Método de extracción y detección de antígenos de Anisakis en alimentos destinados al consumo humano o animal

Método de extracción y detección de antígenos de Anisakis en alimentos destinados al consumo humano o animal. La presente invención se refiere a un método de extracción y detección de alérgenos de parásitos de pescado en muestras alimentarias para el consumo humano o animal. La extracción se basa en aplicar soluciones con baja fuerza iónica, homogeneización, sonicación y diferentes pH a diversos tipos de pescado ya sean frescos o tratados. La detección se basa en métodos inmunoquímicos mediante el uso de anticuerpos policlonales que permiten detectar proteínas antigénicas del parásito así como anticuerpos policlonales que permiten detectar el alérgeno Ani s 4, que por sus características físico-químicas resiste el tratamiento térmico del alimento. El método es sensible, ya que se puede detectar Ani s 4 en cantidades inferiores a 1ppm con tasas de recuperación mayores a un 65%. El método descrito es específico ya que no muestra reactividad cruzada con componentes de las distintas matrices ensayadas.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Fundación para la Investigación Biomédica del Hospital Carlos IIIB1 Patente sin examen previ

Método de extracción y detección de antígenos de Anisakis en alimentos destinados al consumo humano o animal

Método de extracción y detección de antígenos de Anisakis en alimentos destinados al consumo humano o animal. La presente invención se refiere a un método de extracción y detección de alérgenos de parásitos de pescado en muestras alimentarias para el consumo humano o animal. La extracción se basa en aplicar soluciones con baja fuerza iónica, homogeneización, sonicación y diferentes pH a diversos tipos de pescado ya sean frescos o tratados. La detección se basa en métodos inmunoquímicos mediante el uso de anticuerpos policlonales que permiten detectar proteínas antigénicas del parásito así como anticuerpos policlonales que permiten detectar el alérgeno Ani s 4, que por sus características físico-químicas resiste el tratamiento térmico del alimento. El método es sensible, ya que se puede detectar Ani s 4 en cantidades inferiores a 1ppm con tasas de recuperación mayores a un 65%. El método descrito es específico ya que no muestra reactividad cruzada con componentes de las distintas matrices ensayadas.Consejo Superior de Investigaciones Científicas (España), Fundación para la Investigación Biomédica del Hospital Carlos IIIA1 Solicitud de patente con informe sobre el estado de la técnic

Usefulness of bone turnover markers as predictors of mortality risk, disease progression and skeletal-related events appearance in patients with prostate cancer with bone metastases following treatment with zoledronic acid: TUGAMO study

Owing to the limited validity of clinical data on the treatment of prostate cancer (PCa) and bone metastases, biochemical markers are a promising tool for predicting survival, disease progression and skeletal-related events (SREs) in these patients. The aim of this study was to evaluate the predictive capacity of biochemical markers of bone turnover for mortality risk, disease progression and SREs in patients with PCa and bone metastases undergoing treatment with zoledronic acid (ZA). Methods: This was an observational, prospective and multicenter study in which ninety-eight patients were included. Patients were treated with ZA (4mg every 4 weeks for 18 months). Data were collected at baseline and 3, 6, 9, 12, 15 and 18 months after the beginning of treatment. Serum levels of bone alkaline phosphtase (BALP), aminoterminal propeptide of procollagen type I (P1NP) and beta-isomer of carboxiterminal telopeptide of collagen I (b-CTX) were analysed at all points in the study. Data on disease progression, SREs development and survival were recorded. Results: Cox regression models with clinical data and bone markers showed that the levels of the three markers studied were predictive of survival time, with b-CTX being especially powerful, in which a lack of normalisation in visit 1 (3 months after the beginning of treatment) showed a 6.3-times more risk for death than in normalised patients. Levels of these markers were also predictive for SREs, although in this case BALP and P1NP proved to be better predictors. We did not find any relationship between bone markers and disease progression. Conclusion: In patients with PCa and bone metastases treated with ZA, b-CTX and P1NP can be considered suitable predictors for mortality risk, while BALP and P1NP are appropriate for SREs. The levels of these biomarkers 3 months after the beginning of treatment are especially importantThis study was supported by Novartis Oncology Spai

Clonación y caracterización de alérgenos recombinantes de Anisakis simplex. Valoración de su utilidad en el diagnóstico de la Anisakiasis

