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1.
Anal Bioanal Chem ; 411(25): 6733-6743, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31402423

RESUMO

An immunomagnetic optical probe based on a core/shell magnetic nanoparticle-quantum dot was fabricated for detection of Streptococcus agalactiae, the causative agent of pneumonia and meningitis in newborns. The silica-coated magnetic nanoparticles conjugated with anti-S. agalactiae monoclonal antibody provided high specificity for pre-enrichment of bacteria from biological samples with a complex matrix such as milk. Compared with conventional methods such as culture and molecular techniques, the combination of fluorescent quantum dot and magnetic nanoparticle enhanced the sensitivity and speed of bacterial identification. The bio-functionalized fluorescent-magnetic nanoparticles were characterized by TEM, SEM, VSM, XRD, DLS, and FTIR and applied to the detection of S. agalactiae with a limit of 10 and 102 CFU/mL in PBS and milk, respectively. This immunomagnetic optical probe can be used for rapid isolation, sensitive, and specific detection of targeted bacteria without any treatment in clinical and animal samples in the presence of other infectious agents.


Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Nanopartículas de Magnetita/química , Leite/microbiologia , Streptococcus agalactiae/isolamento & purificação , Animais , Anticorpos Imobilizados/química , Análise de Alimentos/métodos , Separação Imunomagnética/métodos , Nanopartículas de Magnetita/ultraestrutura , Microscopia de Fluorescência/métodos , Pontos Quânticos/química , Pontos Quânticos/ultraestrutura , Dióxido de Silício/química , Espectrometria de Fluorescência/métodos , Infecções Estreptocócicas/microbiologia
2.
J Food Sci ; 84(7): 1881-1887, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31264719

RESUMO

Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real-time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real-time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre-enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50 ) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as "almost complete concordance" between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real-time PCR. PRACTICAL APPLICATION: The methodology described in this study significantly reduces the detection time of L. monocytogenes, when compared with culture-based methods, and it requires fewer steps than other molecular methods, making it a reliable and more convenient method for routine testing. Finally, the evaluation of the methodology in spiked food samples, confirms its reliability.


Assuntos
Produtos Pesqueiros/microbiologia , Separação Imunomagnética/métodos , Listeria monocytogenes/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Salmão/microbiologia , Animais , Produtos Pesqueiros/análise , Microbiologia de Alimentos , Listeria monocytogenes/genética , Reação em Cadeia da Polimerase em Tempo Real , Recombinases/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
3.
Anal Bioanal Chem ; 411(23): 6067-6080, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31273413

RESUMO

Rapid detection of trace Salmonella is urgently needed to ensure food safety. We present an innovative pretreatment strategy, based on a two-step enrichment culture and immunomagnetic separation, combined with a chemiluminescence microparticle immunoassay to detect at least one proliferative Salmonella cell in 25 mL (25 g) food. The capture performance of immunomagnetic beads (IMBs) of sizes for Salmonella was investigated, and the IMBs of size 2.8 µm showed a high capture efficiency of 60.7% in 25 mL milk and 74.5% in 25 mL chicken culture filtrate, which ensured the successful capture of trace Salmonella after 2.5 h in situ enrichment even from only one Salmonella cell. The separated Salmonella cells, reaching an amount of 103 colony-forming units (CFU) by a secondary enrichment for 3 h, were detected by a horseradish peroxidase chemiluminescence reaction with 4-(1-imidazolyl)phenol as an enhancer, which evidenced a linear response for Salmonella concentrations ranging from 2.3 × 102 to 7.8 × 104 CFU/mL. The entire detection process was completed within 8 h, with a very low detection limit of 1 CFU/25 mL (25 g), which was verified by colony counting, and a small degree of interference of 0.17-1.06%. Trace Salmonella from five different serovars in milk and chicken was successfully detected without false negative or false positive results. Furthermore, this study provides a basis to develop a fully automated instrument based on IMBs that includes all steps from sample preparation to chemiluminescence microparticle immunoassay for high-throughput screening of foodborne pathogens. Graphical abstract.


