Person: García García, Aina
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First Name
Aina
Last Name
García García
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Veterinaria
Department
Nutrición y Ciencia de los Alimentos
Area
Identifiers
20 results
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Item A novel approach to produce phage single domain antibody fragments for the detection of gluten in foods(Food Chemistry, 2020) García García, Aina; García Lacarra, Teresa; Martín De Santos, María Del Rosario; González Alonso, María Isabel; Madrid García, RaquelIn this study, we demonstrated the feasibility of isolating recombinant phage-antibodies against gluten from a non-immunized library of human single-domain antibodies (dAbs). Phage display technology enabled the selection of affinity probes by successive rounds of biopanning against a biotinylated synthetic peptide comprising repetitive immunogenic gluten motifs. The analysis of a wide representation of heterologous plant species corroborated that two of the isolated clones were specific to wheat, barley and rye proteins. The phage antibody selected as the most appropriate clone for the detection of gluten in foods (dAb8E-phage) was further applied in an indirect ELISA to the analysis of 50 commercial food samples. Although the limit of detection achieved did not improve those of current immunoassays, the proposed methodology could provide promising new pathways for the generation of recombinant antibodies that allow a comprehensive determination of gluten in foods, whilst replacing the need for animal immunization.Item Utilización de técnicas genéticas y anticuerpos recombinantes para la detección de alérgenos alimentarios de origen vegetal(2020) García García, Aina; González Alonso, Isabel; García Lacarra, Teresa; Martín de Santos, RosarioEl correcto etiquetado de los alimentos es un elemento fundamental para garantizar la seguridad alimentaria y prevenir los fraudes comerciales. En este marco, el Reglamento (CE) 178/2002 reconoce a los consumidores el derecho a estar debidamente informados respecto a los alimentos que adquieren para que puedan elegir con conocimiento de causa los productos adecuados a sus necesidades. Este hecho se hace especialmente relevante en el caso de personas que sufren algún tipo de alergia alimentaria dado que, en estos individuos, incluso pequeñas cantidades del alérgeno pueden desencadenar una reacción adversa severa. Para proteger a los consumidores alérgicos, el Reglamento (UE) 1169/2011 establece un total de 14 grupos de ingredientes que obligatoriamente deben declararse en el etiquetado de los productos que los contengan por ser los causantes de una elevada parte de las reacciones alérgicas humanas. Para dar cumplimiento a estas normativas, las industrias y agencias encargadas de la seguridad alimentaria deben disponer de técnicas analíticas sensibles y específicas para identificar aquellos alérgenos que puedan estar presentes, tanto de forma voluntaria como accidental, en los productos alimenticios...Item Multiplex ligation-dependent probe amplification (MLPA) for simultaneous detection of DNA from sunflower, poppy, flaxseed, sesame and soy allergenic ingredients in commercial food products(Food control, 2017) López Calleja, Inés; García García, Aina; Madrid García, Raquel; García Lacarra, Teresa; Martín De Santos, María Del Rosario; González Alonso, María IsabelThis work describes a multiplex ligation-dependent probe amplification (MLPA) technique for the simultaneous detection of five food allergens: sunflower, poppy, flaxseed, sesame and soy. Species-specific MLPA half-probes were designed to detect the DNA from the targeted species. Ligated probes were amplified by polymerase chain reaction (PCR) and amplicons were detected using capillary electrophoresis. The specificity of the MLPA system was assessed against DNA from more than 50 plant and animal species. The limit of detection (LOD) of the assay was determined to be 10 mg/kg in a model cookie experimentally spiked with different concentrations of the target species. The applicability of the MLPA was demonstrated through the analysis of 56 different commercial products (breads, pastries, cereals, chocolates, drinks, etc.), evidencing the presence of one or more undeclared allergenic ingredients in some of the tested samples. Real-time PCR assays were also performed to confirm and complement MLPA results. The MLPA technique has proved as a qualified screening tool for determining the presence of low amounts of sunflower, poppy, flaxseed, sesame and soy in processed foods.Item A sensitive and specific real-time PCR targeting DNA from wheat, barley and rye to track gluten contamination in marketed foods(LWT- Food Science and Technology, 2019) Sohrabi, v; Cruz, Silvia de la; García García, Aina; Madrid García, Raquel; García Lacarra, Teresa; Martín De Santos, María Del Rosario; González Alonso, María IsabelThis work reports a real-time PCR assay to specifically detect the presence of DNA from the main gluten-containing cereals wheat, barley and rye (WBR) in foodstuffs. The WBR real-time PCR uses two specific primers and a Taqman probe for amplification of a 118 bp DNA fragment common to the three target cereals on the ribosomal internal transcribed spacer (ITS) region. In-house validation of the method was performed employing three binary arrays containing different concentrations (100 000–10 mg/kg) of a wheat/barley/rye mixture in maize flour: untreated, soft heat-treated at 160 °C/13 min and intense heat-treated at 200 °C/20 min. Results showed optimal PCR amplification