Anal Bioanal Chem

A VHH antibody (or nanobody) is the antigen binding fragment of heavy chain only antibodies. Discovered nearly 25\uc2\ua0years ago, they have been investigated for their use in clinical therapeutics and immunodiagnostics, and more recently for environmental monitoring applications. A new and valuable immunoreagent for the analysis of small molecular weight environmental chemicals, VHH will overcome many pitfalls encountered with conventional reagents. In the work so far, VHH antibodies often perform comparably to conventional antibodies for small molecule analysis, are amenable to numerous genetic engineering techniques, and show ease of adaption to other immunodiagnostic platforms for use in environmental monitoring. Recent reviews cover the structure and production of VHH antibodies as well as their use in clinical settings. However, no report focuses on the use of these VHH antibodies to detect small environmental chemicals (MW < 1500\uc2\ua0Da). This review article summarizes the efforts made to produce VHHs to various environmental targets, compares the VHH-based assays with conventional antibody assays, and discusses the advantages and limitations in developing these new antibody reagents particularly to small molecule targets. Graphical Abstract Overview of the production of VHHs to small environmental chemicals and highlights of the utility of these new emerging reagents.P30 ES023513/ES/NIEHS NIH HHS/United StatesP42 ES004699/ES/NIEHS NIH HHS/United StatesU54 NS079202/NS/NINDS NIH HHS/United StatesU54 OH007550/OH/NIOSH CDC HHS/United States2017-09-01T00:00:00Z27209591PMC498323

Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection

Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21–99.98) and specificity (100%, 95% CI 96.31–100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.Instituto de BiotecnologíaFil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Llauger, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Monti, Demian Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Monti, Demian Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Aduriz Guerrero, Matí­as. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Aduriz Guerrero, Matí­as. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romão, Ema. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Dumon, Analia Delina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Dumon, Analia Delina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mattio, Maria Fernanda. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Mattio, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wigdorovitz, Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Wigdorovitz, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Muyldermans, Serge. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Muyldermans, Serge. Dalian University of Technology. School of Bioengineering. Liaoning Key Laboratory of Molecular Recognition and Imaging; ChinaFil: Vincke, Cécile. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Vincke, Cécile. VIB Center for Inflammation Research. Myeloid Cell Immunology Lab; BélgicaFil: Parreño, Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Parreño, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Del Vas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Phage Display in the Quest for New Selective Recognition Elements for Biosensors

Phages are bacterial viruses that have gained a significant role in biotechnology owing to their widely studied biology and many advantageous characteristics. Perhaps the best-known application of phages is phage display that refers to the expression of foreign peptides or proteins outside the phage virion as a fusion with one of the phage coat proteins. In 2018, one half of the Nobel prize in chemistry was awarded jointly to George P. Smith and Sir Gregory P. Winter "for the phage display of peptides and antibodies." The outstanding technology has evolved and developed considerably since its first description in 1985, and today phage display is commonly used in a wide variety of disciplines, including drug discovery, enzyme optimization, biomolecular interaction studies, as well as biosensor development. A cornerstone of all biosensors, regardless of the sensor platform or transduction scheme used, is a sensitive and selective bioreceptor, or a recognition element, that can provide specific binding to the target analyte. Many environmentally or pharmacologically interesting target analytes might not have naturally appropriate binding partners for biosensor development, but phage display can facilitate the production of novel receptors beyond known biomolecular interactions, or against toxic or nonimmunogenic targets, making the technology a valuable tool in the quest of new recognition elements for biosensor development.This study was supported by the Ministry of Economy and Competitiveness (Ministerio de Ciencia, Innovación y Universidades RTI2018-096410-B-C21). R.P. acknowledges UCM for a predoctoral grant and R.B. the PI17CIII/00045 grant from the AES-ISCIII program.S

Application of Novel Methods for Mycotoxins Analysis

Crop contamination by mycotoxins is a global problem that poses significant economic burdens due to a number of factors, including the food/feed losses that are caused by reduced production rates; the resulting adverse effects on human and animal health and productivity; and the trade losses associated with the costs incurred by inspection, sampling, and analysis before and after shipments. In this scenario, the development of fit-for-purpose analytical methods for regulated and (re)-emerging mycotoxins continues to be a dynamic research area. Some of the current trends in this research area are presented in this book. The collected contributions address either the need for improved methods for mycotoxin detection addressed by new or incoming regulation (ergot alkaloids and Alternaria toxins) as well as methods for the detection of multiple mycotoxins. New approaches to enhance the performance of well-established methodologies, such as the enzyme-linked immunosorbent assay (ELISA) and fluorescence polarization immunoassays (FPIA), are also addressed

Nuevas estrategias para el desarrollo de biosensores ópticos aplicados al análisis de micotoxinas y hongos toxigénicos en alimentos

