Skip to main content

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

Abstract

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

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Taylor-Papadimitriou J, Burchell J, Miles DW, Dalziel M (1999) Biochim Biophys Acta 1455:301–303

    CAS  Google Scholar 

  2. Jarrard JA, Linnoila RI, Lee HR, Steinberg SM, Witschi H, Szabo E (1998) Cancer Res 58:5582–5589

    CAS  Google Scholar 

  3. Hanisch FG, Muller S (2000) Glycobiology 10:439–449

    Article  CAS  Google Scholar 

  4. Apostolopoulos V, Sandrin MS, McKenzie IF (1999) Vaccine 18:268–275

    Article  CAS  Google Scholar 

  5. Burchell J, Taylor-Papadimitriou J (1984) Int J Cancer 34:763–768

    Article  CAS  Google Scholar 

  6. McGuckin MA, Devine PL (1995) Tumour Diagn Ther 16:1–16

    Google Scholar 

  7. Gendler SJ (2001) J Mammary Gland Biol 6:339–353

    Article  CAS  Google Scholar 

  8. Yan AC, Bell KM, Breeden MM, Ellington AD (2005) AptamersFront Biosci 10:1802–1827

    Article  CAS  Google Scholar 

  9. Ellington A, Szostack JW (1990) Nature 346:818–822

    Article  CAS  Google Scholar 

  10. Tuerk C, Gold L (1990) Science 249:505–510

    Article  CAS  Google Scholar 

  11. Bock LC, Griffin LC, Latham JA, Vermaas EH, Toole JJ (1992) Nature 355:564–566

    Article  CAS  Google Scholar 

  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 98:2688–2692

    CAS  Google Scholar 

  13. Xu W, Ellington AD (1996) Proc Natl Acad Sci USA 93:7475–7480

    Article  CAS  Google Scholar 

  14. Weiss S, Proske D, Neumann M, Groschup MH, Kretzschmar HA, Famulok M, Winnacker EL (1997) J Virol 71:8790–8797

    CAS  Google Scholar 

  15. Lauhon CT, Szostak JW (1994) J Am Chem Soc 14:5798–5811

    Google Scholar 

  16. Lorsch JR, Szostak JW (1994) Biochemistry 33:973–982

    Article  CAS  Google Scholar 

  17. Huizenga DE, Szostak JW (1995) Biochemistry 34:656–665

    Article  CAS  Google Scholar 

  18. Patel DJ, Suri AK, Jiang F, Jiang LC, Fan P, Kumar RA, Nonin S (1997) J Mol Biol 272:645–664

    Article  CAS  Google Scholar 

  19. Kawakami J, Kawase Y, Sugimoto N (1998) Anal Chim Acta 365:95–100

    Article  CAS  Google Scholar 

  20. Brody EN, Gold L (2000) J Biotechnol 74:5–13

    CAS  Google Scholar 

  21. Osborne SE, Matsumura I, Ellington AD (1997) Curr Opin Chem Biol 1:5–9

    Article  CAS  Google Scholar 

  22. Bacher JM, Ellington AD (1998) Drug Discov Today 3:265–273

    Article  CAS  Google Scholar 

  23. German I, Buchanan DD, Kennedy RT (1998) Anal Chem 70:4540–4545

    Article  CAS  Google Scholar 

  24. Gewirtz AM (1999) Curr Opin Mol Ther 1:297–306

    CAS  Google Scholar 

  25. Bianchini M, Radrizzani M, Brocardo MG, Reyes GB, Gonzalez Solveyra C, Santa-Coloma TA (2001) J Immunol Methods 252:191–197

