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

Ferreira, C. S. M.; Papamichael, K.; Guilbault, G.; Schwarzacher, T.; Gariepy, J.; Missailidis, S.
February 2008
Analytical & Bioanalytical Chemistry;Feb2008, Vol. 390 Issue 4, p1039
Academic Journal
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. [Figure not available: see fulltext.]


Related Articles

  • Expression of p16 in Conjunctival Intraepithelial Neoplasia Does Not Correlate with HPV-Infection. Auw-Haedrich, Claudia; Martin, Gottfried; Spelsberg, Helga; Sundmacher, Rainer; Freudenberg, Nikolaus; Maier, Philip; Reinhard, Thomas // Open Ophthalmology Journal;2008, Vol. 2, p48 

    The aim of our study was to identify the frequency of expression of p16INK4a (CDKN2A) and HPV (human papilloma virus) in different grades of conjunctival intraepithelial neoplasia (CIN). Twelve specimens including CIN I (2), II (3), III (5), and CIN with beginning invasion (2), as well as 15...

  • Adenomatoid tumour of the liver. Nagata, S.; Aishima, S.; Fukuzawa, K.; Takagi, H.; Yonemasu, H.; Iwashita, Y.; Kinoshita, T.; Wakasugi, K. // Journal of Clinical Pathology;Jun2008, Vol. 61 Issue 6, p777 

    An unusual primary adenomatoid tumour arising in the normal liver is described. Hepatectomy was performed, and the patient is alive and free of disease 1 year postsurgery. Grossly, the tumour showed a haemorrhagic cut surface with numerous microcystic structures. Histological examination...

  • APOMABH, a La-Specific Monoclonal Antibody, Detects the Apoptotic Tumor Response to Life-Prolonging and DNA-Damaging Chemotherapy. Al-Ejeh, Fares; Darby, Jocelyn M.; Tsopelas, Chris; Smyth, Douglas; Manavis, Jim; Brown, Michael P. // PLoS ONE;2009, Vol. 4 Issue 2, p1 

    Background: Antineoplastic therapy may impair the survival of malignant cells to produce cell death. Consequently, direct measurement of tumor cell death in vivo is a highly desirable component of therapy response monitoring. We have previously shown that APOMABH representing the DAB4 clone of a...

  • Autoantibody profiling in systemic lupus erythematosus. Song-Chou Hsieh; Chia-Li Yu // Current Biomarker Findings;2013, Vol. 3, p55 

    Systemic lupus erythematosus (SLE) is an archetype of systemic autoimmune disease characterized by the production of a broad spectrum of autoantibodies. More than 100 autoantibodies have been found in the sera of patients with SLE, including antibodies against nuclear, cytoplasmic,...

  • Antibody technology in proteomics. Saerens, Dirk; Hassanzadeh, Gholamreza; Muyldermans, Ghassabeh; Muyldermans, Serge // Briefings in Functional Genomics & Proteomics;Jul2008, Vol. 7 Issue 4, p275 

    Today's proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which...

  • Use of Cell-SELEX to Generate DNA Aptamers as Molecular Probes of HPV-Associated Cervical Cancer Cells. Graham, Jessica C.; Zarbl, Helmut // PLoS ONE;Apr2012, Vol. 7 Issue 4, p1 

    Background: Disease-specific biomarkers are an important tool for the timely and effective management of pathological conditions, including determination of susceptibility, diagnosis, and monitoring efficacy of preventive or therapeutic strategies. Aptamers, comprising single-stranded or...

  • XRCC1 Deficiency Sensitizes Human Lung Epithelial Cells to Genotoxicity by Crocidolite Asbestos and Libby Amphibole. Pietruska, Jodie R.; Johnston, Tatiana; Zhitkovich, Anatoly; Kane, Agnes B. // Environmental Health Perspectives;Dec2010, Vol. 118 Issue 12, p1707 

    Background: Asbestos induces DNA and chromosomal damage, but the DNA repair pathways protecting human cells against its genotoxicity are largely unknown. Polymorphisms in XRCC1 have been associated with altered susceptibility to asbestos-related diseases. However, it is unclear whether oxidative...

  • Mitochondrial D310 mutations in the early development of breast cancer. Xu, C; Tran-Thanh, D; Ma, C; May, K; Jung, J; Vecchiarelli, J; Done, S J // British Journal of Cancer;Apr2012, Vol. 106 Issue 9, p1506 

    Background:The role of mitochondrial DNA (mtDNA) mutations in the development of breast cancer is largely unknown. In this study, we investigated the frequency and pattern of mutations in the D310 region, the most commonly mutated region in mtDNA, in a series of breast lesions.Methods:Using...

  • Benign, Premalign ve Malign Prostat Lezyonlarında Ki-67, Bcl-2 ve C-erbB2 Belirteçlerinin Önemi. SAYAR, İlyas; GELİNCİK, İbrahim; BOZKURT, Aliseydi; BAYRAM, İrfan // Journal of Kartal Training & Research Hospital / Kartal Egitim v;2012, Vol. 23 Issue 3, p123 

    Background: In this study, we studied the importance of Ki-67, bcl-2 and c-erbB2 markers in benign, premalign and malign prostate lesions, and the usefulness of these markers forhisto-logic grading of carcinoma. Methods: We applied Ki-67, bcl-2 and c-erbB2 as immuno-histochemical markers to the...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics