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24 results on '"Montañez, M. I."'

1. Antigenic nanostructures based on amoxicilloyl-bidendrons for effector cell activation to study allergic reactions to amoxicillin

Author

Ayane, Amene, Benedé, Sara, Morgado, Anjara, Vida, Yolanda, Rodríguez-Nogales, Alba, Fernández, Tahia, Paris, Juan L., Jiménez, Isabel, Bogas, Gador, Mayorga, Cristobalina, Pérez-Inestrosa, Ezequiel, Torres, María J., Montañez, M. I., Ayane, Amene, Benedé, Sara, Morgado, Anjara, Vida, Yolanda, Rodríguez-Nogales, Alba, Fernández, Tahia, Paris, Juan L., Jiménez, Isabel, Bogas, Gador, Mayorga, Cristobalina, Pérez-Inestrosa, Ezequiel, Torres, María J., and Montañez, M. I.

Published
2023

3. Identification of an antigenic determinant of clavulanic acid responsible for IgE-mediated reactions

Author

Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (España), Junta de Andalucía, Mayorga, Cristobalina [0000-0001-8852-8077], Martín-Serrano, Ángela [0000-0002-2908-8910], Nájera, Francisco [0000-0002-1635-5514], Salas, María [0000-0002-0583-9492], Pérez-Sala, Dolores [0000-0003-0600-665X], Pérez-Inestrosa, Ezequiel [0000-0001-7546-5273], Fernández, Tahia [0000-0003-0625-2156], Montañez, M. I. [0000-0001-6641-5979], Torres, María J. [0000-0003-4499-840X], Barbero, Nekane, Fernández-Santamaría, Rubén, Mayorga, Cristobalina, Martín-Serrano, Ángela, Salas, María, Bogas, Gador, Nájera, Francisco, Pérez-Sala, Dolores, Pérez-Inestrosa, Ezequiel, Fernández, Tahia, Montañez, M. I., Torres, María J., Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (España), Junta de Andalucía, Mayorga, Cristobalina [0000-0001-8852-8077], Martín-Serrano, Ángela [0000-0002-2908-8910], Nájera, Francisco [0000-0002-1635-5514], Salas, María [0000-0002-0583-9492], Pérez-Sala, Dolores [0000-0003-0600-665X], Pérez-Inestrosa, Ezequiel [0000-0001-7546-5273], Fernández, Tahia [0000-0003-0625-2156], Montañez, M. I. [0000-0001-6641-5979], Torres, María J. [0000-0003-4499-840X], Barbero, Nekane, Fernández-Santamaría, Rubén, Mayorga, Cristobalina, Martín-Serrano, Ángela, Salas, María, Bogas, Gador, Nájera, Francisco, Pérez-Sala, Dolores, Pérez-Inestrosa, Ezequiel, Fernández, Tahia, Montañez, M. I., and Torres, María J.

Abstract

BACKGROUND: Selective reactions to clavulanic acid (CLV) account for around 30% of immediate reactions after administration of amoxicillin-CLV. Currently, no immunoassay is available for detecting specific IgE to CLV, and its specific recognition in patients with immediate reactions has only been demonstrated by basophil activation testing, however with suboptimal sensitivity. The lack of knowledge regarding the structure of the drug that remains bound to proteins (antigenic determinant) is hampering the development of in vitro diagnostics. We aimed to identify the antigenic determinants of CLV as well as to evaluate their specific IgE recognition and potential role for diagnosis., METHODS: Based on complex CLV degradation mechanisms, we hypothesized the formation of two antigenic determinants for CLV, AD-I (N-protein, 3-oxopropanamide) and AD-II (N-protein, 3-aminopropanamide), and designed different synthetic analogs to each one. IgE recognition of these structures was evaluated in basophils from patients with selective reactions to CLV and tolerant subjects. In parallel, the CLV fragments bound to proteins were identified by proteomic approaches., RESULTS: Two synthetic analogs of AD-I were found to activate basophils from allergic patients. This determinant was also detected bound to lysines 195 and 475 of CLV-treated human serum albumin. One of these analogs was able to activate basophils in 59% of patients whereas CLV only in 41%. Combining both results led to an increase in basophil activation in 69% of patients, and only in 12% of controls., CONCLUSION: We have identified AD-I as one CLV antigenic determinant, which is the drug fragment that remains protein-bound.

Published
2019

4. Role of nanostructures in allergy: Diagnostics, treatments and safety

Author

Mayorga, Cristobalina, Pérez-Inestrosa, Ezequiel, Rojo, Francisco Javier, Ferrer, Marta, Montañez, M. I., Mayorga, Cristobalina, Pérez-Inestrosa, Ezequiel, Rojo, Francisco Javier, Ferrer, Marta, and Montañez, M. I.

Abstract

Nanotechnology is science, engineering and technology conducted at the nanoscale, which is about 1–100 nm. It has led to the development of nanomaterials, which be have very differently from materials with larger scales and can have a wide range of applications in biomedicine. The physical and chemical properties of materials of such small compounds depend mainly on the size, shape, composition and func tionalization of the system. Nanoparticles, carbon nanotubes, liposomes, polymers, dendrimers and nanogels, among others, can be nanoengineeried for controlling all parameters, including their functionalization with ligands, which provide the desired interaction with the immunological system, that is dendritic cell receptors to acti vate and/or modulate the response, as well as specific IgE, or effector cell receptors. However, undesired issues related to toxicity and hypersensitivity responses can also happen and would need evaluation. There are wide panels of accessible structures, and controlling their physico-chemical properties would permit obtaining safer and more efficient compounds for clinical applications goals, either in diagnosis or treat ment. The application of dendrimeric antigens, nanoallergens and nanoparticles in al lergy diagnosis is very promising since it can improve sensitivity by increasing specific IgE binding, mimicking carrier proteins or enhancing signal detection. Additionally, in the case of immunotherapy, glycodendrimers, liposomes, polymers and nanoparti cles have shown interest, behaving as platforms of allergenic structures, adjuvants or protectors of allergen from degradation or having a depot capacity. Taken together, the application of nanotechnology to allergy shows promising facts facing important goals related to the improvement of diagnosis as well as specific immunotherapy