Assuntos
Contaminação de Alimentos/análise , Medições Luminescentes/métodos , Leite/microbiologia , Aves Domésticas/microbiologia , Salmonella/isolamento & purificação , Animais , Galinhas/microbiologia , Contaminação de Alimentos/economia , Inocuidade dos Alimentos/métodos , Imunoensaio/economia , Imunoensaio/métodos , Separação Imunomagnética/economia , Separação Imunomagnética/métodos , Limite de Detecção , Medições Luminescentes/economia , Infecções por Salmonella/microbiologia , Fatores de Tempo
4.
Analyst ; 144(13): 4086-4092, 2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31169832

RESUMO

Given that Helicobacter pylori (H. pylori) generally infects people in early childhood and that such persons when not treated with antibiotics remain infected for the rest of their lives, it is quite important to detect H. pylori in children, and convenient to do so using non-invasive methods. Stool antigen tests constitute such an effective non-invasive method. In the current work, a novel fecal test was developed to detect H. pylori based on immunomagnetic beads (IMBs) with monoclonal antibodies sensitively recognizing and capturing the H. pylori, coupled with a polyclonal antibody-conjugating quantum dot probe, and ultrasensitive detection was achieved by using a fluorescence spectrometer. The detection method took 120 min to perform, and showed a limit of detection of 102 CFU mL-1 and a linear range of 10 to 106 CFU mL-1 (R2 = 0.9962). Most importantly, this method can be effectively applied to real samples. This study provided a novel method for the non-invasive detection of the fecal antigen H. pylori.


Assuntos
Fezes/microbiologia , Helicobacter pylori/isolamento & purificação , Pontos Quânticos/química , Espectrometria de Fluorescência/métodos , Animais , Anticorpos Monoclonais Murinos/imunologia , Cádmio/química , Helicobacter pylori/imunologia , Humanos , Separação Imunomagnética/métodos , Limite de Detecção , Camundongos , Coelhos , Selênio/química , Sulfetos/química , Compostos de Zinco/química
5.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1122-1123: 83-89, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31173996

RESUMO

For magnetic control of cells for biomedical applications such as targeting of immune cells to tumors, cells must be magnetizable. For that, cells are incubated with superparamagnetic iron oxide nanoparticles (SPIONs) to take them up and thus become magnetizable. When using adherent cells, non-ingested SPIONs can be easily removed by rinsing of the particles regardless of their colloidal stability in cell culture medium. However, if suspension cells such as T cells are to be loaded with SPIONs, established methods to separate excess nanoparticles from cells are based on physicochemical parameters such as density, size or magnetizability. Thus, colloidal stability of the particles is of great importance, since only colloidally stable SPIONs can be completely separated from the cells due to their physicochemical differences. Aggregates of colloidally meta- or unstable particles cannot, however, be separated from cells due to their overlapping sizes and densities. Thus, development of an alternative method for the separation of nanoparticle aggregates from suspension cells is urgently needed. Here, we present an affinity chromatographic separation method based on immunohistochemical properties of the respective cells. A desthiobiotinylated antibody against a cellular surface antigen (here CD90.2 receptor on EL4 T cells) is immobilized on a streptavidin agarose column optimized for cell purification. Subsequently the column is loaded with the particle/cell suspension so that the cells bind to the column. After removing the particles by washing, the cells can be gently eluted with biotin solution under physiological conditions. This allows >95% of the excess iron concentration to be removed while maintaining cell viability.


Assuntos
Cromatografia de Afinidade/métodos , Separação Imunomagnética/métodos , Nanopartículas de Magnetita/química , Animais , Anticorpos/metabolismo , Biotina/química , Linhagem Celular , Sobrevivência Celular/fisiologia , Coloides/química , Camundongos , Estreptavidina/química
6.
Anal Chim Acta ; 1067: 98-106, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31047154