efficiency, reproducibility and sensitivity with an overall limit of detection of 10–50 mg/kg of the target cereals, theoretically equating to approximately 1–5 mg/kg of gluten. Applicability of the assay was further assessed through a market analysis of 220 food products by the real-time PCR assay and the official R5-based ELISA. Comparative results highlighted the ability of the proposed methodology as a highly sensitive and robust procedure to detect the presence of potentially harmful concentrations of undeclared gluten-containing cereals in some of the tested samples, supporting results of the immunological assays currently used for gluten determination in foods.Item Survey of Commercial Food Products for Detection of Walnut (Juglans regia) by Two ELISA Methods and Real Time PCR(Foods, 2021) Madrid García, Raquel; García García, Aina; Cabrera, Pablo; González, Isabel; Martín, Rosario; García, TeresaLabeling of food allergens in accordance with legal regulations is important to protect the health of allergic consumers. The requirements for detecting allergens in foods involve adequate specificity and sensitivity to identify very small amounts of the target allergens in complex food matrices and processed foods. In this work, one hundred commercial samples were analyzed for walnut detection using three different methods: a sandwich enzyme-linked immunosorbent assay (ELISA) kit based on polyclonal antibodies, a direct ELISA using a recombinant multimeric scFv, and a real time PCR. The most sensitive method was real time PCR followed by sandwich ELISA kit and multimeric scFv ELISA. There was agreement between the three methods for walnut detection in commercial products, except for some heat-treated samples or those that contained pecan. The walnut ELISA kit was less affected by sample processing than was the multimeric scFv ELISA, but there was cross-reactivity with pecan, producing some false positives that must be confirmed by real time PCR. According to the results obtained, 7.0 to 12.6% of samples (depending on the analytical method) contained walnut but did not declare it, confirming there is a risk for allergic consumers. Moreover, there was one sample (3.7%) labelled as containing walnut but that tested negative for this tree nut. Genetic and immunoenzymatic techniques offer complementary approaches to develop a reliable verification for walnut allergen labeling.Item Identification and characterisation of the proteins bound by specific phage‐displayed recombinant antibodies (scFv) obtained against Brazil nut and almond extracts(Journal of the Science of Food and Agriculture, 2017) Cruz. Silvia de la; Madrid García, Raquel; García García, Aina; Alcocer, Marcos; Martín De Santos, María Del Rosario; González Alonso, María Isabel; García Lacarra, TeresaBACKGROUND: Almonds and Brazil nuts are widely consumed allergenic nuts whose presence must be declared according to food labelling regulations. Their detection in food products has been recently achieved by ELISA methods with recombinant antibodies (scFv) isolated against complete Brazil nut and almond protein extracts. The screening of phage-scFv libraries against complete protein extracts confers a series of advantages over the use of purified proteins, as recombinant proteins might alter their native folding. However, using this strategy, the nature of the target detected by phage-displayed antibodies remains unknown, and requires further research to identify whether they are nut allergens or other molecules present in the extract, but not related to their allergenic potential. RESULTS: Electrophoretic, chromatographic, immunological and spectrometric techniques revealed that the Brazil nut (BE95) and almond (PD1F6 and PD2C9) specific phage-scFvs detected conformational epitopes of the Brazil nut and almond 11S globulins, recognised by WHO/IUIS as Ber e 2 and Pru du 6 major allergens. Circular dichroism data indicated that severe heat treatment would entail loss of epitope structure, disabling scFv for target detection. CONCLUSIONS: The presence of important Brazil nut and almond allergens (Ber e 2 and Pru du 6) in foodstuffs can be determined by using phage-display antibodies BE95, PD1F6 and PD2C9 as affinity probes in ELISA.Item Development of a new recombinant antibody, selected by phage-display technology from a celiac patient library, for detection of gluten in foods(Current Research in Food Science, 2023) García Calvo, Eduardo Rafael; García García, Aina; Rodríguez Gómez, Santiago; Farrais, Sergio; Martín De Santos, María Del Rosario; García Lacarra, TeresaGluten, a group of ethanol-soluble proteins present in the endosperm of cereals, is extensively used in the food industry due to its ability to improve dough properties. However, gluten is also associated with a range of gluten-related diseases (GRDs), such as wheat allergies, celiac disease, and gluten intolerance. The recommended treatment for GRDs patients is a gluten-free diet. To monitor adherence to this diet, it is necessary to develop gluten-detection systems in food products. Among the available methods, immunodetection systems are the most popular due to their simplicity, reproducibility, and accuracy. The aim of this study was to generate novel high-affinity antibodies against gluten to be used as the primary reactant in an enzyme-linked immunosorbent assay (ELISA) test. These antibodies were developed by constructing an immune library from mRNA obtained from two celiac patients with a high humoral response to gluten-related proteins. The resulting library (composed by 1.1x107) was subjected to selection against gliadin using phage display technology. Following several rounds of selection, the Fab-C was selected, and demonstrated good functionality in ELISA tests, presenting a limit of detection of 15 mg/kg for detection of gluten in spiked mixtures and food products. The methodology can discriminate gluten-free products according to the current legislation.- Project number: 262