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 03-11-200

Isolation of a heat-resistant allegen from the fish parasite Anisakis simplex

5 páginas, 5 figuras -- PAGS nros. 285-289The thermal stability of allergenic peptides from the fish parasite Anisakis simplex has not been fully elucidated. This is of special relevance for physicians who should clearly indicate if sensitized patients should avoid ingestion of raw fish only or whether well-cooked fish should also be avoided, if allergenic peptides derived from the parasite remain immunologically detectable. An allergen was purified after heating a crude parasite extract for 30 min. The allergen was further purified by an ethanol fractionation procedure followed by a reversed-phase HPLC. The N-terminal amino acid sequence was obtained. This allergen was detected by 27% of sensitized subjects. The N-terminal amino acid sequence of the 9 kDa allergen showed no similarities to other known proteins. A minor low molecular weight allergen from A. simplex is highly resistant to heating and it could therefore have significant clinical relevanceThis work was supported by grant PI031442 of FIS, Ministry of HealthPeer reviewe

Changes over Time in IgE Sensitization to Allergens of the Fish Parasite Anisakis spp

© 2016 Carballeda-Sangiao et al.[Background]: Sensitization to Anisakis spp. can produce allergic reactions after eating raw or undercooked parasitized fish. Specific IgE is detected long after the onset of symptoms, but the changes in specific IgE levels over a long follow-up period are unknown; furthermore, the influence of Anisakis spp. allergen exposure through consumption of fishery products is also unknown.[Objective]: To analyse the changes in IgE sensitization to Anisakis spp. allergens over several years of follow-up and the influence of the consumption of fishery products in IgE sensitization. [Methods]: Total IgE, Anisakis spp.-specific IgE, anti-Ani s 1 and anti-Ani s 4 IgE were repeatedly measured over a median follow-up duration of 49 months in 17 sensitized patients.[Results]: Anisakis spp.-specific IgE was detected in 16/17 patients throughout the follow-up period. The comparison between baseline and last visit measurements showed significant decreases in both total IgE and specific IgE. The specific IgE values had an exponential or polynomial decay trend in 13/17 patients. In 4/17 patients, an increase in specific IgE level with the introduction of fish to the diet was observed. Three patients reported symptoms after eating aquaculture or previously frozen fish, and in two of those patients, symptom presentation was coincident with an increase in specific IgE level.[Conclusions]: IgE sensitization to Anisakis spp. allergens lasts for many years since specific IgE was detectable in some patients after more than 8 years from the allergic episode. Specific IgE monitoring showed that specific IgE titres increase in some allergic patients and that allergen contamination of fishery products can account for the observed increase in Anisakis spp.-specific IgE level. Clinical Relevance: Following sensitization to Anisakis spp. allergens, the absence of additional exposure to those allergens does not result in the loss of IgE sensitization. Exposure to Anisakis spp. allergens in fishery products can increase the specific IgE level in some sensitized patients.The research leading to these results received funding from the European Union’s Seventh Framework Programme for Research, Technological Development and Demonstration under grant agreement n° 312068. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer Reviewe

Effect of high hydrostatic pressure on mortality and allergenicity of Anisakis simplex L3 and on muscle properties of infested hake

Background: High pressure (HP) ranging from 100 to 350 MPa (1-15 min) was applied to Anisakis simplex larvae and parasitised hake (Merluccius merluccius) muscle. The aim of the study was to kill the larvae to prevent human anisakidosis, to evaluate the effect on A. simplex allergens and to minimally alter fish muscle quality. Results: The larvae were killed at pressures ≥200 MPa and times ≥1 min, producing alterations in the larva body and ruptures in the cuticle when observed by scanning electron microscopy. Nevertheless, Ani s 4 and A. simplex crude antigens were recognised by immunoblotting and immunohistochemistry at all HPs assayed. Small changes in colour and texture were observed in fish muscle under all pressure/time conditions. Major changes were observed visually at 300 MPa, where the muscle appeared as slightly cooked. Apparent viscosity of muscle homogenates decreased significantly at longer times or higher applied pressure. No changes were detected at 200 MPa in the electrophoretic pattern of proteins treated with or without β-mercaptoethanol, suggesting that disulfide bonds were not formed. Conclusion: Application of HP at 200 MPa for up to 5 min would kill A. simplex larvae, avoiding infestation of the consumer and causing small changes in the hake muscle perceived sensorially. However, HP-treated A. simplex-parasitised fish would still be a potential hazard for consumers allergic to the larvae. © 2009 Society of Chemical Industry.This work has been financed by the Spanish project Plan Nacional de I+D+i AGL2005-05699-C02-01/02 ALI, and CSIC projects: PIE 2004 7 0E 160 CSIC and PIE 2004 7 0E 340Peer Reviewe