RESUMO

The quest for detecting bacteria has gained momentum in food and beverage industry for preventing spoilage of products to maintain requisite quality. The present paper describes the development of a SERS immunosensor for the detection of model pathogen, S. typhimurium using strategically synthesized functionalized polymeric magnetic nanoparticles (FPMNPs) as effective capture probe and immunomagnetic separator. The synthesized probe contains surface diketonic functionalities which covalently link with amino groups of antibodies against Salmonella common structural antigen (CSA-1-Ab) and hence specifically captured the target bacteria. Magnetic core of nanoparticles facilitated easy separation of target bacteria from the milieu of non-specific molecules. Gold nanoparticles (GNPs) modified with CSA-1-Ab and external Raman reporter molecules (RRM) were used as signal probes. We compared the signalling attributes of 4-mercapto benzoic acid (MBA) and 5,5'-dithiobis(succinimidyl-2-nitrobenzoate) (DSNB) as RRMs. Capture and signal probes sandwich the target bacteria upon its addition, generating Raman signal from the 'hot-spots' created by signal probe. Under optimal conditions, the SERS intensities of MBA and DSNB at 1588 and 1336 cm-1 respectively were used to measure the concentration of the pathogen in the range of 101-107 cells mL-1. Limit of detection (LOD) of MBA and DSNB based immunosensor was measured as 100 cells mL-1, and 10 cells mL-1 respectively. Moreover, appreciable recovery (82-114%) was recorded for sensing method for different spiked food products. Thus, the developed magnetically assisted SERS immunosensor is sensitive, specific and has strong potential to be used for detecting contamination in food samples in field conditions.


Assuntos
Contaminação de Alimentos/análise , Separação Imunomagnética , Nanopartículas de Magnetita/química , Polímeros/química , Salmonella typhimurium/isolamento & purificação , Salmonella typhimurium/citologia , Análise Espectral Raman , Propriedades de Superfície
7.
IET Nanobiotechnol ; 13(1): 6-11, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30964030

RESUMO

Circulating tumour cells (CTCs) draw significant attention as a promising biomarker for cancer prognosis, status monitoring, and metastasis diagnosis. However, the concentration of CTCs in peripheral blood is usually extremely low, thereby requiring enrichment followed by isolation of CTCs prior to detection. An immunomagnetic separation is a promising tool for CTCs enrichment. In this study, a cost-effective magnetic separation method, based on streptavidin-biotin complexation, was developed and the effects of magnetic beads' size in CTCs capture were compared. Magnetic nanobeads which were 25 nm in diameter lead to highest capture efficiency (82.2%) compared with 150 nm magnetic beads and 1 µm microbeads. Based on the streptavidin-biotin system, 25 nm magnetic nanobeads could capture model CTCs over 80% efficiency even at concentrations as low as ∼25 cells/mL that may represent the actual level of CTCs in peripheral blood of cancer patients. Furthermore, the isolated cells remained robust and healthy showing insignificant changes in morphology and behaviour when cultured for 24 h immediately after capture and isolation. The magnetic nanobeads based on streptavidin-biotin complexation showed promise for the easy and efficient capture and isolation of healthy CTCs for further diagnosis and analysis.


Assuntos
Proteínas de Bactérias/química , Biotina/análogos & derivados , Separação Imunomagnética/métodos , Nanopartículas de Magnetita/química , Células Neoplásicas Circulantes , Proteínas de Bactérias/metabolismo , Biotina/química , Biotina/metabolismo , Citometria de Fluxo , Humanos , Células K562 , Células MCF-7 , Tamanho da Partícula
8.
Anal Chim Acta ; 1062: 156-164, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-30947992

RESUMO

This study reports on the conception of magneto-Capillary Electrophoresis (magneto-CE), an approach integrating immuno-capture on circulating bio-functionalized magnetic beads into a unique capillary for preconcentration and electrokinetic separation. This hybrid mode is an evolution of in-capillary magnetic bead-based operation from static cluster format to dynamic configuration where beads are allowed to controllably circulate inside a CE capillary for interaction improvement. To implement the magneto-CE operation, a purpose-made instrument was constructed, allowing visual observation of the movement of the magnetic beads. We applied a new methodological strategy for determination of the amyloid ß peptide (Aß 1-42), which is as an established biomarker for molecular diagnosis of Alzheimer's disease (AD). The methodology is based on magneto-immuno-capture of fluorescently labeled Aß 1-42 followed by a chemical elution with a basic solution prior to CE separation with laser induced fluorescent (LIF) detection. The superiority of this dynamic configuration of magneto-CE was demonstrated for this target analyte, with sample pretreatment and separation being performed in-capillary without any delay in between and without any waste of pretreated sample, which otherwise would not be the case with offline/batch-wise operation.