Published
2021

11. Adduct formation and context factors in drug hypersensitivity: insight from proteomic studies

Author

Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, Junta de Andalucía, González-Morena, Juan M., Montañez, M. I., Aldini, Giancarlo, Sánchez-Gómez, Francisco J., Pérez-Sala, Dolores, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, Junta de Andalucía, González-Morena, Juan M., Montañez, M. I., Aldini, Giancarlo, Sánchez-Gómez, Francisco J., and Pérez-Sala, Dolores

Abstract

Drug hypersensitivity reactions result from the activation of the immune system by drugs or their metabolites. The clinical presentations of drug hypersensitivity can range from relatively mild local manifestations to severe systemic syndromes that can be life-threatening. As in other allergic reactions, the causes are multifactorial as genetic, metabolic and concomitant factors may influence the occurrence of drug hypersensitivity. Formation of drug protein adducts is considered a key step in drug adverse reactions, and in particular in the immunological recognition in drug hypersensitivity reactions. Nevertheless, non-covalent interactions of drugs with receptors in immune cells or with MHC clefts and/or exposed peptides can also play an important role. In recent years, development of proteomic approaches has allowed the identification and characterization of the protein targets for modification by drugs in vivo and in vitro, the nature of peptides exposed on MHC molecules, the changes in protein levels induced by drug treatment, and the concomitant modifications induced by danger signals, thus providing insight into context factors. Nevertheless, given the complexity and multifactorial nature of drug hypersensitivity reactions, understanding the underlying mechanisms also requires the integration of knowledge from genomic, metabolomic and clinical studies.

Published
2016

12. The influence of the carrier molecule on amoxicillin recognition by specific IgE in patients with immediate hypersensitivity reactions to betalactams

Author

Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (España), Junta de Andalucía, European Commission, Ariza, Adriana, Mayorga, Cristobalina, Salas, María, Doña, Inmaculada, Martín-Serrano, Ángela, Pérez-Inestrosa, Ezequiel, Pérez-Sala, Dolores, Guzmán, Antonio E., Montañez, M. I., Torres, María J., Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (España), Junta de Andalucía, European Commission, Ariza, Adriana, Mayorga, Cristobalina, Salas, María, Doña, Inmaculada, Martín-Serrano, Ángela, Pérez-Inestrosa, Ezequiel, Pérez-Sala, Dolores, Guzmán, Antonio E., Montañez, M. I., and Torres, María J.

Abstract

The optimal recognition of penicillin determinants, including amoxicillin (AX), by specific IgE antibodies is widely believed to require covalent binding to a carrier molecule. The nature of the carrier and its contribution to the antigenic determinant is not well known. Here we aimed to evaluate the specific-IgE recognition of different AX-derived structures. We studied patients with immediate hypersensitivity reactions to AX, classified as selective or cross-reactors to penicillins. Competitive immunoassays were performed using AX itself, amoxicilloic acid, AX bound to butylamine (AXO-BA) or to human serum albumin (AXO-HSA) in the fluid phase, as inhibitors, and amoxicilloyl-poli-L-lysine (AXO-PLL) in the solid-phase. Two distinct patterns of AX recognition by IgE were found: Group A showed a higher recognition of AX itself and AX-modified components of low molecular weights, whilst Group B showed similar recognition of both unconjugated and conjugated AX. Amoxicilloic acid was poorly recognized in both groups, which reinforces the need for AX conjugation to a carrier for optimal recognition. Remarkably, IgE recognition in Group A (selective responders to AX) is influenced by the mode of binding and/or the nature of the carrier; whereas IgE in Group B (cross-responders to penicillins) recognizes AX independently of the nature of the carrier.

Published
2016

13. Study of protein haptenation by amoxicillin through the use of a biotinylated antibiotic

Author

Ariza, Adriana, Collado, Daniel, Vida, Yolanda, Montañez, M. I., Pérez-Inestrosa, Ezequiel, Blanca, Miguel, Torres, María J., Cañada, F. Javier, Pérez-Sala, Dolores, Ariza, Adriana, Collado, Daniel, Vida, Yolanda, Montañez, M. I., Pérez-Inestrosa, Ezequiel, Blanca, Miguel, Torres, María J., Cañada, F. Javier, and Pérez-Sala, Dolores

Abstract

Allergic reactions towards b-lactam antibiotics pose an important clinical problem. The ability of small molecules, such as a b-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 b-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 b-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 b-lactams.

Published
2014

14. Proteomics in immunological reactions to drugs

Author

Ariza, Adriana, Montañez, M. I., Pérez-Sala, Dolores, Ariza, Adriana, Montañez, M. I., and Pérez-Sala, Dolores

Abstract

Purpose of review: To discuss the avenues that proteomic techniques are opening for the study of the chemical basis and cellular mechanisms of immunological reactions to drugs. Recent findings: Technical developments in recent years are allowing a detailed characterization of drug–protein interactions. In addition, novel metabolic pathways for drug biotransformation are being uncovered and potential targets for protein haptenation are being proposed that may help in the understanding of these complex processes. Summary: Immunological reactions to drugs pose important clinical problems. Since early works exploring drug–protein interactions, there has been steady progress in this field. However, the mechanisms involved remain incompletely understood. The availability of proteomic techniques with high resolution and sensitivity presents a unique opportunity to tackle this subject from a broad perspective, integrating work in model systems and in patients. Chemical and metabolic characterization of immunological reactions to drugs may also help in the prevention, diagnosis and/or treatment of these processes

Published
2011

15. In vitro tests for drug hypersensitivity reactions: an ENDA/ EAACI Drug Allergy Interest Group position paper.

Author

Mayorga, C., Celik, G., Rouzaire, P., Whitaker, P., Bonadonna, P., Rodrigues‐Cernadas, J., Vultaggio, A., Brockow, K., Caubet, J. C., Makowska, J., Nakonechna, A., Romano, A., Montañez, M. I., Laguna, J. J., Zanoni, G., Gueant, J. L., Oude Elberink, H., Fernandez, J., Viel, S., and Demoly, P.

