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1.
J Biochem ; 166(3): 205-212, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31251348

RESUMO

Signal enhancing systems have been introduced to enable detection of cell surface antigens by flow cytometry. Cell surface antigens are important targets that describe the function and lineage of cells. Although flow cytometry is an effective tool for analysing cell surface antigens, this technique has poor sensitivity, which prohibits the detection of many important antigens on cell membranes. Thus, signal amplification is essential for developing practical tools for evaluating cell surface antigens by flow cytometry. Using a bright fluorophore or fluorescent polymer incorporated into antibodies is a straightforward strategy to improve flow cytometry sensitivity but may affect the functional characteristics of the labelled antibody. In contrast, enzymatic signal amplification is a more practical and efficient strategy to improve sensitivity that should not affect antibody activity. Although enzymatic signal amplification still has a number of drawbacks, this approach is a promising strategy to analyse cell surface antigens.


Assuntos
Antígenos de Superfície/análise , Citometria de Fluxo , Anticorpos/química , Anticorpos/imunologia , Anticorpos/metabolismo , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Polímeros/química , Polímeros/metabolismo
2.
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
3.
Int J Nanomedicine ; 14: 2451-2464, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31040668

RESUMO

Background: Acute myocardial infarction (AMI), usually caused by atherosclerosis of coronary artery, is the most severe manifestation of coronary artery disease which results in a large amount of death annually. A new diagnosis approach with high accuracy, reliability and low measuring-time-consuming is essential for AMI quick diagnosis. Purpose: The objective of this study was to develop a new point-of-care testing system with high accuracy and reliability for AMI quick diagnosis. Patients and methods: 50 plasma samples of acute myocardial infarction patients were analyzed by developed Smartphone-Assisted Pressure-Measuring-Based Diagnosis System (SPDS). The concentration of substrate was firstly optimized. The effect of antibody labeling and matrix solution on measuring result were then evaluated. And standard curves for cTnI, CK-MB and Myo were built for clinical sample analysis. The measuring results of 50 clinical samples were finally evaluated by comparing with the measuring result obtained by CLIA. Results: The concentration of substrate H2O2 was firstly optimized as 30% to increase measuring signal. A commercial serum matrix was chosen as the matrix solution to dilute biomarkers for standard curve building to minimize matrix effect on the accuracy of clinical plasma sample measuring. The standard curves for cTnI, CK-MB and Myo were built, with measuring dynamic range of 0-25 ng/mL, 0-33 ng/mL and 0-250 ng/mL, and limit of detection of 0.014 ng/mL, 0.16 ng/mL and 0.85 ng/mL respectively. The measuring results obtained by the developed system of 50 clinical plasma samples for three biomarkers matched well with the results obtained by chemiluminescent immunoassay. Conclusion: Due to its small device size, high sensitivity and accuracy, SPDS showed a bright potential for point-of-care testing (POCT) applications.


Assuntos
Pressão Sanguínea , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/fisiopatologia , Smartphone , Anticorpos/metabolismo , Biomarcadores/sangue , Catálise , Feminino , Humanos , Peróxido de Hidrogênio/análise , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Nanopartículas/química , Platina/química , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Eletricidade Estática
4.
Int J Mol Sci ; 20(9)2019 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-31083520

RESUMO

Alpha-synuclein is considered the major pathological protein associated with Parkinson's disease, but there is still no effective immunotherapy which targets alpha-synuclein. In order to create a safer and more effective therapy against PD, we are targeting an epitope of alpha-synuclein rather than full-length alpha-synuclein. We have selected several antigenic domains (B-cell epitope) through antigenicity prediction, and also made several recombinant protein fragments from alpha-synuclein upon antigenicity prediction in an E. coli system. We then tested the function of each of the peptides and recombinant fragments in aggregation, their toxicity and antigenicity. We have discovered that the full-length recombinant (aa1-140) can aggregate into oligomers or even fibrils, and fragment aa15-65 can promote the aggregation of aa1-140. It is worth noting that it not only promotes whole protein aggregation, but also self-aggregates as seen by western blotting and silver staining assays. We have tested all candidates on primary neurons for their toxicity and discovered that aa15-65 is the most toxic domain compared to all other fragments. The antibody targeting this domain also showed both anti-aggregation activity and some therapeutic effect. Therefore, we believe that we have identified the most potent therapeutic domain of alpha synuclein as a therapeutic target.


