Analytical and Bioanalytical Chemistry

, Volume 390, Issue 4, pp 1039–1050 | Cite as

DNA aptamers against the MUC1 tumour marker: design of aptamer–antibody sandwich ELISA for the early diagnosis of epithelial tumours

  • C. S. M. Ferreira
  • K. Papamichael
  • G. Guilbault
  • T. Schwarzacher
  • J. Gariepy
  • S. MissailidisEmail author
Original Paper


Aptamers are functional molecules able to bind tightly and selectively to disease markers, offering great potential for applications in disease diagnosis and therapy. MUC1 is a well-known tumour marker present in epithelial malignancies and is used in immunotherapeutic and diagnostic approaches. We report the selection of DNA aptamers that bind with high affinity and selectivity an MUC1 recombinant protein containing five repeats of the variable tandem repeat region. Aptamers were selected using the SELEX methodology from an initial library containing a 25-base-long variable region for their ability to bind to the unglycosylated form of the MUC1 protein. After ten rounds of in vitro selection and amplification, more than 90% of the pool of sequences consisted of target-binding molecules, which were cloned, sequenced and found to share no sequence consensus. The binding properties of these aptamers were quantified using ELISA and surface plasmon resonance. The lead aptamer sequence was subsequently used in the design of an aptamer–antibody hybrid sandwich ELISA for the identification and quantification of MUC1 in buffered solutions. Following optimisation of the operating conditions, the resulting enzyme immunoassay displayed an EC50 value of 25 μg/ml, a detection limit of 1 μg/ml and a linear range between 8 and 100 μg/ml for the MUC1 five tandem repeat analyte. In addition, recovery studies performed in buffer conditions resulted in averaged recoveries between 98.2 and 101.7% for all spiked samples, demonstrating the usability of the aptamer as a receptor in microtitre-based assays. Our results aim towards the formation of new diagnostic assays against this tumour marker for the early diagnosis of primary or metastatic disease in breast, bladder and other epithelial tumours.


An aptamer-antibody two-dimentional immunoassay for MUC1


MUC1 Aptamers ELISA Diagnostic assay Immunoassay 



The authors would like to acknowledge Alan Perkins, University of Nottingham, for kindly donating the C595 anti-MUC1 control. Mark Kreuzer is acknowledged for his advice on the immunoassay development. C.S.M.F. acknowledges The Open University for financial support during her PhD. S.M. acknowledges the support of The Open University and the Breast Cancer Campaign for the further development of this project.