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Química Analítica, leída el 15-07-2019Mycotoxins are a diverse group of low molecular weight compounds produced as secondary metabolites by numerous species of filamentous fungi. This assemblage is chemically and toxigenically rather heterogeneous, but generally these toxins are known to cause disease and death in human and other vertebrates even at low concentrations. Mycotoxigenic fungi grow on a wide range of conditions and they can produce mycotoxins into the matrices on which they grow, often food intended for human consumption or animal feed. As a result of the ubiquitous nature of mycotoxigenic fungi, particularly in temperate and tropical regions of the world, mycotoxin contamination is often inevitable, and some calculations have estimated that approximately 25–50% of world crops are contaminated with these toxins. Although the awareness related to the hazards of mycotoxins as food and feed contaminants is growing, there are no absolute measures available for eliminating mycotoxins from agricultural products. While mycotoxin occurrence in the field can be decreased by good agronomic practices and planting resistant varieties, in the end, analytical methods capable of detecting mycotoxins and toxigenic fungi even at low concentration are of key importance for ensuring food safety...Las micotoxinas son metabolitos secundarios tóxicos producidos por algunas cepas de hongos que contaminan alimentos, especialmente cereales y hortalizas. Estos compuestos de bajo peso molecular son químicamente y toxigénicamente heterogéneos; sin embargo, muchas de estas toxinas pueden originar enfermedades, y en ocasiones la muerte, tanto en humanos como en otros vertebrados. Los hongos toxigénicos crecen en muchas condiciones muy diversas, lo que puede dar lugar a la aparición de micotoxinas en los alimentos destinados tanto al consumo humano como al animal. Los hongos toxigénicos están ampliamente distribuidos por todo el mundo, particularmente en las regiones templadas y tropicales, por lo que la contaminación natural por micotoxinas es casi inevitable. De hecho, se estima que aproximadamente el 25–50% de los cultivos mundiales están contaminados por estas toxinas, y la preocupación sobre los peligros asociados a su presencia en alimentos es cada día mayor. Actualmente, no existen alternativas viables para su eliminación en los productos agrícolas; aunque el empleo de buenas prácticas agrícolas o la plantación de variedades resistentes a los hongos, pueden ayudar a mejorar este problema. En cualquier caso, se requieren métodos analíticos sensibles y selectivos para la detección de micotoxinas y hongos toxigénicos, a bajas concentraciones, afin de garantizar la seguridad alimentaria...Depto. de Química AnalíticaFac. de Ciencias QuímicasTRUEunpu

Detection, Control and Contamination of Mycotoxins

The objective of this collection is to illustrate the most recent research on the development of novel and/or rapid methods for mycotoxin determination, and to propose new strategies for monitoring and/or reducing mycotoxin contamination. Innovative sample preparation techniques or protocols and the possibility of multiclass mycotoxin detection will be very positively considered for possible inclusion in this Special Issue. Both methods based on (bio)sensors and chromatography with various detectors (including mass spectrometry) are welcome. Applications of already published methods on new matrices without any modification will not be accepted. However, extensive studies and monitoring on the spread of contamination through the food production chain could be of interest for this collection

Ocratoxina A (OTA), zearalenona (ZEN), desoxinivalenol (DON) e aflatoxina total (TAF) em produtos à base de cereais : revisão sistemática e metanálise