    Article  CAS  Google Scholar 

  26. Hélène C (1996) Ther Dev Eur J Cancer 32(S2):3

    Article  Google Scholar 

  27. Kimoto M, Shirouzu M, Mizutani S, Koide H, Kaziro Y, Hirao I, Yokoyama S (2002) Eur J Biochem 269:697–704

    Article  CAS  Google Scholar 

  28. Lupold SE, Hicke J, Lin Yun C, Donald S (2002) Cancer Res 62:4029–4033

    CAS  Google Scholar 

  29. Hicke BJ, Marion C, Chang YF, Gould T, Lynott CK, Parma D, Schmidt PG, Warren S (2001) J Biol Chem 276:48644–48654

    Article  CAS  Google Scholar 

  30. Daniels DA, Chen H, Hicke BJ, Swiderek KM, Gold L (2003) Proc Natl Acad Sc USA 100:15416–15421

    Article  CAS  Google Scholar 

  31. Uhrbom L, Hesselager G, Ostman A, Nister M, Westermark B (2000) Int J Cancer 85:398–406

    Article  CAS  Google Scholar 

  32. Akhtar S, Dunnion D, Poyner D, Ackroyd J, Bibby M, Double J (2002) Biochem Pharmacol 63:2187–2195

    Article  CAS  Google Scholar 

  33. Bell C, Lynam E, Landfair DJ, Janjic N, Wiles ME (1999) In Vitro Cell Dev Biol Anim 35:533–542

    Article  CAS  Google Scholar 

  34. Ostendorf T, Kunter U, Eitner F, Loos A, Regele H, Kerjaschki D, Henninger DD, Janjic N, Floege J (1999) J Clin Invest 104:913–923

    CAS  Google Scholar 

  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 9:573–582

    Article  CAS  Google Scholar 

  36. Romig TS, Bell C, Drolet DW (1999) J Chromatogr B 731:275–284

    Article  CAS  Google Scholar 

  37. Smith RG, Missailidis S, Price MR (2002) J Chromatogr B 766:13–26

    Article  CAS  Google Scholar 

  38. Murray A, Spencer DIR, Missailidis S, Denton G, Price MR (1998) J Pept Res 52:375–383

    CAS  Article  Google Scholar 

  39. Ferreira CSM, Matthews CS, Missailidis S (2006) Tumor Biol 27:289–301

    Article  CAS  Google Scholar 

  40. Brokx RD, Revers L, Zhang QH, Yang SX, Mal TK, Ikura M, Gariepy J (2003) Biochemistry 42:13817–13825

    Article  CAS  Google Scholar 

  41. Fitzwater T, Polisky B (1996) Method Enzymol 267:275–301

    CAS  Article  Google Scholar 

  42. Fujino T, Sato Y, Une M, Kanayasu-Toyoda T, Yamaguchi T, Shudo K, Inoue K, Nishimaki-Mogamiet T (2003) J Steroid Biochem 97:247–252

    Google Scholar 

  43. Missailidis S, Thomaidou D, Borbas KE, Price MR (2005) J Immunol Methods 296:45–62

    Article  CAS  Google Scholar 

  44. Griffin LC, Toole JJ, Leung LLK (1993) Gene 137:25–31

    Article  CAS  Google Scholar 

  45. Ulrich H, Magdesian MH, Alves MJM, Colli W (2002) J Biol Chem 277:20756–20762

    Article  CAS  Google Scholar 

  46. Karanikas V, Patton K, Jamieson G, Pietersz G, McKenzie I (1998) Tumor Biol 19:71–78

    Article  CAS  Google Scholar 

  47. Rye PD, McGuckin MA (2001) Tumor Biol 22:269–272

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to S. Missailidis.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ferreira, C.S.M., Papamichael, K., Guilbault, G. et al. DNA aptamers against the MUC1 tumour marker: design of aptamer–antibody sandwich ELISA for the early diagnosis of epithelial tumours. Anal Bioanal Chem 390, 1039–1050 (2008). https://doi.org/10.1007/s00216-007-1470-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-007-1470-1

Keywords

  • MUC1
  • Aptamers
  • ELISA
  • Diagnostic assay
  • Immunoassay