGoogle Scholar
  44. 44.
    Tissot J-D, Hohlfeld P, Hochstrasser DF, Tolsa J-F, Calme A, Schneider P (1993) Clonal imbalances of plasma/serum immunogloblin production in infants. Electrophoresis 14:245–247CrossRefGoogle Scholar
  45. 45.
    Tissot J-D, Pietrogrande M, Testoni L, Invernizzi F (1998) Clinical implications of the types of cryoglobulins determined by two-dimensional polyacrylamide gel electrophoresis. Haematologica 83:693–700Google Scholar
  46. 46.
    Tissot J-D, Spertini F (1995) Analysis of immunoglobulins by two-dimensional gel electrophoresis. J Chromatogr A 698:225–250CrossRefGoogle Scholar
  47. 47.
    Rockberg J, Lofblom J, Hjelm B, Uhlén M, Stahl S (2008) Epitope mapping of antibodies using bacterial surface display. Nature Methods 5:1039–1045CrossRefGoogle Scholar
  48. 48.
    Magnusson K, de Wit M, Brennan D, Johnson LB, McGee SF, Lundberg E, Naicker K, Klinger R, Kampf C, Asplund A, Wester K, Gry M, Bjartell A, Gallagher WM, Rexhepaj E, Kilpinen S, Kallioniemi O-P, Belt E, Goos J, Meijer G, Birgisson H, Glimelius B, Borrebaeck CAK, Navani S, Uhlén M, O’Connor DP, Jirstrom K, Pontén F (2011) SATB2 in combination with cytokeratin 20 identifies over 95% of all colorectal carcinomas. Am J Surg Pathol 35:937–1091CrossRefGoogle Scholar

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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