Orientador: Anderson de Souza Sant'AnaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: Devido a aspectos culturais, religiosos, históricos e econômicos, os cereais e seus derivados foram classificados como produtos alimentícios essenciais em todo o mundo. As micotoxinas são metabólitos secundários produzidos por fungos micotoxigênicos em diferentes alimentos durante as etapas de produção, processamento, transporte e armazenamento. Dentre as mais de 300 micotoxinas identificadas, as mais estudadas em cereais e derivados são ocratoxina A (OTA), zearalenona (ZEN), deoxinivalenol (DON) e aflatoxina (AF). Adicionalmente, o estudo da distribuição, prevalência e concentração das micotoxinas em produtos à base de cereais merece destaque, assim como o efeito que as operações unitárias causam nesses produtos durante o processamento. Diversos estudos têm sido realizados com o objetivo de avaliar a taxa de degradação das micotoxinas em cereais e derivados após processamento convencional e após o uso de novas tecnologias, embora a maioria das micotoxinas possa ser considerada estável durante algumas operações unitárias. Por outro lado, a demanda por métodos analíticos de elevado desempenho tem sido um fator determinante e desafiador na análise de micotoxinas. Além disso, a presença de micotoxinas mascaradas, como o deoxinivalenol-3-glucosídeo (DON-3-Glc) e o deoxinivalenol-15-ß-d-glicopiranosídeo (DON-15-glucoside), pode ser considerada um desafio dentro da área de segurança de alimentos. Nesse contexto, o presente estudo objetivou o desenvolvimento de uma revisão sistêmica e uma meta-análise da prevalência, concentração, impacto das operações unitárias e métodos de identificação utilizados para OTA, ZEN, DON e aflatoxinas totais (AFT) em pães, flocos de milho, cereais matinais, macarrão e outros derivados de cereais a partir da literatura publicada entre 1983 e junho de 2017 disponível em bases de dados como Google Scholar, Scopus e PubMed. A ordem de classificação dos derivados de cereais em relação à prevalência de AFT foi biscoitos > flocos de milho > pães > macarrão > cereais matinais; para DON foi flocos de milho > macarrão > biscoitos > pães > cereais matinais; para OTA foi macarrão ~ pães > flocos de milho > biscoitos > cereais matinais; enquanto que para ZEN foi flocos de milho > pães, cereais matinais > biscoitos > macarrão. Adicionalmente, a classificação geral para a prevalência de micotoxinas em derivados de cereais foi DON > OTA > ZEN > TAF > 15-ADON > 3-ADON. A classificação dos derivados de cereais quanto à concentração de DON foi cereais matinais > pães > biscoitos > macarrão > flocos de milho; ZEN, flocos de milho > pães > cereais matinais > biscoitos > macarrão; AFT, macarrão > flocos de milho > pães > biscoitos > cereais matinais; e para OTA, pães > flocos de milho > cereais matinais > biscoitos > macarrão. A concentração de micotoxinas em produtos à base de cereais seguiu a seguinte classificação: DON > ZEN > 15-ADON > OTA > 3-ADON > AFT. Operações unitárias como moagem e fermentação causaram um aumento na concentração de DON e AFT nesses produtos, enquanto que ocorreu redução na concentração de ZEN e OTA. Embora o processamento térmico (cozimento) provocou uma diminuição em DON, OTA e AFT e um aumento na concentração de ZEN em pães, as concentrações de DON e ZEN foram reduzidas em biscoitos. Entretanto, o cozimento de macarrão causou a redução de DON e o aumento de AFT em macarrão. AFT e DON são as micotoxinas que apresentaram o menor e o maior número de estudos, respectivamente, enquanto que a Alemanha representa o primeiro país a iniciar esses estudos. Além disso, a cromatografia líquida com ionização por eletrospray acoplada à espectrometria de massas (LC-ESI/MS) é a técnica de detecção de micotoxinas mais utilizada. A classificação em relação ao número de estudos envolvendo derivados de cereais é cereais em grãos > flocos de milho > pães > cereais matinais > farinha > produtos infantis > macarrão > outros derivados. Assim, a revisão sistêmica e meta-análise realizadas mostraram que as micotoxinas e o que acontece com elas durante o processamento tem relação direta com as operações unitárias utilizadas na preparação dos diferentes tipos de produtos à base de cereais. Os resultados obtidos no presente trabalho podem ser utilizados em estudos de exposição/análise de risco de micotoxinas visando à geração de medidas com embasamento científico a serem implementadas na cadeia de produção de derivados de cereais a fim de assegurar a saúde públicaAbstract: Due to cultural, religious, historical as well as economic aspects, cereal and cereal-based products were categorized as a crucial group of food products around the world. Mycotoxins are secondary metabolites produced by mycotoxigenic molds in food products during production, processing, transportation, and storage. Among more than 300 identified mycotoxins, ochratoxin A (OTA), zearalenone (ZEN), deoxynivalenol (DON) and aflatoxin (AF) are the most studied in cereal-based products. Also, the study of the distribution, prevalence, and concentration of mycotoxins in cereal products deserves notable attention. In addition, the effect of unit operations during manufacturing on the concentration of mycotoxins was the point of interest. Some investigations were conducted in order to calculate the degradation rate of mycotoxins among the conventional as well as novel procedures used for cereal-based production, although most of the mycotoxins can be considered stable during many processing operations. On the other hand, the demand for well-developed analytical methods as one of the main proposed issues among the mycotoxins analysis can be considered an important challenge. Also, the presence of masked mycotoxins, such as the conjugate deoxynivalenol-3-glucoside (DON-3-Glc) and the deoxynivalenol-15-ß-d-glucopyranoside (DON-15-glucoside), can be accounted as a challenge in food safety. In this context, the current study aimed to perform a systematic review and meta-analysis regarding the prevalence, concentration, impact of unit operations and available identification methods for OTA, ZEN, DON and total aflatoxin (TAF) in bread, cornflakes, breakfast meal, pasta and other cereal-based products among the published literature from 1983 through June 2017 in databases (Google Scholar, Scopus and PubMed). The rank order of cereal-based food products on prevalence of TAF was biscuit > cornflakes > bread > pasta > breakfast cereals; for DON was cornflakes > pasta > biscuit > bread > breakfast cereals; for OTA was pasta ~ bread > cornflakes > biscuit > breakfast cereals; and for ZEN was cornflakes > bread > breakfast cereals > biscuit > pasta. Also, the overall rank order of prevalence of mycotoxins in the cereal foods was DON > OTA > ZEN > TAF > 15-ADON > 3-ADON. The rank order of cereals based food products based on concentration of DON was breakfast cereals > bread > biscuit > pasta > cornflakes; ZEN, cornflakes > bread > breakfast > biscuit > pasta; TAF, pasta > cornflakes > bread > biscuit > breakfast; and OTA, bread > cornflakes > breakfast > biscuit > pasta. The overall rank order of concentration of mycotoxins in the cereal-based products was DON > ZEN > 15-ADON > OTA > 3-ADON > TAF. Some of the investigated processing such as milling and fermentation caused an increase in the concentration of DON and TAF; while reduced the concentration of ZEN and OTA. Although heat processing (cooking) caused a decrease in DON, OTA, and TAF and an increase in the concentration of ZEN in bread, it reduced the concentration of DON and ZEN in the biscuit. However, while cooking of pasta reduced the content of DON, it could increase TAF concentration. The lowest and highest numbers of investigations were associated with TAF and DON in cereal-based products, respectively. Among the countries, Germany was ranked as the first in establishing the investigations. Moreover, the liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI/MS) was categorized as the most implemented technique for mycotoxins detection. The rank order of cereal foods based on number of studies was cereal grain > cornflakes > bread > breakfast > flour > infant product > pasta > other products. The outcomes of this systematic review and meta-analysis showed that the mycotoxins and their fate were influenced differently by the unit operations involved in the preparation of the different cereal-based products. The obtained results of the current study can be used in exposure/risk assessment mycotoxin studies aiming the generation of scientific-based measures to be implemented in the production chain of cereal-based products to safeguard public healthDoutoradoCiência de AlimentosDoutor em Ciência de Alimentos190178/2013-2CNP