Effect of successive washes of infested muscle in the extraction and detection of Anisakis allergens

[ES]: Aunque la adecuada congelación o cocción produzcan la muerte de las larvas de Anisakis y se evite la infestación de los consumidores, las larvas muertas conservan su capacidad alergénica y los alérgenos son capaces de inducir una respuesta inmune en pacientes previamente sensibilizados, especialmente si son termo-resistentes como Ani s 4. En la fabricación de surimi, el lavado del músculo de pescado es uno de los pasos clave y de acuerdo a las características de algunos de los alérgenos de Anisakis descritos, se ha considerado que en esta operación se podrían eliminar algunas proteínas alergénicas del músculo infestado. El objetivo del estudio fue evaluar la reducción o eliminación de las proteínas alergénicas del músculo por solubilizaciones selectivas aplicando el método tradicional japonés de obtención de surimi. Se tuvieron en cuenta los pesos moleculares y puntos isoeléctricos de los principales alérgenos de Anisakis a fin de poder eliminarlos en las soluciones residuales de cada lavado. Se utilizó músculo de merluza con infestación natural (@20 larvas por cada 100 g músculo) que fue sometido a 3 lavados sucesivos con buffer fosfato [1:4 (p:v); 10 minutos; 5ºC; pH 7-7,5]. Se evaluó la concentración y perfil de las proteínas extraídas con las soluciones de lavado (Proteína bruta y electroforesis en gel de poliacrilamida) y la presencia de alérgenos de A. simplex (Inmunoblotting). Ani s 4 se mantuvo presente en el músculo después del tercer lavado, aunque su eliminación fue progresiva y se reflejó en las soluciones residuales de cada lavado, quedando en algunos casos por debajo del límite de detección de la técnica (<1 ppm). Es necesario continuar con los trabajos de investigación a fin de mejorar las condiciones de detección de alérgenos de Anisakis e incluir técnicas de concentración de proteínas que permitan detectar su presencia a bajas concentraciones para determinar con exactitud el efecto de los lavados sucesivos en distintas condiciones de infestación y tratamiento.[EN]: Although the adequate freezing or cooking results in the death of Anisakis larvae and this will prevent consumer infestation, dead larvae retain their allergenic capacity and allergens are capable of inducing an immune response in previously sensitized patients, especially if they are thermo-resistant as Ani s 4. In the manufacture of surimi, the washing of fishmuscle is one of the key steps and according to the characteristics of some of the described Anisakis allergens, it has been considered that this operation could eliminate some allergenic proteins from the infested muscle. The objective of the study was to evaluate the reduction or elimination of the allergenic proteins from the muscle by selective solubilizations applying the traditional Japanese method of surimi production. The molecular weights and isoelectric points of major allergens of Anisakis were taken into consideration in order to remove them from waste solutions. Hake muscle with natural infestation (@20 larvae per 100 g muscle) was subjected to three successive washes with phosphate buffer[1:4 (w: v), 10 minutes, 5°C, pH 7-7.5]. The concentration and profileof the proteins extracted with the washing solutions (Crude protein and polyacrylamide gel electrophoresis) and the presence of A. simplex allergen (Immunoblotting) were evaluated in all samples. Ani s 4 remained present in the muscle after the third wash, although its elimination was progressive and was reflectedin each waste solution, being in some cases below the detection limit of the technique (<1 ppm). It is necessary to continue the research to improve the detection of Anisakis allergens, including protein concentration techniques to detect their presence at low concentrations to accurately determine the effectof successive washes under different conditions of infestation and treatment.El presente trabajo ha sido realizado en el ICTAN-CSIC de Madrid y ha sido financiado por el proyecto del Plan Nacional I+D+i español AGL2009-12485-C03-01/03 (ANIDET). Fabiola Olivares ha realizado el trabajo en el ICTAN mediante una beca de estudios concedida por el Programa de Ciencia y Tecnología del Gobierno de Perú (FINCyT) administrada por LASPAU.Peer reviewe

Detección de alergenos de Anisakis simplex en pescado en conserva y pescado de acuicultura

Trabajo presentado a la 8ª Reunión de la Sociedad Española de Seguridad Alimentaria, celebrada en Zaragoza, los días 29 y 30 de septiembre de 2011.Peer reviewe