Subjects
DRUG allergy, ROUTINE diagnostic tests, SKIN tests, CELL analysis, DIAGNOSIS, ALLERGY treatment
Abstract

Drug hypersensitivity reactions ( DHRs) are a matter of great concern, both for outpatient and in hospital care. The evaluation of these patients is complex, because in vivo tests have a suboptimal sensitivity and can be time-consuming, expensive and potentially risky, especially drug provocation tests. There are several currently available in vitro methods that can be classified into two main groups: those that help to characterize the active phase of the reaction and those that help to identify the culprit drug. The utility of these in vitro methods depends on the mechanisms involved, meaning that they cannot be used for the evaluation of all types of DHRs. Moreover, their effectiveness has not been defined by a consensus agreement between experts in the field. Thus, the European Network on Drug Allergy and Drug Allergy Interest Group of the European Academy of Allergy and Clinical Immunology has organized a task force to provide data and recommendations regarding the available in vitro methods for DHR diagnosis. We have found that although there are many in vitro tests, few of them can be given a recommendation of grade B or above mainly because there is a lack of well-controlled studies, most information comes from small studies with few subjects and results are not always confirmed in later studies. Therefore, it is necessary to validate the currently available in vitro tests in a large series of well-characterized patients with DHR and to develop new tests for diagnosis. [ABSTRACT FROM AUTHOR]

Published
2016
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16. Amoxicillin haptenation of α-enolase is modulated by active site occupancy and acetylation

Author

Juan M. González-Morena, Francisco J. Sánchez-Gómez, Yolanda Vida, Ezequiel Pérez-Inestrosa, María Salas, María I. Montañez, Alessandra Altomare, Giancarlo Aldini, María A. Pajares, Dolores Pérez-Sala, Ministerio de Ciencia e Innovación (España), Instituto de Salud Carlos III, Junta de Andalucía, Universidad de Málaga, CSIC - Unidad de Recursos de Información Científica para la Investigación (URICI), González-Morena, Juan M., Vida, Yolanda, Pérez-Inestrosa, Ezequiel, Salas, María, Montañez, M. I., Altomare, Alessandra, Aldini, Giancarlo, Pajares, María A., Pérez-Sala, Dolores, González-Morena, Juan M. [0000-0003-2932-0756], Vida, Yolanda [0000-0002-7004-4629], Pérez-Inestrosa, Ezequiel [0000-0001-7546-5273], Salas, María [0000-0002-0583-9492], Montañez, M. I. [0000-0001-6641-5979], Altomare, Alessandra [0000-0002-9906-6098], Aldini, Giancarlo [0000-0002-2355-6744], Pajares, María A. [0000-0002-4714-9051], and Pérez-Sala, Dolores [0000-0003-0600-665X]

Subjects
Pharmacology, allergic responses to drugs, posttranslational modification, beta-lactam antibiotics, Acetylation, RM1-950, Beta-lactam antibiotics, Protein modification by drugs, Pharmacology (medical), Therapeutics. Pharmacology, Posttranslational modification, protein modification by drugs, Mass spectrometry, Allergic responses to drugs, Original Research, acetylation, mass spectrometry
Abstract

17 p.-8 fig.-1 tab., Allergic reactions to antibiotics are a major concern in the clinic. ß-lactam antibiotics are the class most frequently reported to cause hypersensitivity reactions. One of the mechanisms involved in this outcome is the modification of proteins by covalent binding of the drug (haptenation). Hence, interest in identifying the corresponding serum and cellular protein targets arises. Importantly, haptenation susceptibility and extent can be modulated by the context, including factors affecting protein conformation or the occurrence of other posttranslational modifications. We previously identified the glycolytic enzyme α-enolase as a target for haptenation by amoxicillin, both in cells and in the extracellular milieu. Here, we performed an in vitro study to analyze amoxicillin haptenation of α-enolase using gel-based and activity assays. Moreover, the possible interplay or interference between amoxicillin haptenation and acetylation of α-enolase was studied in 1D- and 2D-gels that showed decreased haptenation and displacement of the haptenation signal to lower pI spots after chemical acetylation of the protein, respectively. In addition, the peptide containing lysine 239 was identified by mass spectrometry as the amoxicillin target sequence on α-enolase, thus suggesting a selective haptenation under our conditions. The putative amoxicillin binding site and the surrounding interactions were investigated using the α-enolase crystal structure and molecular docking. Altogether, the results obtained provide the basis for the design of novel diagnostic tools or approaches in the study of amoxicillin-induced allergic reactions., This work was supported by grants from the Ministerio de Ciencia e Innovación cofunded by ERDF (SAF2015-68590-R and RTI2018-097624-B-I00 to DPS and PCI2019-111825-2 Proyectos de I+D+I “Programación Conjunta Internacional” EuroNanoMed 2019 and PID2019-104293GB-I00 to EI), the Instituto de Salud Carlos III ERDF (RETIC ARADyAL RD16/0006/0021 to DPS, RETIC ARADyAL RD16/0006/0001 to MS and MIM, CPII20/00028 to MIM and RETIC ARADyAL RD16/0006/0012 to EPI, and CPII20/00028 to MIM), Junta de Andalucía and Universidad de Málaga (UMA18-FEDERJA-007 to EPI), Consejería de Transformación Económica, Industria, Conocimiento y Universidades of Junta de Andalucía (PY20_00384 to EPI). The funders had no role in study design, analysis of the data or decission to publish. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Published
2022

17. Biotin-Labelled Clavulanic Acid to Identify Proteins Target for Haptenation in Serum: Implications in Allergy Studies