Assuntos
Peptídeos beta-Amiloides/isolamento & purificação , Eletroforese Capilar , Separação Imunomagnética , Campos Magnéticos , Fragmentos de Peptídeos/isolamento & purificação , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/química , Humanos , Fragmentos de Peptídeos/química
9.
Biosens Bioelectron ; 135: 137-144, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31005765

RESUMO

Foodborne illnesses are a major contributor to misery and health challenges in both rich and poor nations. Illnesses from pathogens such as Escherichia coli and Cryptosporidium parvum oocysts account for most of the cases of diarrhea in the world. Many standard methods exist for detecting these pathogens in water. However, these standard methods do not readily translate to the detection of the same pathogens in food. Detection techniques for pathogens in food are often inadequate, due to their inability to completely separate pathogens from food matrices. In this paper, we present a technique to separate and detect both Escherichia coli cells and Cryptosporidium parvum oocysts that have been embedded in ground meat. We achieve this objective by combining enzymatic digestion of the meat, hydrodynamic cavitation to disassemble pathogens from the meat, immunomagnetic separation to purify meat samples and indirect electrochemical detection of the target pathogens. Our use of hydrodynamic cavitation to separate pathogens is compared against an industry standard separation technique. Results indicate that the use of hydrodynamic cavitation amplifies the detection capabilities of our sensing technique and is overall comparable to or better than conventional stomacher sample preparation.


Assuntos
Cryptosporidium parvum/isolamento & purificação , Escherichia coli O157/isolamento & purificação , Análise de Alimentos/métodos , Carne Vermelha/microbiologia , Animais , Técnicas Biossensoriais/economia , Técnicas Biossensoriais/métodos , Bovinos , Criptosporidiose/diagnóstico , Criptosporidiose/microbiologia , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Análise de Alimentos/economia , Contaminação de Alimentos/análise , Doenças Transmitidas por Alimentos/diagnóstico , Doenças Transmitidas por Alimentos/microbiologia , Hidrodinâmica , Separação Imunomagnética/economia , Separação Imunomagnética/métodos , Fatores de Tempo
10.
Food Chem ; 290: 255-262, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31000045

RESUMO

Two small Lagocephalus sceleratus juveniles were captured in picarel targeting catches from North Aegean Sea (Greece) in the autumn of 2017. An electrochemical immunosensing tool using magnetic beads as immobilisation support was developed and applied to the rapid screening of tetrodotoxins (TTXs), potent neurotoxins that constitute a food safety hazard when present in seafood. This tool revealed the presence of TTXs in both individuals. Results were compared with those provided by mELISA and LC-HRMS, the latter confirming the presence of TTX. Some of the tissues contained TTX contents close to or above 2 mg/kg. L. sceleratus juveniles had been considered as non-toxic and, to our knowledge, this is the first report of high TTX levels in small L. sceleratus individuals. Such specimens can be mistaken with other edible species, posing a threat to consumers. The availability of low-cost and user-friendly tools for TTXs detection will contribute to guarantee seafood safety.


Assuntos
Técnicas Eletroquímicas/métodos , Tetraodontiformes/metabolismo , Tetrodotoxina/análise , Animais , Bactérias/isolamento & purificação , Grécia , Separação Imunomagnética/métodos , Oceanos e Mares , Alimentos Marinhos/análise , Alimentos Marinhos/microbiologia , Tetraodontiformes/crescimento & desenvolvimento , Tetrodotoxina/isolamento & purificação
11.
Anal Bioanal Chem ; 411(19): 4951-4961, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30982928

RESUMO

Immunomagnetic separation (IMS) was combined with flow-based chemiluminescence sandwich immunoassays (CL-SIA) for the quantification of Staphylococcal enterotoxin B in milk. Therefore, iron oxide-shell silica-core magnetic nanocomposites were conjugated to biotinylated anti-SEB antibodies (MNC-IgGs). MNC-IgGs were applied successfully for (i) capturing SEB in milk samples by an affinity reaction, (ii) magnetophoretic collection on antibody spots in a channel of a flow-based immunochip, and (iii) sensitive enzymatic chemiluminescence detection of biotin labels by poly(horseradish peroxidase)-streptavidin. IMS was performed in 0.6 mL and 100 mL milk samples resulting in detection limits of 50 ng L-1 and 0.39 ng L-1, respectively, for the combined analytical method. It was shown that the assay sensitivity was dramatically improved by the combination of IMS with flow-based CL-SIA compared to CL-SIA directly applied with milk samples (detection limit 130 ng L-1). The IMS-CL-SIA has a time-to-result of 2-3 h. The reported combined analytical method can be used for a rapid control of SEB in complex food matrices such as milk. In future, even the monitoring of multiple contaminants in food or water may be performed by IMS-CL-SIA. Graphical abstract.