Assuntos
Doença de Parkinson/tratamento farmacológico , alfa-Sinucleína/química , alfa-Sinucleína/uso terapêutico , Animais , Anticorpos/metabolismo , Mapeamento de Epitopos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/toxicidade , Agregados Proteicos , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidade
5.
Int J Nanomedicine ; 14: 2829-2846, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31114197

RESUMO

Background: Vitamin D3 possesses anti-inflammatory and modulatory properties in addition to its role in calcium and phosphate homeostasis. Upon activation, macrophages (M) can initiate and sustain pro-inflammatory cytokine production in inflammatory disorders and play a pathogenic role in certain cancers. Purpose: The main purpose of this study was to encapsulate and specifically target calcitriol to macrophages and investigate the anti-inflammatory properties of calcitriol in vitro and in vivo. Methods: In this study we have designed and developed near-infrared calcitriol PEGylated nanoparticles (PEG-LNP(Cal)) using a microfluidic mixing technique and modified lipid nanoparticles (LNPs) to target the M specific endocytic receptor CD163. We have investigated LNP cellular uptake and anti-inflammatory effect in LPS-induced M in vitro by flow cytometry, confocal microscopy and gene expression analyses. LNP pharmacodynamics, bio-distribution and organ specific LNP accumulation was also investigated in mice in vivo. Results: In vitro, we observed the specific uptake of PEG-LNP(Cal)-hCD163 in human M, which was significantly higher than the non-specific uptake of control PEG-LNP(Cal)-IgG(h) in M. Pretreatment with encapsulated calcitriol was able to attenuate intracellular TNF-expression, and M surface marker HLA-DR expression more efficiently than free calcitriol in LPS-induced M in vitro. Encapsulated calcitriol diminished mRNA gene levels of TNF-, NF-B, MCP-1 and IL-6, while upregulating IL-10. TNF- and IL-6 protein secretion also decreased. In mice, an in vivo pharmacodynamic study of PEG-LNP(Cal) showed a rapid clearance of IgG and CD163 modified LNPs compared to PEG-LNP(Cal). Antibody modified PEG-LNP(Cal) accumulated in the liver, spleen and kidney, whereas unmodified PEG-LNP(Cal) accumulation was only observed in the liver. Conclusion: Our results show that calcitriol can be effectively targeted to M. Our data confirms the anti-inflammatory properties of calcitriol and this may be a potential way to deliver high dose bioactive calcitriol to M during inflammation in vivo.


Assuntos
Anti-Inflamatórios/farmacologia , Calcitriol/administração & dosagem , Calcitriol/farmacologia , Lipídeos/química , Macrófagos/metabolismo , Nanopartículas/química , Animais , Anticorpos/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Calcitriol/farmacocinética , Quimiocinas/metabolismo , Composição de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Tamanho da Partícula , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismo , Distribuição Tecidual/efeitos dos fármacos
6.
Yonsei Med J ; 60(6): 509-516, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31124333