Author

Ángela Martín-Serrano, Juan M. Gonzalez-Morena, Nekane Barbero, Adriana Ariza, Francisco J. Sánchez Gómez, Ezequiel Pérez-Inestrosa, Dolores Pérez-Sala, Maria J. Torres, María I. Montañez, [Martín-Serrano,A, Ariza,A, Torres,MJ, Montañez,MI] Allergy Research Group, Instituto de Investigación Biomédica de Málaga-IBIMA, Málaga, Spain. [Martín-Serrano.A, Barbero,N, Pérez-Inestrosa,E, Montañez,MI] Centro Andaluz de Nanomedicina y Biotecnología-BIONAND, Málaga, Spain. [Gonzalez-Morena,JM] Department of Structural and Chemical Biology, Centro de Investigaciones Biológicas Margarita Salas (CSIC), Madrid, Spain. [Barbero,N, Pérez-Inestrosa,E] Department Química Orgánica, Universidad de Málaga-IBIMA, Málaga, Spain. [Torres,MJ] Allergy Unit, Hospital Regional Universitario de Málaga, Málaga, Spain. [Torres,MJ] Department of Medicina, Universidad de Málaga, Málaga, Spain., Work at MJT and MIM laboratory was supported by Instituto de Salud Carlos III (ISCIII) of MICINN (grants cofunded by ERDF: 'Una manera de hacer Europa' (PI17/01237, PI18/00095, RETIC ARADYAL RD16/0006/0001 and Euronanomed Program AC19/ 00082), Miguel Servet I program (CP15/00103) and Sara Borrell program (CD17/00146)), Andalusian Regional Ministry of Health (PI-0179-2014, PE-0172-2018). Work at EP-I laboratory was supported by the Spanish Ministerio de Economía, Industria y Competitividad (CTQ 2016-75870-P), Ministerio de Ciencia y Educación (PID 2019-104293GB-I00), Ministerio de Ciencia e Innovación [Proyectos de I+D+I Programación Conjunta Internacional, EuroNanoMed 2019 (PCI 2019-111825-2)], ISCIII RETIC ARADYAL RD16/0006/0012 and Junta de Andalucía (UMA18-FEDERJA-007). Work at DP-S laboratory was supported by Grants from Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación (MICINN, Spain) and European Regional Development Fund, SAF 2015-68590-R and RTI 2018-097624-B-I00, ISCIII RETIC ARADyAL RD16/0006/0021., Instituto de Salud Carlos III, European Commission, Junta de Andalucía, Ministerio de Economía, Industria y Competitividad (España), Ministerio de Ciencia e Innovación (España), Agencia Estatal de Investigación (España), Martín-Serrano, Ángela [0000-0002-2908-8910], González-Morena, Juan M. [0000-0003-2932-0756], Pérez-Inestrosa, Ezequiel [0000-0001-7546-5273], Pérez-Sala, Dolores [0000-0003-0600-665X], Montañez, M. I. [0000-0001-6641-5979], Torres, María J. [0000-0003-4499-840X], Martín-Serrano, Ángela, González-Morena, Juan M., Pérez-Inestrosa, Ezequiel, Pérez-Sala, Dolores, Montañez, M. I., and Torres, María J.

Subjects
0301 basic medicine, Phenomena and Processes::Chemical Phenomena::Physicochemical Phenomena::Solubility [Medical Subject Headings], Chemicals and Drugs::Organic Chemicals::Amides::Lactams::beta-Lactams::Clavulanic Acids::Clavulanic Acid [Medical Subject Headings], Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Blood Proteins::Immunoproteins [Medical Subject Headings], Peptide, Biotina, Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::Humans [Medical Subject Headings], 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Ácido clavulánico, Biotin, Peptide mass fingerprinting, Chemicals and Drugs::Heterocyclic Compounds::Heterocyclic Compounds, 1-Ring::Azoles::Imidazoles::Biotin [Medical Subject Headings], Phenomena and Processes::Chemical Phenomena::Biochemical Phenomena::Biochemical Processes::Biotinylation [Medical Subject Headings], medicine, Chemicals and Drugs::Organic Chemicals::Amides::Lactams::beta-Lactams [Medical Subject Headings], Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Peptides [Medical Subject Headings], Pharmacology (medical), biotinylation, Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Globulins::Serum Globulins::Alpha-Globulins::Haptoglobins [Medical Subject Headings], Original Research, chemistry.chemical_classification, Pharmacology, biology, betalactam, haptenation, lcsh:RM1-950, Anatomy::Hemic and Immune Systems::Immune System [Medical Subject Headings], Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Albumins::Ovalbumin::Avidin [Medical Subject Headings], Human serum albumin, Beta-lactamas, Blood proteins, clavulanate, lcsh:Therapeutics. Pharmacology, 030104 developmental biology, Diseases::Chemically-Induced Disorders::Drug-Related Side Effects and Adverse Reactions::Drug Hypersensitivity [Medical Subject Headings], 030228 respiratory system, chemistry, Biochemistry, Biotinylation, biology.protein, Hipersensibilidad a las drogas, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Chemistry Techniques, Analytical::Mass Spectrometry [Medical Subject Headings], Linker, biotin tag, drug allergy, Avidin, medicine.drug
Abstract

