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Development of aptamer-based technology to detect residues of veterinary drugs, and constituents of vaccines - VM02162

Aptamers are artificial nucleic acid sequences generated against specific targets with the ability to bind to non-nucleic acid target molecules. Aptamers can be derived from either DNA or RNA typically consisting of short sequences of single stranded oligonucleotides. These three-dimensional (3D) shapes possess specific structural, ligand-binding and catalytic properties (Huang and Szostak, 2003). Due to this property aptamers can be produced to bind tightly to various molecular targets such as small molecules, proteins, other nucleic acids and even whole cells, tissues and organisms. Aptamers have been used in a variety of biotechnological and therapeutical applications e.g. diagnostics, biosensors, anti-angiogenic therapeutic agents and for combating infectious agents (Hwang et al., 2007). The identification of novel aptamers is based on a combinatorial approach, nucleic acid molecules are created by selection from a large random sequence pool via repeated rounds of in vitro selection to bind to a specific molecular target, termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Advances in the area of in vitro selection and amplification technology over the last decade have permitted the facile identification of unique RNA folds from random libraries that can potentially target any ligand of interest with affinities in the micromolar to nanomolar range (Patel, 1997). Contrary to antibodies, aptamers are produced by a totally in vitro process, avoiding the use of animals. Batch-to-batch variations are also avoided, as the production process is a tightly controlled chemical synthesis. The products are purified under denaturing conditions to achieve a high degree of purity. The timescale for new aptamer selections is much shorter compared to conventional antibody production, typically weeks vs. months.

Aptamers have been successfully used as alternatives to antibodies to detect various biological compounds such as viruses or cancerous cells. They are also showing great promise in their ability to detect illegal residues in foods of animal origin e.g. malachite green residues in fish (Defra project VMO2150). The aim of this follow-on project is to identify RNA aptamer sequences for (i) specific veterinary drugs and (ii) contaminants in vaccine preparations. The selection of aptamers, which will then be evaluated in prototype screening assays, does not require the use of animals, thus directly contributing to the `3-Rs in research` initiative. This project will be divided into two research phases focusing initially on the development and characterisation of aptamers for target veterinary drugs (antibiotics) and secondly on constituents present in the rabies vaccine. More details on each phase are given below:

Phase 1. – Veterinary drugs.
Between 2003 and 2007 there were c. 700 EU notifications under the Rapid Alert System for Food and Feed (RASFF) system relating to veterinary medicines (primarily antibiotics) in foods of animal origin. Therefore, there is a need for the development of rapid screening assays capable of multiplexing to efficiently monitor the increasing number the food samples for the presence of a wide variety of antibiotics to better safeguard the consumer. The currently available screening procedures for aminoglycosides are limited in capability due to either analyte specificity profile e.g. ELISA (Eurodiagnostica produce individual kits for streptomycin, gentamicin and neomycin) or sensitivity e.g. the STAR test (Gaudin et al., 2002). Therefore, the aminoglycoside class of compounds are suggested for study within this project.

Phase 2. - Aptamer production for inactivated rabies glycoprotein for vaccine potency testing.
The quality control processes that are required for the release of a new batch of vaccine are prescribed in various guidelines. Vaccine manufacturers are required to check each batch for safety, inactivation, pyrogenicity and efficacy. At present the procedures involved in performing these checks involve large numbers of mice, c. 2000 mice /year are used to test the rabies vaccine for veterinary use within the EU alone (Bruckner et al., 2002). In vitro assays based on antibodies have been previously developed in attempts to replace the standard rabies vaccine in vivo potency test developed at the National Institutes of Health, USA (Wilbur and Aubert, 1996). Production of antibodies to viruses and certain contaminants can be problematic, as an alternative approach, aptamers will be selected for these targets within this project with the ultimate aim of developing in vitro vaccine tests for use within control laboratories (outside the scope of this project).

In this project the new aptamer sequences will be evaluated in a suitable multiplex format assay e.g. using fluorescence resonance energy transfer (FRET). This format incorporates a fluorescence probe / quencher pair attached on the 5’ and 3’ terminals of the oligonucleotide sequence. Binding of the target molecule leads to a conformation change in the aptamer and thus, interaction with the target analyte leads to either the loss of fluorescence resonance energy transfer (FRET) or de-quenching of the fluorophore. Such optical signals can be readily detected in a multiplex format e.g. flow cytometry
Time-Scale and Cost
From: 2009

To: 2015

Cost: £621,755
Contractor / Funded Organisations
Central Science Laboratory
Animal Health              
Plants and Animals              
Residues Testing              
Veterinary Medicines              
Fields of Study
Animal Health
Veterinary Medicine