RESUMO

PURPOSE: This study was conducted to verify the induction and mechanism of selective apoptosis in G361 melanoma cells using anti-HER2 antibody-conjugated gold nanoparticles (GNP-HER2). MATERIALS AND METHODS: Following GNP-HER2 treatment of G361 cells, cell cycle arrest and apoptosis were measured by WST-1 assay, Hemacolor staining, Hoechst staining, immunofluorescence staining, fluorescence-activated cell sorting analysis, and Western blotting. RESULTS: G361 cells treated with GNP-HER2 showed condensation of nuclei, which is an apoptotic phenomenon, and translocation of apoptosis-inducing factor and cytochrome c from mitochondria into the nucleus and cytoplasm, respectively. Increases in BAX in cells undergoing apoptosis, activation of caspase-3 and -9, and fragmentation of poly (ADP-ribose) polymerase and DNA fragmentation factor 45 (inhibitor of caspase-activated DNase) were observed upon GNP-HER2 treatment. Following GNP-HER2 treatment, an increase of cells in sub-G1 phase, which is a signal of cell apoptosis, was observed. This resulted in the down-regulation of cyclin A, cyclin D1, cyclin E, cdk2, cdk4, and cdc2 and the up-regulation of p21. Thus, GNP-HER2 treatment was confirmed to induce the cessation of cell cycle progression. Also, decreases in phospho-focal adhesion kinase and phospho-human epidermal growth factor receptor, which activate cellular focal adhesion, and decreases in phospho-paxillin, which stimulates the disassembly of filamentous actin, were observed. Reduced cell adhesion and disassembly of the intracellular structure indicated cell deactivation. CONCLUSION: GNP-HER2 can selectively kill G361 melanoma cells without affecting normal cells. The mechanism of G361 cell death upon treatment with GNP-HER2 was apoptosis accompanied by activation of caspases.


Assuntos
Anticorpos/metabolismo , Apoptose , Ouro/química , Melanoma/patologia , Nanopartículas Metálicas/química , Receptor ErbB-2/metabolismo , Actinas/metabolismo , Fator de Indução de Apoptose/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Forma Celular , Citocromos c/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Fase G1 , Humanos , Nanopartículas Metálicas/ultraestrutura
7.
Mater Sci Eng C Mater Biol Appl ; 100: 424-432, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30948078

RESUMO

This work reports on the development of a label-free immunosensor technology, based on nanoplasmonic Au-TiO2 thin films. The Au-TiO2 thin films were prepared by cost-effective reactive DC magnetron sputtering, followed by a thermal annealing procedure. The latter promoted the growth of the Au nanoparticles throughout the TiO2 matrix and induced some morphological changes, which are the base for the immunosensor device functionality. A posterior plasma etching treatment was required to partially expose the nanoparticles to the biological environment. It gave rise to a 6-fold increase of the total area of gold exposed, allowing further possibilities for the sensor sensitivity enhancement. Experimental results demonstrated the successful functionalization of the films' surface with antibodies, with the immobilization occurring preferentially in the exposed nanoparticles and negligibly on the TiO2 matrix. Antibody adsorption surface coverage studies revealed antibody low affinity to the film's surface. Nevertheless, immunoassay development experiments showed a strong and active immobilized antibody monolayer at an optimized antibody concentration. This allowed a 236 signal-to-noise-ratio in a confocal microscope, using mouse IgG and 100 ng/ml of Fab-specific anti-mouse IgG-FITC conjugated. Label-free detection of the optimized antibody monolayer on Au-TiO2 thin films was also tested, revealing an expected redshift in the LSPR band, which demonstrates the suitability for the development of cost-effective, label-free LSPR based immunosensor devices.


Assuntos
Técnicas Biossensoriais/métodos , Ouro/química , Imunoensaio/métodos , Coloração e Rotulagem , Titânio/química , Adsorção , Animais , Anticorpos/metabolismo , Proteínas Imobilizadas/metabolismo , Camundongos , Nanopartículas/química , Nanopartículas/ultraestrutura , Fenômenos Ópticos , Propriedades de Superfície
8.
Mater Sci Eng C Mater Biol Appl ; 100: 23-29, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30948057

RESUMO

Emulsions are crucial in the treatment of snake bites to bust the antibody response of the inmunogen. The widely used Freund's emulsion typically combines 50/50 water-oil (W/O) phase. However, its use is limited because it is associated with tissue damage. We formulated and characterized a Pickering Emulsion 70/30 (W/O) that uses a chemically modified hydrophobic hydroxyapatite as surfactant. This Pickering emulsion has similar rheologic behavior to Freund's emulsion 50/50, but with lower oil and surfactant concentration. Evaluation of cell recruitment, antibody response and adhering tissue in mice immunized with B. asper of Pacific venom and treated with Freund's and Pickering 70/30 emulsions resulted in similar adjuvant activity (only 18% lower in Pickering 70/30 emulsion). However, Pickering 70/30 emulsions minimized negative side effects in the host animals and showed better ease of flow that favors injection of the host. Our results open up room for optimization and improvement of Pickering emulsion based on modified nanoparticles for medical applications.