16 p.-6 fig., Clavulanic acid (CLV) and amoxicillin, frequently administered in combination, can be independently involved in allergic reactions. Protein haptenation with β-lactams is considered necessary to activate the immune system. The aim of this study was to assess the suitability of biotinylated analogues of CLV as probes to study protein haptenation by this β-lactam. Two synthetic approaches afforded the labeling of CLV through esterification of its carboxylic group with a biotin moiety, via either direct binding (CLV-B) or tetraethylenglycol linker (CLV-TEG-B). The second analogue offered advantages as solubility in aqueous solution and potential lower steric hindrance for both intended interactions, with the protein and with avidin. NMR reactivity studies showed that both CLV and CLV-TEG-B reacts through β-lactam ring opening by aliphatic amino nitrogen, however with different stability of resulting conjugates. Unlike CLV conjugates, that promoted the decomposition of clavulanate fragment, the conjugates obtained with the CLV-TEG-B remained linked, as a whole structure including biotin, to nucleophile and showed a better stability. This was a desired key feature to allow CLV-TEG-B conjugated protein detection at great sensitivity. We have used biotin detection and mass spectrometry (MS) to detect the haptenation of human serum albumin (HSA) and human serum proteins. MS of conjugates showed that HSA could be modified by CLV-TEG-B. Remarkably, HSA preincubation with CLV excess only reduced moderately the incorporation of CLV-TEG-B, which could be attributed to different protein interferences. The CLV-TEG-B fragment with opened β-lactam was detected bound to the 404–430HSA peptide of the treated protein. Incubation of human serum with CLV-TEG-B resulted in the haptenation of several proteins that were identified by 2D-electrophoresis and peptide mass fingerprinting as HSA, haptoglobin, and heavy and light chains of immunoglobulins. Taken together, our results show that tagged-CLV keeps some of the CLV features. Moreover, although we observe a different behavior in the conjugate stability and in the site of protein modification, the similar reactivity indicates that it could constitute a valuable tool to identify protein targets for haptenation by CLV with high sensitivity to get insights into the activation of the immune system by CLV and mechanisms involved in β-lactams allergy., Work at MJT and MIM laboratory was supported by Instituto de Salud Carlos III (ISCIII) of MICINN (grants cofunded by ERDF: “Una manera de hacer Europa” (PI17/01237, PI18/00095, RETIC ARADYAL RD16/0006/0001 and Euronanomed Program AC19/00082), Miguel Servet I program (CP15/00103) and Sara Borrell program (CD17/00146)), Andalusian Regional Ministry of Health (PI-0179-2014, PE-0172-2018). Work at EP-I laboratory was supported by the Spanish Ministerio de Economía, Industria y Competitividad (CTQ 2016-75870-P), Ministerio de Ciencia y Educación (PID 2019-104293GB-I00), Ministerio de Ciencia e Innovación [Proyectos de I+D+I Programación Conjunta Internacional, EuroNanoMed 2019 (PCI 2019-111825-2)], ISCIII RETIC ARADYAL RD16/0006/0012 and Junta de Andalucía (UMA18-FEDERJA-007). Work at DP-S laboratory was supported by Grants from Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación (MICINN, Spain) and European Regional Development Fund, SAF 2015-68590-R and RTI 2018-097624-B-I00, ISCIII RETIC ARADyAL RD16/0006/0021.

Published
2020

18. Amoxicillin inactivation by thiol-catalyzed cyclization reduces protein haptenation and antibacterial potency

Author

María A. Pajares, Tahl Zimmerman, Francisco J. Sánchez-Gómez, Adriana Ariza, María J. Torres, Miguel Blanca, F. Javier Cañada, María I. Montañez, Dolores Pérez-Sala, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, Junta de Andalucía, European Commission, CSIC - Unidad de Recursos de Información Científica para la Investigación (URICI), Pajares, María A., Zimmerman, T., Blanca, Miguel, Cañada, F. Javier, Montañez, M. I., Pérez-Sala, Dolores, Pajares, María A. [0000-0002-4714-9051], Zimmerman, T. [0000-0001-5939-6394], Blanca, Miguel [0000-0003-1631-4621], Cañada, F. Javier [0000-0003-4462-1469], Montañez, M. I. [0000-0001-6641-5979], Pérez-Sala, Dolores [0000-0003-0600-665X], [Pajares,MA, Zimmerman,T, Sánchez-Gómez,FJ, Cañada,FJ, Pérez-Sala,D] Department of Structural and Chemical Biology, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain. [Ariza,A, Torres,MJ, Montañez,MI] Allergy Research Group, Instituto de Investigación Biomédica de Málaga-IBIMA, Hospital Civil, Málaga, Spain. [Ariza,A, Montañez,MI] Nanostructures for Diagnosing and Treatment of Allergic Diseases Laboratory, Andalusian Center for Nanomedicine and Biotechnology- BIONAND, Málaga, Spain. [Torres,MJ] Allergy Unit, Hospital Regional Universitario de Málaga, Hospital Civil, Málaga, Spain. [Blanca,M] Servicio de Alergología, Hospital Infanta Leonor, Madrid, Spain., This work was supported by grant SAF2015-68590-R from MINECO/FEDER, RTI2018-097624-B-I00 and RETIC Aradyal from ISCIII/FEDER RD16/0006/0021 to DP-S, RD16/0006/0001 to MT, RD16/0006/0024 to MB, and grants CP15/00103 and PI17/01237 from ISCIII/ERDF and PI-0179-2014 from Andalusian Regional Ministry Health to MM. AA holds a 'Sara Borrell' research contract (CD17/0146) supported by ISCIII from MINECO [confounded by the European Social Fund (ESF)]. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Subjects
0301 basic medicine, Metabolite, Antibiotics, Crecimiento bacteriano, Benzylpenicillin, Dithiothreitol, chemistry.chemical_compound, 0302 clinical medicine, Chemicals and Drugs::Organic Chemicals::Alcohols::Sugar Alcohols::Dithiothreitol [Medical Subject Headings], Inactivation mechanism, Ampicillin, polycyclic compounds, Pharmacology (medical), Protein adducts, Thiol-containing molecules, Amoxicilina, Original Research, Chemistry, Chemicals and Drugs::Organic Chemicals::Amides::Lactams::beta-Lactams::Cephalosporins::Cephalexin::Cefaclor [Medical Subject Headings], Beta-lactamas, Human serum albumin, Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Peptides::Oligopeptides::Glutathione [Medical Subject Headings], B-lactam antibiotics, Biochemistry, 030220 oncology & carcinogenesis, Chemicals and Drugs::Chemical Actions and Uses::Pharmacologic Actions::Therapeutic Uses::Anti-Infective Agents::Anti-Bacterial Agents [Medical Subject Headings], medicine.drug, Chemicals and Drugs::Chemical Actions and Uses::Specialty Uses of Chemicals::Laboratory Chemicals::Indicators and Reagents::Reducing Agents [Medical Subject Headings], medicine.drug_class, Diseases::Immune System Diseases::Hypersensitivity [Medical Subject Headings], Phenomena and Processes::Chemical Phenomena::Chemical Processes::Physicochemical Processes::Oxidation-Reduction [Medical Subject Headings], Chemicals and Drugs::Heterocyclic Compounds::Heterocyclic Compounds, 1-Ring::Piperazines::Diketopiperazines [Medical Subject Headings], Chemicals and Drugs::Organic Chemicals::Amides::Lactams::beta-Lactams::Penicillins::Penicillin G::Ampicillin::Amoxicillin [Medical Subject Headings], 03 medical and health sciences, thiol-containing molecules, β-lactam antibiotics, Chemicals and Drugs::Organic Chemicals::Amides::Lactams::beta-Lactams [Medical Subject Headings], medicine, otorhinolaryngologic diseases, Thiol groups, Pharmacology, Bacterial growth, lcsh:RM1-950, Beta lactam antibiotic, Amoxicillin, Glutathione, Metabolism, lcsh:Therapeutics. Pharmacology, 030104 developmental biology, Redox regulation, Chemicals and Drugs::Organic Chemicals::Sulfur Compounds::Amino Acids, Sulfur::Cysteine::Acetylcysteine [Medical Subject Headings]
Abstract

