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1.
Sci Rep ; 11(1): 15745, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344917

RESUMO

In Europe, Ixodes ricinus is the most important vector of human infectious diseases, most notably Lyme borreliosis and tick-borne encephalitis virus. Multiple non-natural hosts of I. ricinus have shown to develop immunity after repeated tick bites. Tick immunity has also been shown to impair B. burgdorferi transmission. Most interestingly, multiple tick bites reduced the likelihood of contracting Lyme borreliosis in humans. A vaccine that mimics tick immunity could therefore potentially prevent Lyme borreliosis in humans. A yeast surface display library (YSD) of nymphal I. ricinus salivary gland genes expressed at 24, 48 and 72 h into tick feeding was constructed and probed with antibodies from humans repeatedly bitten by ticks, identifying twelve immunoreactive tick salivary gland proteins (TSGPs). From these, three proteins were selected for vaccination studies. An exploratory vaccination study in cattle showed an anti-tick effect when all three antigens were combined. However, immunization of rabbits did not provide equivalent levels of protection. Our results show that YSD is a powerful tool to identify immunodominant antigens in humans exposed to tick bites, yet vaccination with the three selected TSGPs did not provide protection in the present form. Future efforts will focus on exploring the biological functions of these proteins, consider alternative systems for recombinant protein generation and vaccination platforms and assess the potential of the other identified immunogenic TSGPs.


Assuntos
Antígenos/isolamento & purificação , Ixodes/imunologia , Doença de Lyme/transmissão , Glândulas Salivares/imunologia , Proteínas e Peptídeos Salivares/imunologia , Picadas de Carrapatos/imunologia , Infestações por Carrapato/imunologia , Animais , Antígenos/sangue , Antígenos/imunologia , Borrelia burgdorferi/isolamento & purificação , Bovinos , Técnicas de Visualização da Superfície Celular/métodos , Feminino , Humanos , Imunização , Doença de Lyme/sangue , Doença de Lyme/parasitologia , Masculino , Fragmentos de Peptídeos/imunologia , Biblioteca de Peptídeos , Coelhos , Saccharomyces cerevisiae , Infestações por Carrapato/parasitologia
2.
Methods Mol Biol ; 2183: 95-118, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32959243

RESUMO

Several vaccines are already produced using the baculovirus expression vector system (BEVS). This chapter describes methods for generating recombinant baculoviral DNA (also called bacmid) for cultivating Spodoptera frugiperda Sf-9 cells and producing a baculovirus stock from the recombinant bacmid and for producing a protein-based vaccine with the BEVS in a stirred tank reactor.


Assuntos
Antígenos/biossíntese , Antígenos/genética , Baculoviridae/genética , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Vetores Genéticos/genética , Proteínas Recombinantes , Animais , Antígenos/isolamento & purificação , Técnicas de Cultura de Células , Clonagem Molecular , Expressão Gênica , Engenharia Genética , Células Sf9 , Transfecção , Fluxo de Trabalho
3.
Sensors (Basel) ; 20(6)2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-32183234

RESUMO

The continuous advances of Nanofluidics have been stimulating the development of novel nanostructures and strategies to accumulate very diluted analytes, for implementing a new class of high sensitivity miniaturized polymeric sensors. We take advantage of the electrokinetic properties of these structures, which allow accumulating analytes inside asymmetric microfluidic structures to implement miniaturized sensors able to detect diluted solutions down to nearly 1.2 pg/mL. In particular, exploiting polydimethylsiloxane devices, fabricated by using the junction gap breakdown technique, we concentrate antigens inside a thin microfunnel functionalized with specific antibodies to favor the interaction and, if it is the case, the recognition between antigens in solution and antibodies anchored to the surface. The transduction mechanism consists in detecting the fluorescence signal of labeled avidin when it binds to biotinylated antigens. Here, we demonstrate that exploiting these electrokinetic phenomena, typical of nanofluidic structures, we succeeded in concentrating biomolecules in correspondence of a 1 pL sensing region, a strategy that grants to the device performance comparable to standard immunoassays.


