Ariza, AdrianaCollado, DanielVida, YolandaMontañez, María IPérez-Inestrosa, EzequielBlanca, MiguelTorres, María JoséCañada, F JavierPérez-Sala, Dolores2015-09-112015-09-112014-03-03Ariza A, Collado D, Vida Y, Montañez MI, Pérez-Inestrosa E, Blanca M, et al. Study of protein haptenation by amoxicillin through the use of a biotinylated antibiotic. PLoS ONE. 2014; 9(3):e90891http://hdl.handle.net/10668/1985Journal Article; Research Support, Non-U.S. Gov't;Allergic reactions towards β-lactam antibiotics pose an important clinical problem. The ability of small molecules, such as a β-lactams, to bind covalently to proteins, in a process known as haptenation, is considered necessary for induction of a specific immunological response. Identification of the proteins modified by β-lactams and elucidation of the relevance of this process in allergic reactions requires sensitive tools. Here we describe the preparation and characterization of a biotinylated amoxicillin analog (AX-B) as a tool for the study of protein haptenation by amoxicillin (AX). AX-B, obtained by the inclusion of a biotin moiety at the lateral chain of AX, showed a chemical reactivity identical to AX. Covalent modification of proteins by AX-B was reduced by excess AX and vice versa, suggesting competition for binding to the same targets. From an immunological point of view, AX and AX-B behaved similarly in RAST inhibition studies with sera of patients with non-selective allergy towards β-lactams, whereas, as expected, competition by AX-B was poorer with sera of AX-selective patients, which recognize AX lateral chain. Use of AX-B followed by biotin detection allowed the observation of human serum albumin (HSA) modification by concentrations 100-fold lower that when using AX followed by immunological detection. Incubation of human serum with AX-B led to the haptenation of all of the previously identified major AX targets. In addition, some new targets could be detected. Interestingly, AX-B allowed the detection of intracellular protein adducts, which showed a cell type-specific pattern. This opens the possibility of following the formation and fate of AX-B adducts in cells. Thus, AX-B may constitute a valuable tool for the identification of AX targets with high sensitivity as well as for the elucidation of the mechanisms involved in allergy towards β-lactams.enAnimalesAntibacterianosBiotinilaciónButilaminasHaptenosHipersensibilidadAmoxicilinaInmunoglobulina EMacrófagosEstructura molecularBeta-lactamasMedical Subject Headings::Organisms::Eukaryota::AnimalsMedical Subject Headings::Chemicals and Drugs::Chemical Actions and Uses::Pharmacologic Actions::Therapeutic Uses::Anti-Infective Agents::Anti-Bacterial AgentsMedical Subject Headings::Phenomena and Processes::Chemical Phenomena::Biochemical Phenomena::Biochemical Processes::Binding, CompetitiveMedical Subject Headings::Phenomena and Processes::Chemical Phenomena::Biochemical Phenomena::Biochemical Processes::BiotinylationMedical Subject Headings::Chemicals and Drugs::Organic Chemicals::Amines::ButylaminesMedical Subject Headings::Chemicals and Drugs::Biological Factors::Antigens::Epitopes::HaptensMedical Subject Headings::Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::HumansMedical Subject Headings::Diseases::Immune System Diseases::HypersensitivityMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Globulins::Serum Globulins::Immunoglobulins::Antibodies::Immunoglobulin Isotypes::Immunoglobulin EMedical Subject Headings::Anatomy::Hemic and Immune Systems::Immune System::Phagocytes::MacrophagesMedical Subject Headings::Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Rodentia::Muridae::Murinae::MiceMedical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Microscopy::Microscopy, ConfocalMedical Subject Headings::Phenomena and Processes::Chemical Phenomena::Molecular StructureMedical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Albumins::Serum AlbuminMedical Subject Headings::Chemicals and Drugs::Organic Chemicals::Amides::Lactams::beta-LactamsMedical Subject Headings::Chemicals and Drugs::Organic Chemicals::Amides::Lactams::beta-Lactams::Penicillins::Penicillin G::Ampicillin::Amoxicillinresearch article24595455open access10.1371/journal.pone.00908911932-6203PMC3940954