RESUMEN
BACKGROUND AND OBJECTIVES: The isolation of neutrophils and subsequent detection of anti-human neutrophil antigens (HNA) antibodies are crucial in clinical medicine for the diagnosis of autoimmune neutropenia, neonatal alloimmune neutropenia (NAIN) and transfusion-related acute lung injury (TRALI). This study reports two cases of maternal anti-Fc-gamma-receptor-IIIb (FcγRIIIb) isoimmunization without NAIN symptoms and compares the efficiency of immunomagnetic negative selection (IMNS) with traditional dextran/Ficoll for neutrophil isolation in HNA serological assays. MATERIALS AND METHODS: Investigating two cases of maternal anti-FcγRIIIb isoimmunization, neutrophils from three donors were isolated from 8 mL of whole blood using IMNS and dextran/Ficoll. Serological assays included the granulocyte agglutination and immunofluorescence test, monoclonal antibody immobilization of granulocyte antigens and the LABScreen Multi (One Lambda). IMNS and dextran/Ficoll were compared in terms of cell yield, viability, time, cost and purity. RESULTS: Maternal anti-FcγRIIIb isoantibodies with FCGR3B gene deletion were detected in both cases. Newborns and fathers exhibited specific gene combinations: FCGR3B*02/FCGR3B*02 (Case 1) and FCGR3B*02/FCGR3B*03 (Case 2). IMNS outperformed dextran/Ficoll, yielding four times more neutrophils (average neutrophil counts: 18.5 × 103/µL vs. 4.5 × 103/µL), efficiently removing non-neutrophil cells and reducing processing time (30-40 min vs. 70-90 min), although it incurred a higher cost (2.7 times). CONCLUSION: Two cases of maternal anti-FcγRIIIb isoantibodies, unrelated to NAIN, were identified. Although neutropenia has not been described in these cases, we emphasize the importance of identifying asymptomatic cases with the potential for severe neutropenia. Additionally, IMNS is introduced as a rapid, high-yield, high-purity neutrophil isolation technique, beneficial for serological assays detecting anti-HNA antibodies.
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Isoanticuerpos , Neutrófilos , Receptores de IgG , Humanos , Neutrófilos/inmunología , Femenino , Receptores de IgG/inmunología , Isoanticuerpos/inmunología , Isoanticuerpos/sangre , Recién Nacido , Proteínas Ligadas a GPI/inmunología , Masculino , Separación Inmunomagnética/métodos , Adulto , Embarazo , Neutropenia/inmunología , Neutropenia/sangreRESUMEN
Over the last two decades, the variable domains from heavy chain-only antibodies in camelids (nanobodies) have emerged as valuable immunoreagents for analytical and diagnostic applications. One prominent use of nanobodies is for the detection of small molecules due to their ease of production, resistance to solvents used in sample extraction, facile genetic manipulation, and small size. These last two properties make it possible to produce biotinylated nanobodies in vivo, which can be loaded in an orientated manner on magnetic beads covered with avidin, creating high-density immunoadsorbenpi twbch ""ts. The method described here details the use of nanobody-based adsorbents to concentrate small molecular weight analytes for subsequent quantitative analysis by MALDI-TOF mass spectrometry. Quantitation requires the inclusion of an internal standard (IS), a compound with properties similar to those of the analyte, enabling compensation for uneven distribution during crystallization of the MALDI-TOF matrix. Since nanobody generation against small compounds requires conjugation to carrier proteins, the same conjugation chemistry can be used to synthesize the IS. By design the IS cross reacts with the capture nanobody and can be preloaded in the immunoadsorbent, facilitating quantitative detection of the target compound.
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Anticuerpos de Dominio Único , Cadenas Pesadas de Inmunoglobulina , Separación Inmunomagnética , Fenómenos Magnéticos , Anticuerpos de Dominio Único/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
In spite of a current increasing trend in the development of miniaturized, standalone point-of-care (PoC) biosensing platforms in the literature, the actual implementation of such systems in the field is far from being a reality although deeply needed. In the particular case of the population screenings for local or regional diseases related to specific pathogens, the diagnosis of the presence of specific antibodies could drastically modify therapies and even the organization of public policies. The aim of this work was to develop a fast, cost-effective detection method based on the manipulation of functionalized magnetic beads for an efficient diagnosis of hypersensitivity pneumonitis (HP), looking for the presence of anti-pigeon antigen antibodies (APAA) in a patient's serum. We presented a Diagnostic Biosensor Method (DBM) in detail, with validation by comparison with a traditional high-throughput platform (ELISA assay). We also demonstrated that it was compatible with a microfluidic chip that could be eventually incorporated into a PoC for easy and broad deployment using portable optical detectors. After standardization of the different reaction steps, we constructed and validated a plastic chip that could easily be scaled to high-volume manufacturing in the future. The solution proved comparable to conventional ELISA assays traditionally performed by the clinicians in their laboratory and should be compatible with other antibody detection directly from patient samples.