Assuntos
Antígenos/isolamento & purificação , Técnicas Biossensoriais , Imunoensaio/métodos , Dispositivos Lab-On-A-Chip , Anticorpos/química , Antígenos/química , Dimetilpolisiloxanos/química , Humanos , Nanomedicina/tendências
4.
Nanomedicine (Lond) ; 14(19): 2535-2548, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31603382

RESUMO

Aim: Traditional antigenicity assay requires antigen recovery from the particulate adjuvants prior to analysis. An in situ method was developed for interrogating vaccine antigens with monoclonal antibodies while being adsorbed on adjuvants. Materials & methods: The fluorescence imaging-based high content analysis was used to visualize the antigen distribution on adjuvant agglomerates and to analyze the antigenicity for adsorbed antigens. Results: Simultaneous visualization and quantitation were achieved for dual antigens in a bivalent human papillomavirus vaccine with uniquely labeled antibodies. Good agreement was observed between the in situ multiplexed assays with well-established sandwich enzyme-linked immunosorbent assays. Conclusion: The streamlined procedures and the amenability for multiplexing make the in situ antigenicity analysis a favorable choice for in vitro functional assessment of bionanoparticles as vaccine antigens.


Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos/isolamento & purificação , Imagem Óptica/métodos , Vacinas contra Papillomavirus/imunologia , Adsorção/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Antígenos/imunologia , Antígenos/farmacologia , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/imunologia , Humanos , Imunogenicidade da Vacina/genética , Imunogenicidade da Vacina/imunologia , Vacinas contra Papillomavirus/farmacologia
5.
Biosens Bioelectron ; 144: 111621, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31518787

RESUMO

Immunoassay can be divided into two aspects, one is immobilization of antibodies for the efficient detection of corresponding antigens, and the other is immobilization of antigens to search for antibodies which work against them. In this paper, we demonstrated these two aspects of immunoassay by using the disposable fiber optic biosensors based on surface plasmon resonance (SPR) through surface decoration with half-antibody fragments, which has been scarcely ever reported to the best of our knowledge. We first fabricated the fiber optic SPR biosensor which consists of one gold film coated single-mode fiber sandwiched by two multimode fibers. Then, we decorated the fiber optic SPR biosensors with antibody fragments and antigen fragments, respectively, and compared the specific detection performances of these two kinds of sensors. After surface decoration with half-antibody fragments, the antigen-decorated fiber probe has a demonstrated sensitivity and limit of detection of 0.9771nm/(µg/mL) and 0.1 µg/mL, respectively, which improves by 10 times compared with the performance of the antibody-decorated fiber probe. Additionally, the selective detection results indicate that our proposed biosensor can be employed as a reliable antigen detector or an effective antibody filter. Our proposed sensor has the advantages of miniaturization, low cost, simple usage, label-free detection, high efficiency and sensitivity, and can effectively avoid cross-contamination caused by reuse. Given the reliable and clean detection method for immunoassay, our work should open a new window for the utilization of miniaturized fiber optic sensors in biochemical sensing.


Assuntos
Anticorpos Imobilizados/química , Antígenos/isolamento & purificação , Técnicas Biossensoriais , Imunoensaio , Anticorpos Imobilizados/imunologia , Antígenos/química , Antígenos/imunologia , Tecnologia de Fibra Óptica/métodos , Ouro , Fibras Ópticas , Ressonância de Plasmônio de Superfície
6.
Yakugaku Zasshi ; 139(4): 505-509, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-30930377

RESUMO

Comprehensive identification of antigens in immune complexes (IC-antigens) is beneficial to provide insights into pathophysiology and could form the basis for novel diagnostic and treatment strategies for many immune-related diseases. Immune complexome analysis is a method for comprehensively identifying and profiling IC-antigens in biological fluids (such as serum and cerebrospinal fluid). We applied this strategy to the analysis of circulating ICs in autoimmune diseases (rheumatoid arthritis, Sjögren's syndrome, systemic scleroderma, and systemic lupus erythematosus), infectious diseases, and cancers. Fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) consists of fluorogenic derivatization of proteins, followed by HPLC of the derivatized proteins, isolation of the proteins differentially expressed in a certain group, enzymatic digestion of the isolated proteins followed by LC-tandem MS using a database-searching algorithm for protein identification. We have applied this method to understand the cardioprotective effect of pre-administration of docetaxel in adriamycin/docetaxel combination anti-cancer therapy, and the cellular processes that are affected by non-steroidal anti-inflammatory drugs (NSAIDs) in mouse stomach tissue during ulcer formation.


