title: Preparation of antibodies and development of an enzyme-linked immunosorbent assay (ELISA) for the determination of doxycycline antibiotic in milk samples.
abstract: This paper reports the development of an immunoassay for the specific analysis of doxycycline (DC), a congener of the tetracycline antibiotic family (TCs), in milk samples. This is the first time that DC antibody production is reported, based on a rationally designed and well-characterized immunizing hapten. The chemical structure of the immunizing hapten (13-[(2-carboxyethyl)thiol]-5-hydroxy-6-α-deoxytetracycline, TC1) was designed to maximize recognition of the tetracycline characteristic moiety defined as lower periphery of the TCs plus the region of the upper periphery composed by the hydroxyl group at position C(5) (B ring) and the dimethylamino group in ring A. Polyclonal antibodies raised against TC1 coupled to horseshoe crab hemocianyn (HCH) were used to develop a homologous indirect competitive enzyme-linked immunosorbent assay (ELISA). The microplate ELISA can detect DC in buffer down to 0.1 μg L(-1). The ELISA has been proven to tolerate a wide range of ionic strengths and pH values. The assay is very selective for DC with a minor recognition of methacycline (32% of cross-reactivity). Experiments performed with whole milk samples demonstrate that samples can be directly analyzed after a simple treatment method, reaching detectability values below 5 μg L(-1).
authors.affiliation: Applied Molecular Receptors Group, Department of Chemical and Biomolecular Nanotechnology, Institute for Advanced Chemistry of Catalonia, Spanish Council for Scientific Research, Jordi Girona 18-26, Barcelona, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Adrian
authors.forename: Fátima
authors.initials: F
authors.lastname: Fernández
authors.forename: Francisco
authors.initials: F
authors.lastname: Sánchez-Baeza
authors.forename: M-Pilar
authors.initials: MP
authors.lastname: Marco
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000032:analysis* / Q000276:immunology; D000906:Antibodies / Q000032:analysis / Q000276:immunology; D002417:Cattle; D004318:Doxycycline / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D005506:Food Contamination / Q000032:analysis; D008892:Milk / Q000737:chemistry*; D011817:Rabbits
chemical_list: D000900:Anti-Bacterial Agents; D000906:Antibodies; D004318:Doxycycline