Selection of aptamers with high affinity and high specificity against C595, an anti-MUC1 IgG3 monoclonal antibody, for antibody targeting.
Targeting of antibodies has found a number of applications in assays, anti-idiotypic therapies and vaccine design with a number of anti-idiotypic Abs generated and used in clinical applications, and some currently in clinical trials. Meanwhile, aptamers are a novel and particularly interesting targeting modality, with a unique ability to bind to a variety of targets. Aptamers offer unique benefits compared to other targeting agents, due to their high affinity and selectivity, relatively small size and in vitro synthesis, making them attractive alternatives to Abs and peptides. Aptamers have already been selected against a number of Abs for various applications. We now present a novel methodology for the selection of aptamers against Abs, which minimises the number of steps used and results in molecules that bind to the target Ab with high affinity and specificity. We have used the well-characterised anti-MUC1 monoclonal Ab C595 as an exemplar for raising aptamers against Abs. The methodology is based on the adsorption of the Ab to the surface of a PCR tube and the performance of SELEX selections in the PCR tube, based on elution steps resulting from the denaturation of the Ab on the first PCR amplification cycle. After 10 rounds of selection and amplification, selected aptamers have been characterised using a number of techniques, including fluorescence quenching, ELISA and competition ELISA procedures and a FRET type assay. Aptamers were found to bind their target Ab with a higher affinity than its natural antigenic peptide, as observed in fluorescent quenching and FRET experiments. Furthermore, they were able to displace the antigens from the antibody binding pocket in competition assays. This methodology offers the possibility of rapidly selecting aptamers for antibody targeting that could be used as diagnostic, imaging or therapeutic agents, or as recognition units in immunoassays, and can be potentially useful in raising aptamers against other protein targets.
Missailidis
Sotiris
S
Chemistry Department, The Open University, Walton Hall, Milton Keynes MK7 6AA, UK. s.missailidis@open.ac.uk
Thomaidou
Despina
D
Borbas
K Eszter
KE
Price
Mike R
MR
D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D001667:Binding, Competitive; D020650:Combinatorial Chemistry Techniques; D019020:Directed Molecular Evolution / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D031541:Fluorescence Resonance Energy Transfer; D015723:Gene Library; D006801:Humans; D018396:Mucin-1 / Q000276:immunology*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D018396:Mucin-1; D009838:Oligodeoxyribonucleotides