Assuntos
Enterotoxinas/análise , Imunoensaio/métodos , Separação Imunomagnética/instrumentação , Luminescência , Magnetismo , Leite/química , Nanocompostos/química , Staphylococcus aureus/química , Superantígenos/análise , Animais , Automação , Biotina/análise , Microbiologia de Alimentos/métodos , Peroxidase do Rábano Silvestre/química , Limite de Detecção , Estreptavidina/química
12.
Int J Food Microbiol ; 298: 31-38, 2019 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-30903916

RESUMO

Marine bivalve shellfish are of public health interest because they can accumulate pollutants in their tissues. As they are usually consumed raw or lightly cooked, they are considered to be a possible source of foodborne infections, including giardiosis and cryptosporidiosis. Although data indicating contamination of shellfish with Giardia cysts and Cryptosporidium oocysts have been published, comparing results from different studies is difficult, as there is no standardized protocol for the detection and quantification of these parasites in mussels, and different researchers have used different analytical approaches. The aim of this study was to identify and characterize the most sensitive protocol for the detection of Giardia cysts and Cryptosporidium oocysts in shellfish. In an effort to test the sensitivity and the detection limits of the protocol, every step of the process was investigated, from initial preparation of the mussel matrix through detection of the parasites. Comparative studies were conducted, including several methods previously applied by other researchers, on commercial mussels Mytilus galloprovincialis spiked with a known number of (oo)cysts of both parasites. As preparation of the mussel matrix plays an important role in the sensitivity of the method, different techniques were tested. These included: (ia) removal of the coarse particles from the matrix with sieving, (ib) extraction of the lipids with diethyl ether, and (ic) artificial digestion of the matrix with pepsin digestion solution, and (ii) the use or not of immunomagnetic separation (IMS) for the concentration of the (oo)cysts. Pre-treatment of the mussel homogenate with pepsin digestion solution, followed by IMS, then detection with a direct immunofluorescence assay, achieved the highest sensitivity: 32.1% (SD: 21.1) of Giardia cysts and 61.4% (SD: 26.2) Cryptosporidium oocysts were recovered, with a detection limit of 10 (oo)cysts per g of mussel homogenate. The outcome of the current study was the standardization of a protocol, with defined detection limits, for the detection of these two protozoan transmission stages in mussels, in order to be used as a reference technique in future studies. Further advantages of this protocol are that it uses the whole mussel as a starting material and does not require difficult handling procedures. The method has potential to be applied in larger surveys and, potentially, to other species of shellfish for the detection of these parasites. However, the composition (lipid to protein ratio) may be of relevance for detection efficiency for some other species of shellfish.


Assuntos
Cryptosporidium/isolamento & purificação , Técnica Direta de Fluorescência para Anticorpo/normas , Microbiologia de Alimentos/métodos , Giardia/isolamento & purificação , Mytilus/microbiologia , Oocistos/isolamento & purificação , Frutos do Mar/microbiologia , Animais , Criptosporidiose/prevenção & controle , Giardíase/prevenção & controle , Separação Imunomagnética , Mar Mediterrâneo
13.
Int J Food Microbiol ; 296: 14-20, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-30826538

