1. Study of protein haptenation by amoxicillin through the use of a biotinylated antibiotic.
- Author
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Ariza A, Collado D, Vida Y, Montañez MI, Pérez-Inestrosa E, Blanca M, Torres MJ, Cañada FJ, and Pérez-Sala D
- Subjects
- Amoxicillin chemistry, Animals, Anti-Bacterial Agents chemistry, Binding, Competitive immunology, Biotinylation methods, Butylamines, Haptens immunology, Humans, Immunoglobulin E blood, Macrophages, Mice, Microscopy, Confocal, Molecular Structure, Serum Albumin metabolism, beta-Lactams immunology, Amoxicillin pharmacology, Anti-Bacterial Agents pharmacology, Haptens metabolism, Hypersensitivity immunology, beta-Lactams metabolism
- Abstract
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.
- Published
- 2014
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