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Alveolitis Alérgica Extrínseca , Técnicas Biosensibles , Alveolitis Alérgica Extrínseca/diagnóstico , Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Diseño de Equipo , Humanos , Separación Inmunomagnética , Dispositivos Laboratorio en un Chip , Microfluídica , Sistemas de Atención de PuntoRESUMEN
This study evaluated the technology of detection of Giardia spp. cysts and Cryptosporidium spp. oocysts in environmental matrices obtained after water treatment on a bench scale. Calcium carbonate flocculation with immunomagnetic separation was the selected method to quantify the protozoa, and the importance of the number of acid dissociations in the immunomagnetic separation was assessed. When adding the third acid dissociation, an increase of 71% ± 6 in floated residue and 31.9% ± 28.7 in filter backwash water in cyst recovery was observed, while in oocyst recovery, a non-significant increase was detected. In the filtered water, this increased dissociation was important in the protozoa recovery with increases greater than 33%. The results showed that there is a strong interaction of these target organisms with the magnetic microspheres, since protozoa were still recovered in the third acid dissociation and some of them were still adhered to the magnetic microspheres.
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Cryptosporidium/aislamiento & purificación , Giardia/aislamiento & purificación , Purificación del Agua/métodos , Agua/parasitología , Animales , Carbonato de Calcio/química , Floculación , Separación Inmunomagnética , Oocistos/aislamiento & purificaciónRESUMEN
Clinical manifestations of leptospirosis are diverse and very similar to other febrile diseases, hence early and accurate detection of subclinical infections is a key element in disease control. We evaluated immunomagnetic separation (IMS) capture technology coupled with a standard quantitative PCR (qPCR) system for the detection of pathogenic Leptospira in urine samples from 803 cows from dairy herds with a history of clinical cases of leptospirosis. The urine samples were first processed in a purification step, then subdivided into 2 subsamples, one that continued to DNA extraction and direct qPCR, and one that was pretreated by IMS before continuing to DNA extraction and qPCR. Overall, 133 of 803 (16.6%) samples were IMS-qPCR positive, whereas only 92 of 803 (11.5%) were positive when using direct qPCR. Statistically significant differences were observed between the mean estimated Leptospira load between the IMS-qPCR and the direct qPCR positive urine samples. The IMS-qPCR technology revealed a larger number of positive results and higher bacterial loads than direct qPCR. This difference is most likely the result of the high antigen-binding capacity and capture efficiency of the IMS system. The use of polyclonal antibodies produced by the inoculation of 3 synthetic peptides, which make up the extracellular regions of the LipL32 protein, provided a high detection capacity to the IMS-qPCR technique, resulting in performance superior to direct qPCR.
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Crianza de Animales Domésticos , Enfermedades de los Bovinos/diagnóstico , Leptospira/aislamiento & purificación , Leptospirosis/veterinaria , Animales , Bovinos , Enfermedades de los Bovinos/orina , Chile , Industria Lechera , Femenino , Separación Inmunomagnética/veterinaria , Leptospira/genética , Leptospira/inmunología , Leptospirosis/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Sensibilidad y Especificidad , Urinálisis/veterinariaRESUMEN
The objective of this study was to estimate the performance of the peptide magnetic separation PCR test (PMS-PCR) for the diagnosis of Mycobacterium avium subsp. paratuberculosis (MAP) in sub-clinically infected dairy cattle. Twenty-one herds were randomly selected from a source population of 131 commercial dairy herds with a known history of MAP infection, located in the De Los Rios and De Los Lagos regions, in southern Chile. In the selected herds, all milking cows with ≥2 parities and without any clinical signs were sampled, collecting feces and blood-serum samples. The PMS-PCR test was used to analyze the fecal samples, while serum samples were analyzed using a commercial ELISA kit. A Bayesian latent class model was used to estimate the sensitivity (Se) and specificity (Sp) of the diagnostic tests. A total of 1381 animals were sampled in the 21 selected dairy herds, with an average sample size of 65 animals per herd (range 10-721). The PMS-PCR test had a greater Se than the ELISA test, with a median of 85.5 % (posterior probability interval (PPI) 95 %: 79.3-91.0%), while the ELISA test presented a median of 21.7 % (95 % PPI: 18.3-25.4%). On the other hand, the ELISA test had a better Sp than the PMS-PCR test, with a median of 97.7 % (95 % PPI: 96.6-98.5%), whereas PMS-PCR presented a median of 90.8 % (95 % PPI: 88.3-93.9%). Model results showed that PMS-PCR has a better Se than all available tests for MAP diagnosis in subclinical animals. However, this test should be used with care in herds with high infection rates, where a high MAP environmental load is expected, potentially increasing the frequency of false positive cases due to the pass-through phenomenon.