Nuevos elementos de reconocimiento molecular selectivo y métodos de amplificación para el desarrollo de (bio)sensores ópticos

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, leída el 01-04-2022Mycotoxins are low molecular weight substances produced as secondary metabolites by a wide variety of filamentous fungi that can be found as natural contaminants in many foods and feeds. The number of toxic fungal metabolites currently known exceeds the thousand units, but only a few of them are considered a threat to humans and animal health. Exposure to mycotoxins can be due to the consumption of contaminated foodstuff, but also by inhalation of dust containing mycotoxigenic fungal spores. It has been estimated that nearly 40% of the global crops can be contaminated with mycotoxins. Hence, the development of analytical methods that can detect these mycotoxins in foodstuff are essential. On the other hand, despite their elevated risk infood quality, several mycotoxins present medical applications, for example as immunosuppressant drugs for organ transplantation. However, a limitation for this application is the narrow therapeutic window presented by these drugs, e.g., mycophenolic acid. High doses of these compounds can cause serious adverse health effects in humans, but little doses could not be sufficient to prevent organ rejection in transplanted patients. Thus, it is also of utmost importance to monitor the levels of these compounds in blood to improve the clinical efficacy of the immunosuppressant...Las micotoxinas son sustancias de bajo peso molecular producidas como metabolitos secundarios por una gran variedad de hongos filamentosos que pueden encontrarse como contaminantes naturales en muchos alimentos y piensos. El número de metabolitos fúngicos tóxicos que se conocen en la actualidad supera las mil unidades, pero sólo unas pocas docenas se consideran una amenaza para la salud humana y animal. La exposición a las micotoxinas puede deberse al consumo de alimentos contaminados, pero también a la inhalación de polvo que contiene esporas de hongos micotoxigénicos. Se calcula que aproximadamente el 40% de los cultivos del mundo pueden estar contaminados con micotoxinas, de ahí que sea esencial el desarrollo de métodos analíticos que permitan detectar estas micotoxinas en los alimentos. Por otro lado, a pesar de su elevado riesgo en la calidad de los alimentos, varias micotoxinas presentan aplicaciones médicas, por ejemplo, como fármacos inmunosupresores para el trasplante de órganos. Sin embargo, una limitación para esta aplicación es la estrecha ventana terapéutica que presentan estos fármacos, por ejemplo, el ácido micofenólico. Altas dosis de estos compuestos pueden causar graves efectos adversos para la salud delos seres humanos, pero pequeñas dosis pueden provocar el rechazo de órganos en pacientes trasplantados. Por ello, también es de suma importancia controlar los niveles de estos compuestos en sangre para mejorar la eficacia clínica del inmunosupresor...Fac. de Ciencias QuímicasTRUEunpu