Selection, characterisation and analytical application of dna aptamer against the anaphylactic toxic allergen, b-conglutin, lup an 1

Abstract

Lupin has recently been added to the list of allergens requiring mandatory advisory labelling on foodstuffs sold in the European Union, and since December 2008 all products containing even trace amounts of lupin must be labelled correctly. Lupin globulins consist of two major globulins called α-conglutin (11S and “legumin-like”) and β-conglutin (7S and “vicilin-like”), and another additional two globulins, γ-conglutin and δ-conglutin, which are present in lower amounts. β-conglutin is the only conglutin currently included in the list of the International Union of Immunological Societies (IUIS), designated as Lup an 1. The overall objective of these PhD is the selection of aptamers that can detect this allergen. Nucleic acid aptamers are synthetic ligands selected from vast combinatorial libraries through a process referred to as SELEX – Systematic Evolution of Ligand By Exponential Enrichment. Aptamers possess unique chemical and biochemical characteristics, such as: well known chemistry and remarkable stability, moreover, aptamers can be selected against virtually any target and in non-physiological conditions. In order to achieve the overall objective, a set of subobjectives will be achieved. The first of these involves the elucidation of protocols for the selective extraction of each of the lupin α, β, γ, and δ subunits, resulting in (i) protocols that can be used for selective extraction and isolation of the lupin α, β, γ, and δ proteins from food for subsequent analysis; (ii) standards that can be used in analytical assays and tools; and (iii) target that can be used for the selection of aptamers specific to the β-conglutin subunit. The core of the work is the selection of aptamers against the allergen Lup an 1 using a SELEX procedure, as well the preparation of protocols that can be used to monitor the evolution of aptamer selection. The functionality of the aptamer is demonstratedby exploiting it in an enzyme linked oligonucleotide assay as well as apta-PCR. Finally the resulting aptamer candidates that exhibit high affinity are fully characterised, truncated, and the structure of the final truncated aptamer is elucidated