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Enfermedades de los Bovinos/diagnóstico , Pruebas Diagnósticas de Rutina/veterinaria , Separación Inmunomagnética/veterinaria , Mycobacterium avium subsp. paratuberculosis/aislamiento & purificación , Paratuberculosis/diagnóstico , Reacción en Cadena de la Polimerasa/veterinaria , Animales , Infecciones Asintomáticas/epidemiología , Teorema de Bayes , Bovinos , Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/microbiología , Chile/epidemiología , Industria Lechera , Pruebas Diagnósticas de Rutina/instrumentación , Análisis de Clases Latentes , Paratuberculosis/epidemiología , Paratuberculosis/microbiología , Reacción en Cadena de la Polimerasa/métodos , Prevalencia , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: The diagnosis of active Toxocara canis infections in humans is challenging. Larval stages of T. canis do not replicate in human tissues and disease may result from infection with a single T. canis larva. Recently, we developed a nanobody-based electrochemical magnetosensor assay with superior sensitivity to detect T. canis excretory-secretory (TES) antigens. Here, we evaluate the performance of the assay in children from an Ecuadorian birth cohort that followed children to five years of age. METHODS: Samples were selected based on the presence of peripheral blood eosinophilia and relative eosinophil counts. The samples were analyzed by the nanobody-based electrochemical magnetosensor assay, which utilizes a bivalent biotinylated nanobody as capturing agent on the surface of streptavidin pre-coated paramagnetic beads. Detection was performed by a different nanobody chemically labelled with horseradish peroxidase. RESULTS: Of 87 samples tested, 33 (38%) scored positive for TES antigen recognition by the electrochemical magnetosensor assay. The average concentration of TES antigen in serum was 2.1 ng/ml (SD = 1.1). The positive result in the electrochemical assay was associated with eosinophilia > 19% (P = 0.001). Parasitological data were available for 57 samples. There was no significant association between positivity by the electrochemical assay and the presence of other soil-transmitted helminth infections. CONCLUSIONS: Our nanobody-based electrochemical assay provides highly sensitive quantification of TES antigens in serum and has potential as a valuable tool for the diagnosis of active human toxocariasis.
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Antígenos Helmínticos/sangre , Técnicas Electroquímicas/métodos , Eosinofilia/parasitología , Proteínas del Helminto/sangre , Anticuerpos de Dominio Único/inmunología , Toxocariasis/diagnóstico , Animales , Biotinilación , Camelidae , Preescolar , Ecuador/epidemiología , Eosinofilia/epidemiología , Humanos , Separación Inmunomagnética , Lactante , Población Rural , Toxocara canis , Toxocariasis/epidemiologíaRESUMEN
Immunoprecipitation is a helpful tool to assess interactions between proteins and proteins or nucleic acids (DNA or RNA). Its principle consists in capturing and enriching one or multiple target proteins from a complex sample with a specific antibody conjugated to a solid matrix and isolating the RNA and/or protein molecules associated to those target(s) group of proteins that can be further identified by advanced techniques such as RNA-seq and/or mass spectrometry. Since this technique allows for identifying, mapping, and checking new protein-protein and protein-RNA interactions, its use is very convenient in situations where many proteins remain with their functions uncharacterized, as is the case of the protozoan Trypanosoma cruzi. Here we describe a protocol that is based on the cryogrinding method for cell lysis and the use of antibodies conjugated to magnetic beads to capture and purify protein complexes in a robust and efficient way.
