Person:
García García, Aina

First Name

Aina

Last Name

García García

URI

https://hdl.handle.net/20.500.14352/80389

Affiliation

Universidad Complutense de Madrid

Faculty / Institute

Veterinaria

Department

Nutrición y Ciencia de los Alimentos

Identifiers

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Search Results

Now showing 1 - 8 of 8

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, Raquel
In 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.
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 Isabel
This 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.
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 Isabel
This 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.
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, Teresa
Labeling 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.
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, Teresa
BACKGROUND: 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.
Multimeric recombinant antibody (scFv) for ELISA detection of allergenic walnut. An alternative to animal antibodies
(Journal of Food Composition and Analysis, 2018) Madrid García, Raquel; de la Cruz, Silvia; García García, Aina; Alcocer, Marcos J.C.; González Alonso, María Isabel; García Lacarra, Teresa; Martín De Santos, María Del Rosario
Walnuts are classified as an important allergenic ingredient that can cause severe reactions in sensitized individuals. To prevent unintended exposure to products containing walnut, food manufacturers have the responsibility to declare its presence in packaged foods. Immunochemical methods are widely used to detect walnut proteins. However, available immunoassays rely on the use of antibodies raised in animals. In this work, an affinity probe for walnut proteins has been isolated from the Tomlinson I library, and further engineered in Pichia pastoris to produce the in vivo Juglans regia Biotinylated Soluble Fragment-single chain and multimeric antibody (JrBSF-scFv). The multimeric scFv has been used to develop a direct enzyme-linked immunosorbent assay (ELISA), allowing detection of walnut in a food matrix with a limit of detection (LOD) of 1616 mg kg−1. This is the first recombinant antibody available for detection of walnut proteins. The assay is specific, only cross-reacting to some extent (2.25%) to pecan, thus being useful as a screening tool for detection of walnut in raw or baked food matrices. Multimerization of the scFv with different avidin derivates could be of interest to improve sensitivity of the assay.
Production of in vivo biotinylated scFv specific to almond (Prunus dulcis) proteins by recombinant Pichia pastoris
(Journal of Biotecnology, 2016) Cruz, Silvia de la; Alcocer, Marcos; Martín De Santos, María Del Rosario; González Alonso, María Isabel; García Lacarra, Teresa; García García, Aina; Madrid García, Raquel
The methylotropic yeast Pichia pastoris has demonstrated its suitability for large-scale production of recombinant proteins. As an eukaryotic organism P. pastoris presents a series of advantages at expression and processing of heterologous proteins when compared with Escherichia coli. In this work, P. pastoris has been used to express a scFv from a human synthetic library previously shown to bind almond proteins. In order to facilitate purification and post processing manipulations, the scFv was engineered with a C-terminal tag and biotinylated in vivo. After purification, biotinylated scFv were bound to avidin conjugated with HRP producing a multimeric scFv. The multimeric scFv showed to maintain their ability to recognize almond protein when assayed in ELISA, reaching a LOD of 470mgkg(-1). This study describes an easy method to produce large quantities of in vivo biotinylated scFv in P. pastoris. By substituting the enzyme or fluorochromes linked to avidin, it will be possible to generate a diverse number of multimeric scFv as probes to suit different analytical platforms in the detection of almond in food products.
Use of multiplex ligation-dependent probe amplification (MLPA) for screening of wheat, barley, rye and oats in foods
(Food control, 2018) García García, Aina; Madrid García, Raquel; García Lacarra, Teresa; Martín De Santos, María Del Rosario; González Alonso, María Isabel
A multiplex ligation-dependent probe amplification (MLPA) approach is described for the simultaneous detection of DNA from four common cereal ingredients in foods: wheat, barley, rye and oats. The method uses species-specific MLPA half-probes targeting DNA fragments from the ribosomal internal space transcriber (ITS) region for the detection of barley and oats, and from the genes encoding the low molecular weight (LMW) glutenin and the ω-secalin for wheat and rye, respectively. After hybridization, the probes are ligated and amplified by polymerase chain reaction (PCR) into specific and size-differential amplicons that are simultaneously detected by capillary electrophoresis. The cereal MLPA technique showed an optimal specificity against a representative number of plant and animal species. A sensitive detection of the targets (LOD of 50 mg kg−1) was achieved in a reference model cake experimentally spiked with different levels of each wheat, barley, rye and oats seeds. MLPA applicability was assessed through the analysis of 40 commercial food products with different labeling declarations regarding the targets (contain, may contain and do not declare/gluten free), indicating the presence of non-declared cereal ingredients in some of the tested samples. MLPA results were further confirmed by simplex Taqman real-time PCR assays. The described MLPA technique is a reliable and sensitive tool for screening the presence of low amounts of wheat, barley, rye and oats in processed foodstuffs, contributing to compliance with authenticity legal requirements and protecting consumers from fraudulent commercial practices or adventitious contamination which may lead to allergic reactions associated to cereal proteins ingestion.