RESUMO

Growing demand for fresh, unprocessed food favours the emergence of Cryptosporidium infections in humans. Mainly it is food of plant origin or unpasteurized milk which have been involved in food-borne outbreaks of cryptosporidiosis. So far consumption of shellfish contaminated with Cryptosporidium were not associated with human infections although such as possibility exists. In this study an attempt was undertaken to evaluate the analytical performance of three commonly used methods for recovery of Cryptosporidium oocysts from shellfish: i) pepsin digestion of shellfish in conjunction with immunomagnetic separation (IMS) of oocysts (method A), ii) pepsin-HCl treatment of shellfish homogenate without IMS (method B), and iii) a strainer method with direct oocyst extraction and separation from shellfish tissue using IMS (method C). Each method's performance was assessed according to the ISO standard requirements by testing shellfish homogenates seeded with different numbers of C. parvum oocysts. Two groups of parameters were compared, encompassing precision (coefficient of variation (CV)) and accuracy of measurements. These were described by linear regression models allowing calculation of the methods' limits of detection (LOD) and quantification (LOQ). In addition, oocyst recovery efficiencies from shellfish were calculated for each method. All three compared methods allowed for at least 66% recovery of Cryptosporidium oocysts from the tested samples. The best recovery (83.3-100%) in the whole range of tested suspensions was obtained for method C. The accuracy of method B was better (linearity of r2 = 0.9996 in the full measurement range) than that of method A (r2 = 0.968). Method C showed the best accuracy (r2 = 1) and precision (CV 0.2-14.1). Compared to other methods it was also characterised by the best LOD and LOQ, attaining ≅4 and ≅12 oocysts per 3 g of tested shellfish sample respectively. Despite a lack of the ability of method A to give the proportional results in oocysts recovery (non-linearity of the method) compared to the reference values, it achieved the highest LOD and LOQ values among the tested methods. As demonstrated here, the most efficient method for extraction of Cryptosporidium oocysts from shellfish tissues was method C employing sample homogenisation and separation of oocysts from tissue debris using IMS. Used alone this method does not in fact allow for identification of Cryptosporidium species but delivers quantitative results concerning the level of food contamination by parasites.


Assuntos
Criptosporidiose/prevenção & controle , Cryptosporidium parvum/crescimento & desenvolvimento , Contaminação de Alimentos/análise , Oocistos/isolamento & purificação , Frutos do Mar/parasitologia , Animais , Filtração/métodos , Imunofluorescência , Humanos , Separação Imunomagnética/métodos , Limite de Detecção , Pepsina A/metabolismo
14.
Biosens Bioelectron ; 129: 175-181, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30710755

RESUMO

White blood cells (WBCs) isolated from peripheral blood have been verified as important biomarkers for the diagnosis, treatment and prognosis of cancer. However, it's still under challenge to acquire high-purity WBCs, even by taking advantage of current microfluidic technology. Considering the universality of clinical magnetic activated cell sorting (MACS) method, new developments on microfluidic chip in combination of magnetic cells separation technologies may provide a fascinating approach for high-purity WBCs sorting and widely clinical application. Here, we present a flyover style microfluidic chip which has been elaborately embedded with two-stage magnetic separation in continuous flow for WBCs sorting. Immunomagnetic micro/nano-particles (IMNPs) labeled WBC (WBC@IMNPs) were sequentially separated by a lateral magnetic force and a vertical magnetic force, and the final separation purity of WBCs reached up to 93 ±â€¯1.67% at a flow rate of 20 µL min-1. Furthermore, the WBCs viability was up to 97.5 ±â€¯1.8%. Consequently, this novel flyover style microfluidic-chip with magnetic separation technology has been successfully demonstrated as cut-in-edge method for high-purity WBCs sorting, and obviously it's easy to extend for other types of cells sorting under great potential application in biomedical fields.


Assuntos
Separação Imunomagnética/instrumentação , Dispositivos Lab-On-A-Chip , Leucócitos/citologia , Animais , Sobrevivência Celular , Desenho de Equipamento , Campos Magnéticos , Camundongos Endogâmicos BALB C , Níquel/química
15.
Methods Mol Biol ; 1920: 317-326, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30737700

RESUMO

Germ cell transplantation technology has played a critical role in germline modification and preservation of genetic resources. Several germ cell transplantation systems have been developed, including sperm, oocyte, or germline stem cell transplantation systems in mammals. Meanwhile, in avian species, this has mostly relied on primordial germ cell (PGC) transplantation for efficient germline transmission. In this chapter, we describe how to isolate PGCs from avian embryos and produce germline chimeras through transplantation of donor PGCs to recipient embryos.


Assuntos
Transplante de Células , Galinhas , Células Germinativas/citologia , Células Germinativas/transplante , Animais , Movimento Celular , Separação Celular/métodos , Transplante de Células/métodos , Cruzamentos Genéticos , Embrião não Mamífero , Imunofluorescência , Imuno-Histoquímica , Separação Imunomagnética/métodos
16.
PLoS One ; 14(2): e0211866, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30735560