16 p.-7 fig.-2 tab.-1 graph. abst., Serum and cellular proteins are targets for the formation of adducts with the β-lactam antibiotic amoxicillin. This process could be important for the development of adverse, and in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein adducts formation. Consequently, we show that several thiol-containing compounds, including dithiothreitol, N-acetyl-L-cysteine, and glutathione, perform a nucleophilic attack on the amoxicillin molecule that is followed by an internal rearrangement leading to amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity. Increased diketopiperazine conversion is also observed with human serum albumin but not with L-cysteine, which mainly forms the amoxicilloyl amide. The effect of thiols is catalytic and can render complete amoxicillin conversion. Interestingly, this process is dependent on the presence of an amino group in the antibiotic lateral chain, as in amoxicillin and ampicillin. Furthermore, it does not occur for other β-lactam antibiotics, including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated amoxicillin transformation are exemplified by a reduced bacteriostatic action and a lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of the intracellular redox status through inhibition of glutathione synthesis influenced the extent of amoxicillin adduct formation with cellular proteins. These results open novel perspectives for the understanding of amoxicillin metabolism and actions, including the formation of adducts involved in allergic reactions., This work was supported by grant SAF2015-68590-R from MINECO/FEDER, RTI2018-097624-B-I00 and RETIC Aradyal from ISCIII/FEDER RD16/0006/0021 to DP-S; RD16/0006/0001 to MT, RD16/0006/0024 to MB; grants CP15/00103 and PI17/ 01237 from ISCIII/ERDF and PI-0179-2014 from Andalusian Regional Ministry Health to MM. AA holds a “Sara Borrell” research contract (CD17/0146) supported by ISCIII from MINECO [confounded by the European Social Fund (ESF)]. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)

Published
2020

19. Identification of an antigenic determinant of clavulanic acid responsible for IgE-mediated reactions

Author

Maria Salas, Cristobalina Mayorga, Francisco Najera, Dolores Pérez-Sala, Gador Bogas, Ezequiel Perez-Inestrosa, María José Torres, Angela Martin-Serrano, Tahia D. Fernandez, Nekane Barbero, Ruben Fernandez-Santamaria, Maria I. Montañez, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (España), Junta de Andalucía, Mayorga, Cristobalina, Martín-Serrano, Ángela, Nájera, Francisco, Salas, María, Pérez-Sala, Dolores, Pérez-Inestrosa, Ezequiel, Fernández, Tahia, Montañez, M. I., Torres, María J., Mayorga, Cristobalina [0000-0001-8852-8077], Martín-Serrano, Ángela [0000-0002-2908-8910], Nájera, Francisco [0000-0002-1635-5514], Salas, María [0000-0002-0583-9492], Pérez-Sala, Dolores [0000-0003-0600-665X], Pérez-Inestrosa, Ezequiel [0000-0001-7546-5273], Fernández, Tahia [0000-0003-0625-2156], Montañez, M. I. [0000-0001-6641-5979], and Torres, María J. [0000-0003-4499-840X]

Subjects
0301 basic medicine, Drug, Hypersensitivity, Immediate, Models, Molecular, In vitro test, in vitro test, media_common.quotation_subject, Immunology, Molecular Conformation, Immunoglobulin E, Epitope, Immediate reactions, 03 medical and health sciences, Epitopes, Structure-Activity Relationship, 0302 clinical medicine, Antigen, Tandem Mass Spectrometry, medicine, Immunology and Allergy, Humans, clavulanic acid, immediate reactions, Clavulanic Acid, media_common, biology, medicine.diagnostic_test, Chemistry, Drug allergy, Human serum albumin, In vitro, Basophils, Basophil activation, 030104 developmental biology, 030228 respiratory system, ROC Curve, Immunoassay, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, biology.protein, IgE, drug allergy, medicine.drug, Chromatography, Liquid
Abstract