Assuntos
Adjuvantes Imunológicos/química , Anticorpos/metabolismo , Durapatita/química , Emulsões/química , Nanopartículas/química , Venenos de Serpentes/imunologia , Animais , Camundongos , Venenos de Serpentes/química , Serpentes/metabolismo , Tensoativos/química
9.
IET Nanobiotechnol ; 13(1): 90-99, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30964044

RESUMO

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein involved in cell proliferation and differentiation. Ribosomal inactivating proteins derived from plants specifically target ribosomes and irreversibly inhibit protein synthesis. EpCAM antibody and saporin were conjugated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide chemistry. The mass of the conjugates were characterised using matrix-assisted laser desorption ionisation (MALDI). The saporin-EpCAM (SAP-EpAB) conjugates were tested in-vitro against MCF-7 (breast cancer cells), WERI-Rb1 (retinoblastoma) cells. The flow cytometry and fluorescence microscopy were performed to show the binding efficiency of SAP-EpAB conjugate. Whole transcriptome changes of sap-conjugate treated cells were studied using affymetrix microarrays. MALDI-TOF analysis and polyacrylamide gel electrophoresis confirmed the conjugation of SAP with EpCAM antibody. Flow cytometry and fluorescent microscopy analysis revealed the binding of SAP-EpAB conjugates to the MCF-7, WERI-Rb1 cells. Apoptosis assay by annexin-V has shown an increased apoptotic and necrotic population in conjugate treated cells. MTT assay confirmed the tumour cell death and had shown the IC50 value of 0.8 µg for conjugate in MCF-7 (breast cancer cells), and 1 µg for WERI-Rb1 (retinoblastoma) cells. The microarray analysis revealed downregulation of the tumourigenic genes and upregulation of pro-apoptotic genes leading to apoptosis of tumour cells.


Assuntos
Anticorpos/metabolismo , Antineoplásicos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Molécula de Adesão da Célula Epitelial/metabolismo , Saporinas/química , Anticorpos/química , Anticorpos/imunologia , Anticorpos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/imunologia , Humanos , Células MCF-7 , Saporinas/metabolismo
10.
Mater Sci Eng C Mater Biol Appl ; 101: 88-91, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31029367

RESUMO

Cancer Biomarkers are important biological molecules which provide early detection, diagnosis and classification of cancer cells. In this work, nanoparticle tags with different redox potentials have been used as an electrochemical coding technology for the simultaneous detection of carcinoembryonic antigen (CEA), vascular endothelial growth factor (VEGF) and α-fetoprotein (AFP) biomarkers. As far as we know, this is the only work that covers the simultaneous detections of these biomarkers. For this purpose, basically, an electrochemical sandwich immunosensor, which was fabricated from carbon screen printed electrode, was utilized as a diagnostic platform. As nanoparticle tags, PbAu@É£Fe2O3, CuAu@É£Fe2O3 and ZnAu@É£Fe2O3 hybrid nanolabels were synthesized and used for labelling anti-CEA, anti-VEGF and anti-AFP, respectively. By monitoring differential pulse voltammetric oxidation peaks of labeling metals (Pb, Cu, Zn), CEA, VEGF and AFP biomarkers were detected at the same time.