Asunto(s)
Separación Inmunomagnética/métodos , Inmunoprecipitación/métodos , Sustancias Macromoleculares/aislamiento & purificación , Trypanosoma cruzi/fisiología , Sustancias Macromoleculares/metabolismo , Espectrometría de Masas/métodos , Parasitología/métodos , Mapeo de Interacción de Proteínas , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , ARN Protozoario/aislamiento & purificación , ARN Protozoario/metabolismoRESUMEN
Measuring antimalarial antibodies can estimate transmission in a population. To compare outputs, standardized laboratory testing is required. Here we describe the in-country establishment and quality control (QC) of a multiplex bead assay (MBA) for three sero-surveys in Haiti. Total IgG data against 21 antigens were collected for 32,758 participants. Titration curves of hyperimmune sera were included on assay plates, assay signals underwent 5-parameter regression, and inspection of the median and interquartile range (IQR) for the y-inflection point was used to determine assay precision. The medians and IQRs were similar for Surveys 1 and 2 for most antigens, while the IQRs increased for some antigens in Survey 3. Levey-Jennings charts for selected antigens provided a pass/fail criterion for each assay plate and, of 387 assay plates, 13 (3.4%) were repeated. Individual samples failed if IgG binding to the generic glutathione-S-transferase protein was observed, with 659 (2.0%) samples failing. An additional 455 (1.4%) observations failed due to low bead numbers (<20/analyte). The final dataset included 609,438 anti-malaria IgG data points from 32,099 participants; 96.6% of all potential data points if no QC failures had occurred. The MBA can be deployed with high-throughput data collection and low inter-plate variability while ensuring data quality.
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Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Inmunoglobulina G/sangre , Separación Inmunomagnética/métodos , Malaria Falciparum/diagnóstico , Plasmodium falciparum/inmunología , Control de Calidad , Pruebas Serológicas/métodos , Anticuerpos Antiprotozoarios/inmunología , Especificidad de Anticuerpos , Estudios Transversales , Conjuntos de Datos como Asunto , Haití/epidemiología , Humanos , Inmunoglobulina G/inmunología , Separación Inmunomagnética/instrumentación , Malaria Falciparum/sangre , Malaria Falciparum/epidemiología , Malaria Falciparum/inmunología , Proteínas Recombinantes/inmunología , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Dental stem cells have many applications in medicine, dentistry and stem cell biology in general due to their easy accessibility and low morbidity. A common surgical manoeuvre after a tooth extraction is the dental socket curettage which is necessary to clean the alveolus and favour alveolar bone healing. This procedure can cause very low morbidity compared to bone marrow collection procedures and the collected material is normally discarded. In order to investigate if the tissue obtained by dental socket curettage after a tooth extraction was a feasible alternative source to isolate human stem cells, we isolated and characterized two different stem cell populations based on STRO-1 and CD146 expression. We were able to collect and grow cells from dental socket of vital and non-vital teeth. Both populations were proliferative, clonogenic and expressed STRO-1, CD146, CD90, NG2, PDGFR-ß, which are markers found in stem cells, presented in vitro multiline-differentiation into osteogenic, chondrogenic, and adipogenic tissue, and in vivo transplanted cells formed mineralized tissue. Interestingly, STRO-1+ clonogenic cells presented better multidifferentiation than CD146+ cells. Our results showed that mesenchymal stem cells can be isolated from the tiny tissue collected by dental socket curettage after vital and non-vital tooth extraction and suggest that STRO-1 is an important marker to be used to sort cells with multidifferentiation capacity.