RESUMO

Personalised medicine targeted to specific biomarkers such as BRAF and c-Kit has radically improved the success of melanoma therapy. More recently, further advances have been made using therapies targeting the immune response. In particular, therapies targeting the PD-1/PD-L1 or CTLA-4 axes alone or in combination have shown more sustained responses in 30-60% of patients. However, these therapies are associated with considerable toxicities and useful biomarkers to predict responders and non-responders are slow to emerge. Here we developed a reliable melanoma circulating tumor cell (CTC) detection method with PD-L1 evaluation on CTCs. A set of melanoma cell surface markers was tested as candidates for targeted melanoma CTC isolation and a melanoma specific immunostaining-based CTC identification protocol combined with PD-L1 detection was established. In vitro testing of the effect of exposure to blood cells on melanoma cell PD-L1 expression was undertaken. Immunomagnetic targeting isolated melanoma CTCs in up to 87.5% of stage IV melanoma patient blood samples and 3 8.6% of these had some PD-L1 expressing CTCs. Our in vitro data demonstrate PD-L1 induction on melanoma cells in the blood.This study established a robust, reliable method to isolate melanoma CTCs and detect expression of PD-L1 on these cells.


Assuntos
Antígeno B7-H1/genética , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Separação Imunomagnética/métodos , Neoplasias Pulmonares/diagnóstico , Melanoma/diagnóstico , Neoplasias Cutâneas/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Antígeno B7-H1/sangue , Biomarcadores Tumorais/sangue , Antígeno CTLA-4/sangue , Antígeno CTLA-4/genética , Feminino , Humanos , Imunofenotipagem , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Masculino , Melanoma/sangue , Melanoma/genética , Melanoma/secundário , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Células Neoplásicas Circulantes/imunologia , Células Neoplásicas Circulantes/patologia , Receptor de Morte Celular Programada 1/sangue , Receptor de Morte Celular Programada 1/genética , Neoplasias Cutâneas/sangue , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia
17.
Sheng Wu Gong Cheng Xue Bao ; 35(1): 150-158, 2019 Jan 25.
Artigo em Chinês | MEDLINE | ID: mdl-30756544

RESUMO

Human lipocalin 6 (hLCN6) is an epididymis-specific secretory protein. It binds to sperm and plays important role in sperm maturation. To explore the feasibility for isolating spermatozoa from mixed cells using anti-hLCN6 monoclonal antibody-conjugated immunomagnetic beads (anti-hLCN6 IMBs) and establish a new method for the separation of sperms from mixed stains, 2 sets of 30 cases of cell mixture suspensions and stains containing different proportions of sperm and epithelial cells were prepared. Biotin-labeled anti-hLCN6 monoclonal antibody (mAb) was incubated with the cell mixtures, and the spermatozoa were then isolated with avidin-coated IMBs. Sperm DNA was extracted and analyzed by PCR-STR typing. Differential lysis was also conducted to compare the effect of the two different isolation methods. The dissociation constant (Kd) of anti-hLCN6 mAb was 3.47×10⁻9 mol/L measured by ELISA. Western blotting and immunofluorescence assays showed that hLCN6 was detectable on sperm cells and mainly located on the post-acrosomal region of the sperm head, but not in epithelial cells. Anti-hLCN6 IMBs could capture and separate the sperm cells successfully. Microscopic observation showed that the IMBs could bind to the head of sperm specifically. The success rate of STR typing (more than 13 STR loci, RFU>200) was 90% when the number of sperm cells was 10³/mL and 100% when the sperm cells number was equal to or more than 104/mL. When the number of sperm cells was 10³/mL, 104/mL and 105/mL in mixed stain samples, the success rate of STR typing were 40%, 90% and 100%, respectively. Taken together, the anti-hLCN6 immunomagnetic beads (IMB) method described here could be effective for the isolation of sperm from mixed cells, and the success rate was higher than that of the traditional differential lysis strategy. IMB sorting is a simple and efficient method for the separation of sperms from sperm and epithelial cell mixture, and can be utilized as a supplementary method for forensic mixture samples analysis in sexual assault cases.


Assuntos
Espermatozoides , Separação Celular , DNA , Humanos , Separação Imunomagnética , Lipocalinas , Masculino , Reação em Cadeia da Polimerase
18.
Methods Mol Biol ; 1899: 67-83, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30649766

RESUMO

This SOP describes a closed system for isolating GMP-grade CD4+CD25+ T cells from non-mobilized leukapheresis collections (nMLCs), independent of a clean room in a certified GMP premises, by using CliniMACS format GMP grade reagents (CD25-labeled magnetic beads with/without pre-depletion of CD8+ T cells and CD19+ B cells), a GMP grade-A laminar hood and CliniMACS cell processing system.