Assuntos
Biomarcadores Tumorais/análise , Técnicas Biossensoriais/métodos , Antígeno Carcinoembrionário/análise , Imunoensaio/métodos , Fator A de Crescimento do Endotélio Vascular/análise , alfa-Fetoproteínas/análise , Anticorpos/metabolismo , Espectroscopia Dielétrica , Técnicas Eletroquímicas , Humanos , Nanopartículas/química , Oxirredução
11.
Int J Mol Sci ; 20(4)2019 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-30813492

RESUMO

AGPase catalyzes a key rate-limiting step that converts ATP and Glc-1-p into ADP-glucose and diphosphate in maize starch biosynthesis. Previous studies suggest that AGPase is modulated by redox, thermal and allosteric regulation. However, the phosphorylation of AGPase is unclear in the kernel starch biosynthesis process. Phos-tagTM technology is a novel method using phos-tagTM agarose beads for separation, purification, and detection of phosphorylated proteins. Here we identified phos-tagTM agarose binding proteins from maize endosperm. Results showed a total of 1733 proteins identified from 10,678 distinct peptides. Interestingly, a total of 21 unique peptides for AGPase sub-unit Brittle-2 (Bt2) were identified. Bt2 was demonstrated by immunoblot when enriched maize endosperm protein with phos-tagTM agarose was in different pollination stages. In contrast, Bt2 would lose binding to phos-tagTM when samples were treated with alkaline phosphatase (ALP). Furthermore, Bt2 could be detected by Pro-Q diamond staining specifically for phosphorylated protein. We further identified the phosphorylation sites of Bt2 at Ser10, Thr451, and Thr462 by iTRAQ. In addition, dephosphorylation of Bt2 decreased the activity of AGPase in the native gel assay through ALP treatment. Taking together, these results strongly suggest that the phosphorylation of AGPase may be a new model to regulate AGPase activity in the starch biosynthesis process.


Assuntos
Endosperma/metabolismo , Glucose-1-Fosfato Adenililtransferase/metabolismo , Proteínas de Plantas/metabolismo , Subunidades Proteicas/metabolismo , Proteômica/métodos , Amido/biossíntese , Zea mays/metabolismo , Sequência de Aminoácidos , Anticorpos/metabolismo , Modelos Biológicos , Fosforilação , Proteínas de Plantas/química , Sefarose
12.
Mol Cell ; 73(5): 1075-1082.e4, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30849388

RESUMO

High-throughput DNA sequencing techniques have enabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3 × 104 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a "superFLAG" epitope variant. We also measured the enzymatic activity of 1.56 × 105 molecular variants of full-length human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates revealed amino acid interaction networks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues.


Assuntos
Anticorpos/metabolismo , Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oligopeptídeos/metabolismo , Análise Serial de Proteínas/métodos , Afinidade de Anticorpos , Especificidade de Anticorpos , Automação Laboratorial , Sítios de Ligação de Anticorpos , Catálise , Análise Mutacional de DNA/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Cinética , Mutação , O(6)-Metilguanina-DNA Metiltransferase/genética , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Oligopeptídeos/genética , Análise Serial de Proteínas/instrumentação , Ligação Proteica , Engenharia de Proteínas , Fluxo de Trabalho
13.
Chem Commun (Camb) ; 55(30): 4387-4390, 2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30916085

RESUMO

A novel and versatile platform for single-step amplified fluorescence detection of antibodies via specific proximity-induced hybridization chain assembly is developed.


Assuntos
Anticorpos/sangue , Técnicas Biossensoriais/métodos , DNA/química , DNA/genética , Técnicas de Amplificação de Ácido Nucleico , Anticorpos/metabolismo , DNA/metabolismo , Humanos , Limite de Detecção , Espectrometria de Fluorescência
14.
Anal Bioanal Chem ; 411(12): 2475-2479, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30888467

RESUMO

One-half of the 2018 Nobel Prize in Chemistry was awarded jointly to George P. Smith and Sir Gregory P. Winter "for the phage display of peptides and antibodies". This feature article summarizes significant achievements leading to the development of phage display of peptides and antibodies, where a bacteriophage is genetically modified to display peptides and proteins, with the primary aim of producing new biopharmaceuticals. These significant achievements are proven to be useful for the development of phage-based bioassays and biosensors.


