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.
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Taylor-Papadimitriou J, Burchell J, Miles DW, Dalziel M (1999) Biochim Biophys Acta 1455:301–303
Jarrard JA, Linnoila RI, Lee HR, Steinberg SM, Witschi H, Szabo E (1998) Cancer Res 58:5582–5589
Hanisch FG, Muller S (2000) Glycobiology 10:439–449
Apostolopoulos V, Sandrin MS, McKenzie IF (1999) Vaccine 18:268–275
Burchell J, Taylor-Papadimitriou J (1984) Int J Cancer 34:763–768
McGuckin MA, Devine PL (1995) Tumour Diagn Ther 16:1–16
Gendler SJ (2001) J Mammary Gland Biol 6:339–353
Yan AC, Bell KM, Breeden MM, Ellington AD (2005) AptamersFront Biosci 10:1802–1827
Ellington A, Szostack JW (1990) Nature 346:818–822
Tuerk C, Gold L (1990) Science 249:505–510
Bock LC, Griffin LC, Latham JA, Vermaas EH, Toole JJ (1992) Nature 355:564–566
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
Xu W, Ellington AD (1996) Proc Natl Acad Sci USA 93:7475–7480
Weiss S, Proske D, Neumann M, Groschup MH, Kretzschmar HA, Famulok M, Winnacker EL (1997) J Virol 71:8790–8797
Lauhon CT, Szostak JW (1994) J Am Chem Soc 14:5798–5811
Lorsch JR, Szostak JW (1994) Biochemistry 33:973–982
Huizenga DE, Szostak JW (1995) Biochemistry 34:656–665
Patel DJ, Suri AK, Jiang F, Jiang LC, Fan P, Kumar RA, Nonin S (1997) J Mol Biol 272:645–664
Kawakami J, Kawase Y, Sugimoto N (1998) Anal Chim Acta 365:95–100
Brody EN, Gold L (2000) J Biotechnol 74:5–13
Osborne SE, Matsumura I, Ellington AD (1997) Curr Opin Chem Biol 1:5–9
Bacher JM, Ellington AD (1998) Drug Discov Today 3:265–273
German I, Buchanan DD, Kennedy RT (1998) Anal Chem 70:4540–4545
Gewirtz AM (1999) Curr Opin Mol Ther 1:297–306
Bianchini M, Radrizzani M, Brocardo MG, Reyes GB, Gonzalez Solveyra C, Santa-Coloma TA (2001) J Immunol Methods 252:191–197
Hélène C (1996) Ther Dev Eur J Cancer 32(S2):3
Kimoto M, Shirouzu M, Mizutani S, Koide H, Kaziro Y, Hirao I, Yokoyama S (2002) Eur J Biochem 269:697–704
Lupold SE, Hicke J, Lin Yun C, Donald S (2002) Cancer Res 62:4029–4033
Hicke BJ, Marion C, Chang YF, Gould T, Lynott CK, Parma D, Schmidt PG, Warren S (2001) J Biol Chem 276:48644–48654
Daniels DA, Chen H, Hicke BJ, Swiderek KM, Gold L (2003) Proc Natl Acad Sc USA 100:15416–15421
Uhrbom L, Hesselager G, Ostman A, Nister M, Westermark B (2000) Int J Cancer 85:398–406
Akhtar S, Dunnion D, Poyner D, Ackroyd J, Bibby M, Double J (2002) Biochem Pharmacol 63:2187–2195
Bell C, Lynam E, Landfair DJ, Janjic N, Wiles ME (1999) In Vitro Cell Dev Biol Anim 35:533–542
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
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
Romig TS, Bell C, Drolet DW (1999) J Chromatogr B 731:275–284
Smith RG, Missailidis S, Price MR (2002) J Chromatogr B 766:13–26
Murray A, Spencer DIR, Missailidis S, Denton G, Price MR (1998) J Pept Res 52:375–383
Ferreira CSM, Matthews CS, Missailidis S (2006) Tumor Biol 27:289–301
Brokx RD, Revers L, Zhang QH, Yang SX, Mal TK, Ikura M, Gariepy J (2003) Biochemistry 42:13817–13825
Fitzwater T, Polisky B (1996) Method Enzymol 267:275–301
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
Missailidis S, Thomaidou D, Borbas KE, Price MR (2005) J Immunol Methods 296:45–62
Griffin LC, Toole JJ, Leung LLK (1993) Gene 137:25–31
Ulrich H, Magdesian MH, Alves MJM, Colli W (2002) J Biol Chem 277:20756–20762
Karanikas V, Patton K, Jamieson G, Pietersz G, McKenzie I (1998) Tumor Biol 19:71–78
Rye PD, McGuckin MA (2001) Tumor Biol 22:269–272
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.
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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
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DOI: https://doi.org/10.1007/s00216-007-1470-1