Assuntos
Separação Celular/métodos , Separação Imunomagnética/métodos , Leucaférese/métodos , Linfócitos T/citologia , Antígenos CD19/imunologia , Linfócitos B/imunologia , Antígenos CD4/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Subunidade alfa de Receptor de Interleucina-2/imunologia , Linfócitos T/imunologia
19.
J Sep Sci ; 42(7): 1423-1431, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30667151

RESUMO

Okadaic acid is a marine biotoxin that primarily occurs in shellfish and can cause diarrheic shellfish poisoning in humans. When analyzing biological samples using liquid chromatography with tandem mass spectrometry, the presence of complex matrices is a major issue. Thus, it is crucial to selectively and simply extract the target analyte from samples and minimize matrix effects simultaneously. To meet this need, an immunomagnetic-bead-based liquid chromatography with tandem mass spectrometry method was developed to detect okadaic acid in shellfish. Magnetic beads bound to monoclonal antibody against okadaic acid were used as affinity probes to specifically enrich okadaic acid in samples, which effectively eliminated matrix effects. A magnetic separator was used to aggregate and separate magnetic particles from sample matrices, and methanol was used to elute okadaic acid from the magnetic beads. Standard solution prepared with methanol was employed directly for quantitative analysis. Several experimental conditions were optimized to improve performance. The method is of interest as a rapid (10 min) sample clean-up and selective enrichment tool, and it showed good linearity and sensitivity, with reported limits of detection and quantitation of 3 and 10 µg/kg, respectively. Fifty-three shellfish samples from an aquatic products market were tested using this method, and four samples positive for okadaic acid were found.


Assuntos
Separação Imunomagnética , Ácido Okadáico/análise , Frutos do Mar/análise , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Cromatografia Líquida , Ácido Okadáico/imunologia , Espectrometria de Massas em Tandem
20.
Fish Shellfish Immunol ; 87: 96-104, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30633961

RESUMO

Granulocytes are known as the main immunocompetent hemocytes that play important roles in the immune defense of oyster Crassostrea gigas. In the present study, an alcohol acyltransferase (designed as CgAATase) with specific expression pattern was identified from oyster C. gigas, and it could be employed as a potential marker for the isolation of oyster granulocytes. The open reading frame (ORF) of CgAATase was of 1431 bp, encoding a peptide of 476 amino acids with a typically conserved AATase domain. The mRNA transcripts of CgAATase were highest expressed in hemocytes, lower expressed in hepatopancreas, mantle, gonad, gill, ganglion, adductor muscle, and labial palp. The mRNA expression level of CgAATase in hemocytes was significantly up-regulated at 3-12 h and reached the highest level (27.40-fold compared to control group, p < 0.05) at 6 h after Vibrio splendidus stimulation. The total hemocytes were sorted as granulocytes, semi-granulocytes and agranulocytes by Percoll® density gradient centrifugation. CgAATase transcripts were dominantly observed in granulocytes, which was 8.26-fold (p < 0.05) and 2.80-fold (p < 0.05) of that in agranulocytes and semi-granulocytes, respectively. The monoclonal antibody against CgAATase was produced and employed for the isolation of granulocytes with the immunomagnetic bead. CgAATase protein was mainly detected on the cytomembrane of granulocytes. About 85.7 ±â€¯4.60% of the granulocytes were positive for CgAATase and they could be successfully separated by flow cytometry with immunomagnetic bead coated with anti-CgAATase monoclonal antibody, and 97.7 ±â€¯1.01% of the rest hemocytes (agranulocytes and semi-granulocytes) were negative for CgAATase. The isolated primary granulocytes could maintain cell activity for more than one week in vitro culture that exhibited numerous filopodia. These results collectively suggested that CgAATase was a potential marker of oyster granulocytes, and the granulocytes could be effectively isolated from total circulating hemocytes by immunomagnetic bead coated with the anti-CgAATase monoclonal antibody.


Assuntos
Crassostrea/imunologia , Granulócitos/imunologia , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Crassostrea/citologia , Crassostrea/enzimologia , Citometria de Fluxo/métodos , Granulócitos/citologia , Granulócitos/enzimologia , Hemócitos/citologia , Separação Imunomagnética/métodos , Proteínas/genética , Vibrio/imunologia
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