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Diferenciación Celular , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Alveolo Dental/citología , Animales , Antígenos de Superficie/análisis , Antígeno CD146/análisis , Proliferación Celular , Células Cultivadas , Humanos , Separación Inmunomagnética , Masculino , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
Tristeza is a disease that affects citrus crops in general, caused by the Citrus tristeza virus (CTV). It is considered an economically important virus diseases in citrus, which is present in the main citrus producing regions all around the world. Early detection of CTV is crucial to avoid any epidemics and substantial economic losses for the citrus growers. Consequently, the development of rapid, accurate, and sensitive methods capable of detecting the virus in the early stages of the disease is highly desired. Based on that, a low-cost and rapid magneto-immunoassay methodology to detect the capsid protein from CTV (CP-CTV) was proposed. For this, magnetic beads were decorated with antibodies anti-CP-CTV and horseradish peroxidase enzyme (HRP) and applied for the capture and separation of CP-CTV from the sample solutions. The magnetically captured biomarker was detected using a simple disposable microfluidic electrochemical device (DµFED) constructed by rapid prototyping technique and composed by an array of immunosensors. In DµFED, the electrodes were modified with monoclonal antibody anti-CP-CTV and the detection was carried out using amperometry, based on the hydroquinone/H2O2 catalytic redox reaction due to the presence of HRP label in an immune-sandwich structure. The proposed immunoassay presented excellent linearity with a wide linear range of concentration of 1.95-10.0â¯×â¯103â¯fgâ¯mL-1 and ultralow detection limit of 0.3â¯fgâ¯mL-1. The disposable device was successfully applied for the detection of Citrus tristeza virus in healthy and infected plant samples, where it showed good agreements with the comparative method of enzyme-linked immunosorbent assay (ELISA). The developed immunoassay methodology showed a sensitive and selective way in the detection of CTV. Hence, it can be considered as a promising analytical alternative for rapid and low-cost diagnosis of Tristeza disease in citrus.
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Closterovirus/aislamiento & purificación , Dispositivos Laboratorio en un Chip , Animales , Anticuerpos Inmovilizados/inmunología , Anticuerpos Monoclonales de Origen Murino/inmunología , Proteínas de la Cápside/análisis , Proteínas de la Cápside/inmunología , Citrus/virología , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Electrodos , Oro/química , Inmunoensayo/métodos , Separación Inmunomagnética/métodos , Límite de Detección , Nanopartículas del Metal/química , Ratones , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Enfermedades de las Plantas/virología , Hojas de la Planta/virología , Reproducibilidad de los ResultadosRESUMEN
Detecting bacteria is important in the fields of human health, environmental monitoring, and food safety. Foodborne pathogens alone are estimated to cause 420â¯000 deaths annually, with low-income regions affected most. Despite improvements in bacterial detection, fast, disposable, low-cost, sensitive, and user-friendly methods are still needed. Traditional methods for detecting bacteria rely primarily on cell culturing or polymerase chain reaction (PCR), which require highly trained personnel and a central laboratory and take several hours or even days to deliver results. Low-cost methods like lateral flow immunoassays exist but frequently suffer from poor sensitivity and/or lack quantitative results. Here, a rapid method for detecting bacteria at very low concentrations is presented using two sequential preconcentration steps. In the first preconcentration step, the sample is mixed with antibody-modified magnetic particles and free antibodies conjugated to ß-galactosidase (ß-gal). The target bacteria are isolated and concentrated using immunomagnetic separation. The isolated bacteria are then incubated with chlorophenol red-ß-d-galactopyranoside (CPRG), which reacts with ß-gal to produce chlorophenol red (CPR) in a bacteria concentration-dependent manner. In the second step, CPR and CPRG are separated and focused using an isotachophoretic microfluidic paper-based analytical device, significantly improving the final detection limit relative to paper-based devices lacking the focusing mechanism. Moreover, CPR and CPRG form two visible color bands that act as test and control bands, respectively, improving assay robustness. The method was tested with E. coli DH5-α and successfully detected concentrations as low as 9.2 CFU/mL in laboratory samples and 920 CFU/mL in apple juice samples in â¼90 min.