Assuntos
Anticorpos/metabolismo , Técnicas de Visualização da Superfície Celular/métodos , Química , Prêmio Nobel , Peptídeos/metabolismo , Anticorpos/uso terapêutico , Biofarmácia , História do Século XXI
15.
Mater Sci Eng C Mater Biol Appl ; 99: 1502-1508, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30889686

RESUMO

Nanostructured capacitive biosensors, combined with inexpensive fabrication technologies, may provide simple, sensitive devices for detecting clinically relevant cancer biomarkers. Herein, we report a novel platform for detecting the pancreatic cancer biomarker CA19-9 using low-cost screen-printed interdigitated electrodes (SPIDEs). The SPIDEs were modified by carbon nano-onions (CNOs) and graphene oxide (GO) films, on which a layer of anti-CA19-9 antibodies was immobilized. The modification with CNOs and GO significantly improved the analytical performance of the biosensor, which displayed superior results to those prepared only with GO. The biossensor exhibited high reproducibility and a relatively low limit of detection of 0.12 U mL-1. Using these devices in combination with information visualization methods we were able to detect CA19-9 in whole cell lysates of colorectal adenocarcinoma. The fabrication of these low-cost, disposable immunosensors is a successful attempt to explore CNOs in capacitive biosensors, which may be extended for detection of different cancer biomarkers.


Assuntos
Biomarcadores Tumorais/análise , Antígeno CA-19-9/análise , Carbono/química , Nanoestruturas/química , Anticorpos/metabolismo , Capacitância Elétrica , Eletrodos , Grafite/química , Humanos , Nanoestruturas/ultraestrutura , Impressão
16.
Acta Virol ; 63(1): 88-95, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30879317

RESUMO

The main immunogenic protein of the porcine epidemic diarrhea virus (PEDV) is the spike protein (S protein), which plays an important role in receptor binding, membrane fusion, and viral invasion of the host. In this paper, the linear epitope of the 3F10 non-neutralizing monoclonal antibody that was previously prepared in our laboratory was identified. The expression of truncated forms of the S protein for 3F10 reaction studies proved that the epitope was located between amino acids (aa) 674 and 791. To further locate the core aa of the 3F10 epitope, 12 random peptide libraries were used, and the result showed that the key aa located at aa 685-688 of the S protein (LLAF) were recognized by 3F10. Homology analysis of the regions corresponding to 20 typical strains of different PEDV subtypes showed that the epitope is highly conserved. Identifying the epitope recognized by an antibody helps to improve our understanding of the structure and function of the antigen. Keywords: spike protein; linear epitope; homology analysis.


Assuntos
Epitopos , Vírus da Diarreia Epidêmica Suína , Glicoproteína da Espícula de Coronavírus , Doenças dos Suínos , Animais , Anticorpos/metabolismo , Infecções por Coronavirus/virologia , Epitopos/química , Epitopos/genética , Biblioteca de Peptídeos , Vírus da Diarreia Epidêmica Suína/química , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Suínos , Doenças dos Suínos/virologia
17.
Methods Mol Biol ; 1972: 185-198, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30847792

RESUMO

A chip-based immunoaffinity capillary electrophoresis (ICE) system has been developed for measuring inflammatory mediators in dried blood samples routinely taken from newborn babies. A defined area of each dried blood spot was removed from the sample card and its contents eluted. The recovered eluates were injected into the chip and the analytes of interest isolated by the immunoaffinity disk within the chip. The captured analytes were labeled in-situ with a red light-emitting laser dye and electro-eluted into the chip separation channel. Electrophoretic separation of all of the analytes was achieved within 2.0 min with quantification of each peak being performed by online LIF detection and integration of each peak area. The degree of accuracy and precision achieved by the chip-based system is comparable to conventional immunoassays and the system is robust enough to be applied to the analysis of clinical samples.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Eletroforese Capilar/métodos , Eletroforese em Microchip/métodos , Imunoensaio/métodos , Mediadores da Inflamação/sangue , Anticorpos/metabolismo , Biotinilação , Quimiocinas/sangue , Humanos , Recém-Nascido , Inflamação/sangue
18.
J Immunol Res ; 2019: 9561350, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30906792