12 p.-6 fig.-1 graf. abst., BACKGROUND: Selective reactions to clavulanic acid (CLV) account for around 30% of immediate reactions after administration of amoxicillin-CLV. Currently, no immunoassay is available for detecting specific IgE to CLV, and its specific recognition in patients with immediate reactions has only been demonstrated by basophil activation testing, however with suboptimal sensitivity. The lack of knowledge regarding the structure of the drug that remains bound to proteins (antigenic determinant) is hampering the development of in vitro diagnostics. We aimed to identify the antigenic determinants of CLV as well as to evaluate their specific IgE recognition and potential role for diagnosis., METHODS: Based on complex CLV degradation mechanisms, we hypothesized the formation of two antigenic determinants for CLV, AD-I (N-protein, 3-oxopropanamide) and AD-II (N-protein, 3-aminopropanamide), and designed different synthetic analogs to each one. IgE recognition of these structures was evaluated in basophils from patients with selective reactions to CLV and tolerant subjects. In parallel, the CLV fragments bound to proteins were identified by proteomic approaches., RESULTS: Two synthetic analogs of AD-I were found to activate basophils from allergic patients. This determinant was also detected bound to lysines 195 and 475 of CLV-treated human serum albumin. One of these analogs was able to activate basophils in 59% of patients whereas CLV only in 41%. Combining both results led to an increase in basophil activation in 69% of patients, and only in 12% of controls., CONCLUSION: We have identified AD-I as one CLV antigenic determinant, which is the drug fragment that remains protein-bound., The present study has been supported by Institute of Health “Carlos III” of the Ministry of Economy and Competitiveness (grants cofunded by European Regional Development Fund (ERDF): PI12/02529, PI15/01206, CP15/00103, RETICs RIRAAF RD12/0013/0001, RD12/0013/0003 and RD12/0013/0008, RETIC ARADYAL RD16/0006/0001, RD16/0006/0012 and RD16/0006/0021, Biobank Network RD09/0076/00112, and Biobank platform PT13/0010/0006) and by State Secretariat for Research, Development and Innovation of the Ministry of Economy and Competitiveness (grants cofunded by European Regional Development Fund (ERDF), “Una manera de hacer Europa”: MINECO SAF2012-36519, SAF2015-68590R, CTQ2013-41339P, and CTQ2016-75870P). Andalusian Regional Ministry of Economy and Knowledge (grants cofunded by European Regional Development Fund (ERDF), “Andalucía se mueve con Europa”: CTS-06603); Andalusian Regional Ministry Health (grants: PI-0699-2011, PI-0159-2013, PI-0179-2014, PI-0241-2016, and PI-0250-2016), Merck-Serono Research Grant from Fundación Salud 2000 and “Premio UNICAJA a la innovación en biomedicina y salud.” CM holds a “Nicolas Monardes” research contract by Andalusian Regional Ministry Health: C-0044-2012 SAS 2013. MIM holds a “Miguel Servet I” and GB holds a “Rio Hortega,” both research contracts by Institute of Health “Carlos III” of the Ministry of Economy and Competitiveness (grants cofunded by European Social Fund (ESF)): CP15/00103 and CM16/00067. TDF hold a “Ramon y Cajal” research contract from Ministry of Economy and Competitiveness (RYC-2013-13138).

Published
2018

20. Basophil Histamine Release Induced by Amoxicilloyl-poly-L-lysine Compared With Amoxicillin in Patients With IgE-Mediated Allergic Reactions to Amoxicillin.

Author

Arribas F, Falkencrone S, Sola J, Gomez-Serranillos MP, Laguna JJ, Montañez MI, Fernandez TD, Rodríguez D, Pineda F, Skov PS, Mayorga C, and Torres MJ

Subjects
Adult, Aged, Amoxicillin chemistry, Anaphylaxis diagnosis, Anaphylaxis immunology, Anaphylaxis metabolism, Antibody Specificity immunology, Biomarkers, Drug Hypersensitivity diagnosis, Female, Humans, Male, Middle Aged, Polylysine chemistry, ROC Curve, Skin Tests, Young Adult, Amoxicillin adverse effects, Basophils immunology, Basophils metabolism, Drug Hypersensitivity immunology, Drug Hypersensitivity metabolism, Histamine Release immunology, Immunoglobulin E immunology
Abstract

Background: Amoxicillin (AX) is the ß-lactam most often involved in IgE-mediated reactions. Diagnosis is based mainly on skin testing, although sensitivity is not optimal. We produced a new AX derivative, amoxicilloyl-poly-L-lysine (APL), and analyzed its recognition of IgE using the passive histamine release test (pHRT)., Methods: The study population comprised patients (n=19) with confirmed AX allergy and specific IgE to AX and controls (n=10) with good tolerance to AX. pHRT was performed using "IgE-stripped" blood from a single donor that was sensitized in vitro by patient sera and incubated with AX or APL. Histamine release was determined and expressed as nanograms of histamine released per milliliter of blood., Results: The clinical symptoms were anaphylaxis (n=9), urticaria (n=7), erythema (n=2), and nondefined immediate reactions (n=1). The median (IQR) time interval between reaction and study was 90 (60-240) days and between drug intake and development of symptoms 24 (10-60) minutes. The median sIgE level was 3.37 (0.95-5.89) kUA/L. The sensitivity of pHRT to APL was 79% and the specificity 100%, which were higher than data obtained with pHRT to AX (63% sensitivity and 90% specificity). There was a positive correlation between maximal histamine release levels obtained with AX and APL (r=0.63)., Conclusions: In patients with immediate hypersensitivity reactions to AX, APL showed higher sensitivity and specificity than the culprit drug, AX, when tested in vitro by pHRT. This indicates that APL can improve the in vitro diagnostic accuracy of allergic reactions to AX. Further assessment of skin testing is necessary.

Published
2017
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21. In vitro tests for drug hypersensitivity reactions: an ENDA/EAACI Drug Allergy Interest Group position paper.

Author

Mayorga C, Celik G, Rouzaire P, Whitaker P, Bonadonna P, Rodrigues-Cernadas J, Vultaggio A, Brockow K, Caubet JC, Makowska J, Nakonechna A, Romano A, Montañez MI, Laguna JJ, Zanoni G, Gueant JL, Oude Elberink H, Fernandez J, Viel S, Demoly P, and Torres MJ

Subjects
Biomarkers, Drug Hypersensitivity blood, Drug Hypersensitivity etiology, Drug Hypersensitivity genetics, HLA Antigens genetics, HLA Antigens immunology, Humans, Immunity, Immunoglobulin E blood, Immunoglobulin E immunology, In Vitro Techniques, Practice Guidelines as Topic, T-Lymphocytes immunology, T-Lymphocytes metabolism, Drug Hypersensitivity diagnosis, Skin Tests methods
Abstract

Drug hypersensitivity reactions (DHRs) are a matter of great concern, both for outpatient and in hospital care. The evaluation of these patients is complex, because in vivo tests have a suboptimal sensitivity and can be time-consuming, expensive and potentially risky, especially drug provocation tests. There are several currently available in vitro methods that can be classified into two main groups: those that help to characterize the active phase of the reaction and those that help to identify the culprit drug. The utility of these in vitro methods depends on the mechanisms involved, meaning that they cannot be used for the evaluation of all types of DHRs. Moreover, their effectiveness has not been defined by a consensus agreement between experts in the field. Thus, the European Network on Drug Allergy and Drug Allergy Interest Group of the European Academy of Allergy and Clinical Immunology has organized a task force to provide data and recommendations regarding the available in vitro methods for DHR diagnosis. We have found that although there are many in vitro tests, few of them can be given a recommendation of grade B or above mainly because there is a lack of well-controlled studies, most information comes from small studies with few subjects and results are not always confirmed in later studies. Therefore, it is necessary to validate the currently available in vitro tests in a large series of well-characterized patients with DHR and to develop new tests for diagnosis., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Published
2016
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22. The Addition of Benzylpenicillin Does Not Increase the Skin Test Sensitivity Obtained With Classic β-Lactam Determinants.