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Separación Inmunomagnética/métodos , Isotacoforesis/instrumentación , Isotacoforesis/métodos , Papel , Técnicas Bacteriológicas , Escherichia coli , Microbiología de Alimentos , Jugos de Frutas y Vegetales/microbiología , Malus , Análisis de la Célula IndividualRESUMEN
PURPOSE: EpCAM is a common marker used in the detection of circulating tumor cells (CTC). Disseminated cancer cells display the characteristics of epithelial-to-mesenchymal transition events. The purpose of this study was to assess the potential of epithelial membrane protein 2 (EMP2) as a novel biomarker for CTC retrieval in breast cancer. METHODS: MCF7 and MDA-MB-231 cells were stained with either anti-EpCAM or anti-EMP2 mAbs, respectively, followed by flow cytometric assay to measure their expression levels. PBMCs isolated from healthy donors were used for breast cancer cell spiking. CD45-depleted PBMCs from breast cancer patients' blood were used for CTC capturing. Immunomagnetic separation was used to enrich breast cancer cells. Cytospin centrifugation was performed to concentrate the captured cells, followed by immunofluorescence staining with anti-CD45 mAb, anti-pan cytokeratin mAb and DAPI. Fluorescent images were taken using a confocal microscope for CTC counts. RESULT: MDA-MB-231 cells had 2.56 times higher EMP2 expression than MCF7 cells, and EMP2 had a significantly higher capture efficiency than EpCAM for MCF7 cells. Furthermore, anti-EMP2 was capable of capturing MCF7 cells that escaped in the flow-through of anti-EpCAM. Likewise, EMP2 had a significantly higher capture efficiency on MDA-MB-231 cells when compared to MCF7 cells. Most importantly, EMP2 biomarker was successfully used for CTC capture in patients with primary breast cancer. CONCLUSIONS: EMP2 is superior to EpCAM for capturing both MCF7 and MDA-MB-231 cells. Additionally, EMP2 is a novel biomarker and capable of capturing breast cancer cells in patient blood samples.
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Neoplasias de la Mama/sangre , Neoplasias de la Mama/diagnóstico , Separación Celular/métodos , Separación Inmunomagnética/métodos , Glicoproteínas de Membrana/sangre , Células Neoplásicas Circulantes/metabolismo , Adulto , Anciano , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/inmunología , Neoplasias de la Mama/inmunología , Molécula de Adhesión Celular Epitelial/sangre , Molécula de Adhesión Celular Epitelial/inmunología , Femenino , Humanos , Células MCF-7 , Glicoproteínas de Membrana/inmunología , Persona de Mediana Edad , Células Neoplásicas Circulantes/patologíaRESUMEN
Detecting pathogenic protozoa in drinking-water treatment sludge is a challenge as existing methods are complex, and unfortunately, there are no specific technical standards to follow. Selecting an efficient analytical method is imperative in developing countries, such as Brazil, in order to evaluate the risk of parasite infection. In this context, three methods to detect Giardia spp. cysts and Cryptosporidium spp. oocysts were tested in sludge generated when water with protozoa and high turbidity was treated. Jar testing was carried out using polyaluminium chloride as a coagulant to generate the residue to be analyzed. The results showed that calcium carbonate flocculation with reduced centrifugation and immunomagnetic separation obtained the highest recoveries in the tested matrix showing 60.2% ± 26.2 for oocysts and 46.1% ± 5 for cysts. The other two methods, the first using the ICN 7× cleaning solution and the second considering the acidification of the sample, both followed by the immunomagnetic separation step, also presented high recoveries showing 41.2% ± 43.3 and 37.9% ± 52.9 for oocysts and 11.5% ± 85.5 and 26% ± 16.3 for cysts, respectively. Evidently, these methods and others should be studied in order to make it possible to detect protozoa in settled residue.
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Cryptosporidium/aislamiento & purificación , Agua Potable/parasitología , Monitoreo del Ambiente/métodos , Giardia/aislamiento & purificación , Oocistos/aislamiento & purificación , Hidróxido de Aluminio , Animales , Brasil , Carbonato de Calcio , Floculación , Separación Inmunomagnética , Aguas del Alcantarillado/parasitología , Purificación del Agua/métodos , Calidad del Agua , Abastecimiento de AguaRESUMEN
Abstract Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1 mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18 CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10 h, which is a promising rapid method to detect Salmonella in emergency.
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Animales , Salmonella/aislamiento & purificación , Contaminación de Alimentos , Separación Inmunomagnética/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Microbiología de Alimentos/métodos , Salmonella/genética , Proteínas Bacterianas/inmunología , Sensibilidad y Especificidad , Leche/microbiología , Carne/microbiología , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/metabolismoRESUMEN
microRNA profiling has identified cell-specific expression patterns that could represent molecular signatures triggering the acquisition of a specific phenotype; in other words, of cellular identity and its associated function. Several groups have hypothesized that retinal cell phenotyping could be achieved through the determination of the global pattern of miRNA expression across specific cell types in the adult retina. This is especially relevant for Müller glia in the context of retinal damage, as these cells undergo dramatic changes of gene expression in response to injury, that render them susceptible to acquire a progenitor-like phenotype and be a source of new neurons.We describe a method that combines an experimental protocol for excitotoxic-induced retinal damage through N-methyl-D-aspartate subretinal injection with magnetic-activated cell sorting (MACS) of Müller cells and RNA isolation for microRNA profiling. Comparison of microRNA patterns of expression should allow Müller cell phenotyping under different experimental conditions.