Item Diseño y desarrollo de una herramienta audiovisual para la docencia virtual de la inspección veterinaria oficial de pescados y productos de la pesca en un mercado central(2022) Muñoz Atienza, Estefanía; Yagüe Sánchez, Ángel; Álvarez López, Alberto; Borrero Del Pino, Juan; García García, Aina; García Lacarra, Teresa; Hernández Cruza, Pablo Elpidio; Marín Martínez, María; Martín De Santos, María Del Rosario; Dias Araújo, Carlos; Díaz Formoso, Lara; Cintas Izarra, Luis Miguel; da Silva Serra Contente de Matos, Diogo; Feito Hermida, Javier; García Calvo, Eduardo Rafael; Gómez Sala, Beatriz; Lafuente Orte, Irene; Peña Vidal, Nuria; Rodríguez Gómez, Santiago; Sevillano González, Ester; Beltrán Crespo, Antonio; Caba Manzaneque, Elia; Cabrales Miró-Granada, Ana; Cano Muñoz, Marisa; Cañizares Cooz, Daniela; Celorrio de Ochoa, David; Dorado Nuñez, Gemma; Fernández Silva, Ana; Gutiérrez de Cabiedes de Fuente, Alejandro; Márquez Bayón, Teresa; Martín Martí, Laura; Olmeda García, Patricia; Roncero Fernández, Alejandro Francisco; Sánchez Prada, Raquel; Taberneiro Auiget, DanielEl objetivo global de este Proyecto de Innovación Docente es la creación de vídeos explicativos como una herramienta de aprendizaje incorporada en el Campus Virtual para mejorar el estudio sobre las actividades de higiene, inspección y control alimentario que se realizan en el Mercado Central de Pescados de Mercamadrid. La creación y el empleo de estos vídeos están dirigidos, en un principio, a los estudiantes universitarios de Grado en Veterinaria que cursan la asignatura de Higiene, Inspección y Seguridad Alimentaria. En este Proyecto se han creado vídeos explicativos que tratan sobre: (i) los controles oficiales realizados por los Técnicos Superiores Veterinarios de Mercamadrid; (ii) los riesgos sanitarios asociados al consumo de pescados, crustáceos y moluscos; (iii) la frescura del pescado; (iv) el etiquetado del pescado; (v) la identificación de especies de pescado y marisco; (vi) la prevención de fraudes en la comercialización de pescados y mariscos; y (vii) la autentificación de pescados fileteados mediante técnicas de análisis. Item Exploring Gluten Assessment in Marketed Products through a Sandwich ELISA Methodology Based on Novel Recombinant Antibodies(Foods, 2024) García Calvo, Eduardo Rafael; García García, Aina; Rodríguez Gómez, Santiago; Martín De Santos, María Del Rosario; García Lacarra, TeresaThis study presents the development of a sandwich ELISA method for gluten detection in foods, using recombinant Fab antibody fragments against gliadin. The Fabs were chemically biotinylated and immobilized on streptavidin-coated plates as capture antibodies, while alkaline phosphatase-conjugated Fabs were used as detection antibodies. Four different gliadin-binding Fabs were tested and the Fab pair Fab8E-4 and Fab-C showed the best compatibility. An indirect sandwich immunoassay, using unmodified Fab8E-4 for capture and Fab-C as the detection antibody, achieved a detection limit of 26 ng/mL of gliadin, corresponding to 10 mg/kg of gluten in foods. No cross-reactivity was observed against 60 gluten-free species commonly used in the food industry. Analysis of 50 commercial products demonstrated consistent results compared to the standard method for gluten detection. The complete lack of cross-reactivity of the developed immunoassay with oat products potentially provides an advantage over other gluten detection systems.Item Unraveling the Properties of Phage Display Fab Libraries and Their Use in the Selection of Gliadin-Specific Probes by Applying High-Throughput Nanopore Sequencing(Viruses, 2024) García Calvo, Eduardo Rafael; García García, Aina; Rodríguez Gómez, Santiago; Martín De Santos, María Del Rosario; García Lacarra, TeresaDirected evolution is a pivotal strategy for new antibody discovery, which allowed the generation of high-affinity Fabs against gliadin from two antibody libraries in our previous studies. One of the libraries was exclusively derived from celiac patients’ mRNA (immune library) while the other was obtained through a protein engineering approach (semi-immune library). Recent advances in high-throughput DNA sequencing techniques are revolutionizing research across genomics, epigenomics, and transcriptomics. In the present work, an Oxford Nanopore in-lab sequencing device was used to comprehensively characterize the composition of the constructed libraries, both at the beginning and throughout the phage-mediated selection processes against gliadin. A customized analysis pipeline was used to select high-quality reads, annotate chain distribution, perform sequence analysis, and conduct statistical comparisons between the different selection rounds. Some immunological attributes of the most representative phage variants after the selection process were also determined. Sequencing results revealed the successful transfer of the celiac immune response features to the immune library and the antibodies derived from it, suggesting the crucial role of these features in guiding the selection of high-affinity recombinant Fabs against gliadin. In summary, high-throughput DNA sequencing has improved our understanding of the selection processes aimed at generating molecular binders against gliadin.