Author

Lacombe-Barrios J, Salas M, Gómez F, Doña I, Ariza A, Mayorga C, Blanca M, Montañez MI, and Torres MJ

Subjects
Adolescent, Adult, Aged, Algorithms, Amoxicillin-Potassium Clavulanate Combination administration & dosage, Amoxicillin-Potassium Clavulanate Combination adverse effects, Amoxicillin-Potassium Clavulanate Combination immunology, Anti-Bacterial Agents administration & dosage, Anti-Bacterial Agents immunology, Critical Pathways, Cross Reactions, Drug Hypersensitivity immunology, Humans, Intradermal Tests, Middle Aged, Penicillin G immunology, Predictive Value of Tests, Prospective Studies, Single-Blind Method, Young Adult, beta-Lactams administration & dosage, beta-Lactams immunology, Anti-Bacterial Agents adverse effects, Drug Hypersensitivity diagnosis, Penicillin G administration & dosage, Skin Tests, beta-Lactams adverse effects
Published
2016

23. IgE to penicillins with different specificities can be identified by a multiepitope macromolecule: Bihaptenic penicillin structures and IgE specificities.

Author

Ariza A, Barrionuevo E, Mayorga C, Montañez MI, Perez-Inestrosa E, Ruiz-Sánchez A, Rodríguez-Guéant RM, Fernández TD, Guéant JL, Torres MJ, and Blanca M

Subjects
Adolescent, Adult, Aged, Amoxicillin adverse effects, Antibody Affinity immunology, Antibody Specificity immunology, Drug Hypersensitivity immunology, Epitopes immunology, Female, Haptens immunology, Humans, Hypersensitivity, Immediate immunology, Immunoassay methods, Male, Middle Aged, Penicillin G adverse effects, Penicillins adverse effects, Penicillins immunology, Radioallergosorbent Test methods, Skin Tests, Young Adult, beta-Lactams adverse effects, beta-Lactams immunology, Amoxicillin immunology, Drug Hypersensitivity diagnosis, Hypersensitivity, Immediate diagnosis, Immunoglobulin E blood, Penicillin G immunology
Abstract

Quantitation of specific IgE by immunoassay is a recommended in vitro test for the diagnosis of immediate hypersensitivity reactions to betalactams (BLs), particularly when skin test results are negative. IgE antibodies that recognize the common nuclear structure of all BLs or the specific side chain structure can be mainly distinguished by immunoassays. The aim of this study was to develop an immunoassay system to detect IgE antibodies with different specificities. Cellulose discs conjugated with benzylpenicillin (BP), amoxicillin (AX) or both drugs, with poly-l-lysine (PLL) as carrier molecule, were used as solid phases in the radioallergosorbent test (RAST). Direct and inhibition radioimmunoassay studies were made to verify the structures recognized by serum IgE antibodies from penicillin-allergic patients. Our results indicated that the addition of both haptens did not decrease the capacity to capture IgE when serum specific to either BP or AX was used, at least in terms of sensitivity. In addition, the inclusion of two haptens improved significantly the levels of IgE detection in patients who recognized both BP and AX. Therefore, the use of a solid phase with a carrier molecule conjugated with two determinants (AX and BP) is helpful to recognize IgE antibodies against either of these determinants and is useful for screening sera with different specificities., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Published
2014
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24. Dendrimer-modified solid supports: nanostructured materials with potential drug allergy diagnostic applications.

Author

Ruiz-Sanchez AJ, Montañez MI, Mayorga C, Torres MJ, Kehr NS, Vida Y, Collado D, Najera F, De Cola L, and Perez-Inestrosa E

Subjects
Antibody Specificity, Cellulose chemistry, Humans, Hypersensitivity, Immediate chemically induced, Immunoglobulin E immunology, Penicillins adverse effects, Penicillins immunology, Surface Properties, Dendrimers chemistry, Drug Hypersensitivity diagnosis, Hypersensitivity, Immediate diagnosis, Nanostructures chemistry, Radioallergosorbent Test instrumentation, Radioallergosorbent Test methods, Zeolites chemistry
Abstract

Complex functional materials consisting of bioactive molecules immobilized on solid supports present potential applications in biosensoring. Advances in the fabrication of these surface materials are of growing interest for antibody-based diagnosis. This work exploits dendrimers as versatile nanostructures for templating sensor surfaces and the critical role of the immobilization protocol in the solid supports cellulose and zeolites, of organic and inorganic composition respectively. The fabrication and characterization, including the degree of functionalization and reproducibility, of different nanostructured materials are described. To validate the approach, the fabricated supports were further used as a solid phase for developing a radioimmunoassay to detect immunoglobulin E (IgE) specific to penicillin, the antibody involved in immediate allergy responses to this drug. The dendrimer-modified supports provide assays with significantly enhanced sensitivity, as well as increase the availability of biomolecules for specific interaction and minimize nonspecific adsorptions through appropriate functionalization protocols in each case. The manufacturing methodology involved the use of a long, flexible hydrophilic spacer in the cellulose materials, and a higher surface density of the immobilized dendrimers in the zeolite crystals. The ability of hybrid zeolite materials in such biosensing applications was evaluated for the first time. The assays were validated in human serum samples from patients allergic to penicillin and from non-allergic controls. The specificity and improved sensitivity of the dendrimer- modified supports make these strategies versatile for different bioactive molecules and could have significant implications for the quantification of a wide range of specific IgE antibodies and other biomolecules of diagnostic interest.

Published
2012
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