RESUMO

Neutrophils have recently been proposed as cells with high functional plasticity and are involved in the pathogenesis of infections, malignancy, and autoimmune diseases. However, less is known about the role of neutrophil in humoral response. In this study, we examined the importance of neutrophils and the neutrophil-derived DAMP protein, MRP14, in antibody production. Splenic neutrophils and MRP14 that are present in the splenic peri-MZ region have a close contact with MZ B cells and promote their differentiation into plasma cells. Using neutrophil-depleting mice and an MRP14-blocking compound, we showed that the presence of neutrophil and MRP14 is required for class switch, plasma cell maintenance, and antibody production in the spleen. We found that MRP14 could also be produced by neutrophils in the bone marrow and support the maintenance of bone marrow plasma cells. MRP14 binding could enhance the effect of the BAFF signal and protect primary multiple myeloma cells from doxorubicin-induced apoptosis. Our data demonstrate the effects of neutrophils on neighboring B cells and plasma cells, which provides new insights into the connection between neutrophil and humoral responses.


Assuntos
Alarminas/metabolismo , Linfócitos B/fisiologia , Células da Medula Óssea/fisiologia , Calgranulina B/metabolismo , Imunidade Humoral , Neutrófilos/imunologia , Plasmócitos/fisiologia , Animais , Anticorpos/metabolismo , Comunicação Celular , Diferenciação Celular , Plasticidade Celular , Células Cultivadas , Humanos , Switching de Imunoglobulina , Camundongos , Camundongos Endogâmicos C57BL
19.
BMJ Case Rep ; 12(2)2019 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-30765442

RESUMO

We describe a woman with both central and peripheral nervous system symptoms consistent with Morvan's syndrome who was successfully treated with immunosuppression including rituximab and the new antiepileptic drug lacosamide against peripheral nerve hyperexcitability. Despite being over 8 months in hospital and 4 months in an intensive care unit she recovered fully. It is also the first case where cerebrospinal fluid neurofilament-light (NfL) levels were followed during the disease course. The clinical course resembled that of anti-NMDA receptor encephalitis, where patients often recover surprisingly well despite severe symptoms and an extensive time in intensive care. A possible explanation is the comparatively low levels of NfL, indicating disease processes that are not characterised by extensive neuroaxonal degeneration.


Assuntos
Lacosamida/administração & dosagem , Rituximab/administração & dosagem , Siringomielia/tratamento farmacológico , Adulto , Anticorpos/metabolismo , Cuidados Críticos , Feminino , Humanos , Lacosamida/uso terapêutico , Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/imunologia , Rituximab/uso terapêutico , Siringomielia/imunologia , Resultado do Tratamento
20.
Tissue Cell ; 56: 1-6, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30736897

RESUMO

Cetacean mechanical senses, such as hearing, echolocation, active touch and the perception of water movements, are essential for their survival. Dolphins skin possesses dense packing of dermal papillae associated with the cutaneous ridges that suggests a sensory function, furthermore they are well innervated and very sensitive to touch. This is mediated by mechanoreceptors, abundant in the region of the head and in the dorsal part of the body. Most odontocetes possess vibrissae (i.e., sensory hair) that have been well described in literature and present a microanatomy similar to that of terrestrial mammals. The aim of this study was to characterize Merkel cell through use of specific antibodies: Substance P, Anti-calbindin DK28, Anti-5HT, Leu- enkephalin, Protein Gene Product 9.5 (PGP9.5) and Anti-Human Neuronal Protein, for the first time. Merkel cells (MCs) in the dolphin skin are specialized skin receptors, characterized by their particular location and close association with nerve terminals. The presence of neuroendocrine markers and different neuropeptides confirms that MCs play also neuroendocrine function and are considered as part of the diffuse neuroendocrine system. Furthermore, the presence of Leu-enkephalin in Merkel cells could involve these cells in inflammatory responses in the skin.


Assuntos
Golfinhos/metabolismo , Células de Merkel/metabolismo , Pele/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Humanos , Neurônios/metabolismo
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