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Células Ependimogliales/metabolismo , Perfilación de la Expresión Génica/métodos , Separación Inmunomagnética/métodos , MicroARNs/metabolismo , Enfermedades de la Retina/patología , Animales , Modelos Animales de Enfermedad , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/patología , Transportador 1 de Aminoácidos Excitadores/inmunología , Perfilación de la Expresión Génica/instrumentación , Humanos , Separación Inmunomagnética/instrumentación , Inyecciones Intravítreas , Ratones , Ratones Endogámicos C57BL , MicroARNs/aislamiento & purificación , N-Metilaspartato/administración & dosificación , N-Metilaspartato/toxicidad , Reacción en Cadena en Tiempo Real de la Polimerasa/instrumentación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Enfermedades de la Retina/inducido químicamente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/instrumentaciónRESUMEN
ABSTRACT We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23 CFU for pure culture, whereas 2.3 × 104 or 2.3 × 106 CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3 × 106 CFU, but PCR was negative at the level of 2.3 × 107 CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3 × 103 or 2.3 × 106 CFU, whereas 2.3 × 105 or 2.3 × 107 CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.
Asunto(s)
Humanos , Peste/microbiología , ADN Bacteriano/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Magnetismo/métodos , Yersinia pestis/aislamiento & purificación , Yersinia pestis/clasificación , Yersinia pestis/genética , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/química , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Separación Inmunomagnética , Cartilla de ADN/genética , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Magnetismo/instrumentaciónRESUMEN
We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23CFU for pure culture, whereas 2.3×104 or 2.3×106CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3×106CFU, but PCR was negative at the level of 2.3×107CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3×103 or 2.3×106CFU, whereas 2.3×105 or 2.3×107CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.
Asunto(s)
ADN Bacteriano/genética , Magnetismo/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Peste/microbiología , Yersinia pestis/aislamiento & purificación , Cartilla de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , Humanos , Separación Inmunomagnética , Magnetismo/instrumentación , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Yersinia pestis/clasificación , Yersinia pestis/genéticaRESUMEN
OBJECTIVE: To evaluate the diagnostic performance of two commercially available ELISA kits, Novalisa® and Ridascreen® , for the detection of antibodies to Taenia solium, compared to serological diagnosis of neurocysticercosis (NCC) by LLGP-EITB (electro-immunotransfer blot assay using lentil-lectin purified glycoprotein antigens). METHODS: Archive serum samples from patients with viable NCC (n = 45) or resolved, calcified NCC (n = 45), as well as sera from patients with other cestode parasites (hymenolepiasis, n = 45 and cystic hydatid disease, n = 45), were evaluated for cysticercosis antibody detection using two ELISA kits, Novalisa® and Ridascreen® . All NCC samples had previously tested positive, and all samples from heterologous infections were negative on LLGP-EITB for cysticercosis. Positive rates were calculated by kit and sample group and compared between the two kits. RESULTS: Compared to LLGP-EITB, the sensitivity of both ELISA assays to detect specific antibodies in patients with viable NCC was low (44.4% and 22.2%), and for calcified NCC, it was only 6.7% and 4.5%. Sera from patients with cystic hydatid disease were highly cross-reactive in both ELISA assays (38/45, 84.4%; and 25/45, 55.6%). Sera from patients with hymenolepiasis cross-reacted in five cases in one of the assays (11.1%) and in only one sample with the second assay (2.2%). CONCLUSIONS: The performance of Novalisa® and Ridascreen® was poor. Antibody ELISA detection cannot be recommended for the diagnosis of neurocysticercosis.
Asunto(s)
Antígenos Helmínticos/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Separación Inmunomagnética/métodos , Neurocisticercosis/diagnóstico , Taenia solium/aislamiento & purificación , Animales , Antígenos Helmínticos/sangre , Pruebas Inmunológicas , Neurocisticercosis/sangre , Neurocisticercosis/parasitología , Sensibilidad y Especificidad , Taenia solium/inmunologíaRESUMEN
Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10h, which is a promising rapid method to detect Salmonella in emergency.