  1. 1.
    Taylor-Papadimitriou J, Burchell J, Miles DW, Dalziel M (1999) Biochim Biophys Acta 1455:301–303Google Scholar
  2. 2.
    Jarrard JA, Linnoila RI, Lee HR, Steinberg SM, Witschi H, Szabo E (1998) Cancer Res 58:5582–5589Google Scholar
  3. 3.
    Hanisch FG, Muller S (2000) Glycobiology 10:439–449CrossRefGoogle Scholar
  4. 4.
    Apostolopoulos V, Sandrin MS, McKenzie IF (1999) Vaccine 18:268–275CrossRefGoogle Scholar
  5. 5.
    Burchell J, Taylor-Papadimitriou J (1984) Int J Cancer 34:763–768CrossRefGoogle Scholar
  6. 6.
    McGuckin MA, Devine PL (1995) Tumour Diagn Ther 16:1–16Google Scholar
  7. 7.
    Gendler SJ (2001) J Mammary Gland Biol 6:339–353CrossRefGoogle Scholar
  8. 8.
    Yan AC, Bell KM, Breeden MM, Ellington AD (2005) AptamersFront Biosci 10:1802–1827CrossRefGoogle Scholar
  9. 9.
    Ellington A, Szostack JW (1990) Nature 346:818–822CrossRefGoogle Scholar
  10. 10.
    Tuerk C, Gold L (1990) Science 249:505–510CrossRefGoogle Scholar
  11. 11.
    Bock LC, Griffin LC, Latham JA, Vermaas EH, Toole JJ (1992) Nature 355:564–566CrossRefGoogle Scholar
  12. 12.
    Hicke BJ, Watson SR, Koenig A, Lynott CK, Bargatze RF, Chang YF, Ringquist S, MoonMcDermot L, Jennings S, Fitzwater T, Han HL, Varki N, Albinana I, Willis MC, Varki A, Parma D (1996) J Clin Invest 98:2688–2692Google Scholar
  13. 13.
    Xu W, Ellington AD (1996) Proc Natl Acad Sci USA 93:7475–7480CrossRefGoogle Scholar
  14. 14.
    Weiss S, Proske D, Neumann M, Groschup MH, Kretzschmar HA, Famulok M, Winnacker EL (1997) J Virol 71:8790–8797Google Scholar
  15. 15.
    Lauhon CT, Szostak JW (1994) J Am Chem Soc 14:5798–5811Google Scholar
  16. 16.
    Lorsch JR, Szostak JW (1994) Biochemistry 33:973–982CrossRefGoogle Scholar
  17. 17.
    Huizenga DE, Szostak JW (1995) Biochemistry 34:656–665CrossRefGoogle Scholar
  18. 18.
    Patel DJ, Suri AK, Jiang F, Jiang LC, Fan P, Kumar RA, Nonin S (1997) J Mol Biol 272:645–664CrossRefGoogle Scholar
  19. 19.
    Kawakami J, Kawase Y, Sugimoto N (1998) Anal Chim Acta 365:95–100CrossRefGoogle Scholar
  20. 20.
    Brody EN, Gold L (2000) J Biotechnol 74:5–13Google Scholar
  21. 21.
    Osborne SE, Matsumura I, Ellington AD (1997) Curr Opin Chem Biol 1:5–9CrossRefGoogle Scholar
  22. 22.
    Bacher JM, Ellington AD (1998) Drug Discov Today 3:265–273CrossRefGoogle Scholar
  23. 23.
    German I, Buchanan DD, Kennedy RT (1998) Anal Chem 70:4540–4545CrossRefGoogle Scholar
  24. 24.
    Gewirtz AM (1999) Curr Opin Mol Ther 1:297–306Google Scholar
  25. 25.
    Bianchini M, Radrizzani M, Brocardo MG, Reyes GB, Gonzalez Solveyra C, Santa-Coloma TA (2001) J Immunol Methods 252:191–197CrossRefGoogle Scholar
  26. 26.
    Hélène C (1996) Ther Dev Eur J Cancer 32(S2):3CrossRefGoogle Scholar
  27. 27.
    Kimoto M, Shirouzu M, Mizutani S, Koide H, Kaziro Y, Hirao I, Yokoyama S (2002) Eur J Biochem 269:697–704CrossRefGoogle Scholar
  28. 28.
    Lupold SE, Hicke J, Lin Yun C, Donald S (2002) Cancer Res 62:4029–4033Google Scholar
  29. 29.
    Hicke BJ, Marion C, Chang YF, Gould T, Lynott CK, Parma D, Schmidt PG, Warren S (2001) J Biol Chem 276:48644–48654CrossRefGoogle Scholar
  30. 30.
    Daniels DA, Chen H, Hicke BJ, Swiderek KM, Gold L (2003) Proc Natl Acad Sc USA 100:15416–15421CrossRefGoogle Scholar
  31. 31.
    Uhrbom L, Hesselager G, Ostman A, Nister M, Westermark B (2000) Int J Cancer 85:398–406CrossRefGoogle Scholar
  32. 32.
    Akhtar S, Dunnion D, Poyner D, Ackroyd J, Bibby M, Double J (2002) Biochem Pharmacol 63:2187–2195CrossRefGoogle Scholar
  33. 33.
    Bell C, Lynam E, Landfair DJ, Janjic N, Wiles ME (1999) In Vitro Cell Dev Biol Anim 35:533–542CrossRefGoogle Scholar
  34. 34.
    Ostendorf T, Kunter U, Eitner F, Loos A, Regele H, Kerjaschki D, Henninger DD, Janjic N, Floege J (1999) J Clin Invest 104:913–923Google Scholar
  35. 35.
    Willis MC, Collins BD, Zhang T, Green LS, Sebesta DP, Bell C, Kellogg E, Gill SC, Magallanez A, Knauer S, Bendele RA, Gill PS, Janjic N, Collins B (1998) Bioconjug Chem 9:573–582CrossRefGoogle Scholar
  36. 36.
    Romig TS, Bell C, Drolet DW (1999) J Chromatogr B 731:275–284CrossRefGoogle Scholar
  37. 37.
    Smith RG, Missailidis S, Price MR (2002) J Chromatogr B 766:13–26CrossRefGoogle Scholar
  38. 38.
    Murray A, Spencer DIR, Missailidis S, Denton G, Price MR (1998) J Pept Res 52:375–383CrossRefGoogle Scholar
  39. 39.
    Ferreira CSM, Matthews CS, Missailidis S (2006) Tumor Biol 27:289–301CrossRefGoogle Scholar
  40. 40.
    Brokx RD, Revers L, Zhang QH, Yang SX, Mal TK, Ikura M, Gariepy J (2003) Biochemistry 42:13817–13825CrossRefGoogle Scholar
  41. 41.
    Fitzwater T, Polisky B (1996) Method Enzymol 267:275–301CrossRefGoogle Scholar
  42. 42.
    Fujino T, Sato Y, Une M, Kanayasu-Toyoda T, Yamaguchi T, Shudo K, Inoue K, Nishimaki-Mogamiet T (2003) J Steroid Biochem 97:247–252Google Scholar
  43. 43.
    Missailidis S, Thomaidou D, Borbas KE, Price MR (2005) J Immunol Methods 296:45–62CrossRefGoogle Scholar
  44. 44.
    Griffin LC, Toole JJ, Leung LLK (1993) Gene 137:25–31CrossRefGoogle Scholar
  45. 45.
    Ulrich H, Magdesian MH, Alves MJM, Colli W (2002) J Biol Chem 277:20756–20762CrossRefGoogle Scholar
  46. 46.
    Karanikas V, Patton K, Jamieson G, Pietersz G, McKenzie I (1998) Tumor Biol 19:71–78CrossRefGoogle Scholar
  47. 47.
    Rye PD, McGuckin MA (2001) Tumor Biol 22:269–272CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • C. S. M. Ferreira
    • 1
    • 5
  • K. Papamichael
    • 2
    • 6
  • G. Guilbault
    • 2
  • T. Schwarzacher
    • 3
  • J. Gariepy
    • 4
  • S. Missailidis
    • 1
    Email author
  1. 1.Chemistry DepartmentThe Open UniversityMilton KeynesUK
  2. 2.Chemistry DepartmentNational University of Ireland, University College CorkCorkIreland
  3. 3.Department of BiologyUniversity of LeicesterLeicesterUK
  4. 4.Ontario Cancer InstitutePrincess Margaret HospitalTorontoCanada
  5. 5.Ontario Cancer InstituteUniversity of TorontoTorontoCanada
  6. 6.Tyndall National InstituteProspect RowCorkIreland

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