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 microg/ml, a detection limit of 1 microg/ml and a linear range between 8 and 100 microg/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.

Aptamers in the Diagnosis of Rickettsiosis Sensu Lato

“…However, aptamers can be raised, and have been raised, directly against bacteria; and they have been used in ELISA type assays, such as those described by Ferreira et al (2008), and for the detection of Francisella tularensis in an ALISA. There, B. henselae was used as negative control, to show that aptamers were specific for F. tularensis, but did not bind to the related Bartonella (Vivekananda & Kiel 2006).…”

Aptamer-based radiopharmaceuticals for diagnostic imaging and targeted radiotherapy of epithelial tumors

“…Aptamers vary in size between 25 and 50 bases and are identified from combinatorial libraries through selective targeting and affinity maturation. They offer unique benefits compared to other targeting agents, in that they bind with high affinity and selectivity, are easily and quickly synthesised using in vitro techniques, are stable and consistent (Jayasena, 1999), making them alternatives to antibodies and peptides for diagnostic assays (Hesselberth et al, 2000;Ferreira et al, 2008). As aptamers are not immunogenic or toxic, they also have potential as therapeutic and imaging agents (Borbas et al, 2007).…”

“…The two year safety results demonstrated that all the doses of Pegaptanib were well tolerated and any adverse events were momentary owing to the injection preparation and procedures rather than the Pegaptanib drug (D'Amico et al, 2006). Furthermore, labelled aptamers have been used as antibody mimics in sandwich assays, ELISAs, and Western blots (Bacher and Ellington, 1998;Jayasena, 1999;Morris et al, 1998;Ferreira et al, 2008), and their ability to directly transduce molecular recognition into optical signals. The demonstration that aptamers, either through incorporation of fluorescent bases in the selection process or through post-SELEX modifications to generate aptamer beacons (Potyrailo et al, 1998), can be used for ligand identification based on detectable changes in the fluorescence emission upon ligand binding indicates their potential as a sensitive detection tool.…”

Anti-MUC1 nano-aptamers for triple-negative breast cancer imaging by single-photon emission computed tomography in inducted animals: initial considerations

“…18,19 Aptamers are synthetic oligonucleotides, such as ribonucleic acid (RNA) and single-stranded deoxyribonucleic acid, which can bind to their targets with high affinity and specificity because of their specific secondary and tertiary structures. 20 Compared with antibodies, they offer a great potential in targeting tumor markers like MUC1 because of their small size, lack of immunogenicity, and superior tumor penetration.…”

“…20 Compared with antibodies, they offer a great potential in targeting tumor markers like MUC1 because of their small size, lack of immunogenicity, and superior tumor penetration. 21 In addition, they have been capable of detecting circulating MUC1 in sandwich enzyme-linked immunosorbent assays, improving current detection limits, 19 and have been extensively studied as radiopharmaceuticals for their potential in gamma-camera and single-photon emission computed tomography (SPECT) imaging. [21][22][23] Furthermore, they have been shown to be particularly promising agents in photodynamic therapy as phototoxic agents 24 and delivery agents of standard chemotherapy such as doxorubicin.…”