Assuntos
Cromatografia Líquida , Pesquisa Farmacêutica , Proteoma , Proteômica/métodos , Espectrometria de Massas em Tandem , Animais , Anti-Inflamatórios não Esteroides , Complexo Antígeno-Anticorpo , Antígenos/isolamento & purificação , Antígenos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Doenças Autoimunes/imunologia , Cardiotônicos , Docetaxel/administração & dosagem , Docetaxel/farmacologia , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Humanos , Camundongos , Neoplasias/imunologia
7.
Biosens Bioelectron ; 127: 174-180, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30605806

RESUMO

A label-free electrochemical immunosensor for quantitative detection of human epididymis specific protein 4 antigen (HE4 Ag) was developed by a novel multi-amplification signal system. The multi-amplification signal system was formed by loading bimetallic Au@Pd holothurian-shaped nanoparticles (Au@Pd HSs) on titanium oxide nanoclusters functionalized nitrogen-doped reduced graphene oxide (TiO2-NGO). The Au@Pd HSs were obtained via seed-mediated approach with in-situ grown palladium nanoarms on gold nanorods (Au NRs) surfaces, which possessed good electrocatalysis for hydrogen peroxide (H2O2) reduction and excellent biocompatibility. The TiO2-NGO with the high catalytic activity and large specific surface area was synthesized by hydrothermal method. Using H2O2 as an electrochemically active substrate, the prepared label-free electrochemical immunosensor based on the TiO2-NGO/Au@Pd HSs hetero-nanostructures as the signal amplification platform exhibited excellent selectivity, reproducibility and stability for the detection of HE4 Ag. Meanwhile, the linear range from 40 fM to 60 nM with the detection limit of 13.33 fM (S/N = 3) was obtained, indicating the immunosensor offers a promising method for clinical detection of HE4 Ag.


Assuntos
Antígenos/isolamento & purificação , Técnicas Biossensoriais , Imunoensaio , Proteínas/isolamento & purificação , Anticorpos Imobilizados/química , Antígenos/química , Antígenos/imunologia , Catálise , Ouro/química , Grafite/química , Humanos , Peróxido de Hidrogênio/química , Limite de Detecção , Nanopartículas Metálicas/química , Paládio/química , Proteínas/química , Proteínas/imunologia , Titânio/química , Proteína 2 do Domínio Central WAP de Quatro Dissulfetos
8.
J Histochem Cytochem ; 67(6): 441-452, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30694090

RESUMO

Heat-induced antigen retrieval (HIAR) is routinely employed on aldehyde-fixed tissue sections to enhance the reactivity of antibodies that exhibit weak or no specific interactions with tissue antigens when applied in conventional immunohistochemical protocols. A major drawback of HIAR protocols is, however, the heat-induced detachment of sections from the microscope slide with resultant impaired tissue morphology or loss of the section. We developed a method in which tissue sections mounted on glass slides are temporally coverslipped, and a clamp is used to compress the sections on the microscope slide during HIAR treatment. This "pressurized coverslipping" during HIAR was tested on various formalin-fixed tissues (murine kidneys and temporal bones, human tonsils and temporal bones) that were embedded in paraffin or celloidin. The method reliably kept the sections adherent to the slide, preserved the tissue morphology, and effectively retrieved tissue antigens for improved results in immunohistochemical labeling, even for exceptionally delicate, large, and poorly adhering sections, that is, decalcified human temporal bone sections. In summary, we present a simple method for improved slide adherence and morphological preservation of tissue sections during HIAR treatment that can be combined with all HIAR protocols and that requires only basic lab equipment.


Assuntos
Antígenos/isolamento & purificação , Força Compressiva , Temperatura Alta , Fixação de Tecidos/métodos , Preservação de Tecido/métodos , Animais , Fenômenos Biomecânicos , Humanos , Camundongos , Inclusão em Parafina , Pressão
9.
Methods Mol Biol ; 1855: 461-465, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30426439

RESUMO

Minute amounts of proteins are required for immunization of mice for the development of antibodies including monoclonal antibodies. Here we describe a rapid procedure for the isolation of proteins from polyacrylamide gels after sodium dodecyl sulfate polyacrylamide gel electrophoresis in sufficient amounts for immunization of animals.


Assuntos
Resinas Acrílicas/química , Eletroforese em Gel de Poliacrilamida/métodos , Proteínas/isolamento & purificação , Animais , Antígenos/isolamento & purificação , Eletroforese em Gel de Poliacrilamida/instrumentação , Desenho de Equipamento , Camundongos , Pós , Coloração e Rotulagem/métodos
10.
Sensors (Basel) ; 18(12)2018 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-30477240

RESUMO

Disposable immunosensors are analytical devices used for the quantification of a broad variety of analytes in different areas such as clinical, environmental, agricultural and food quality management. They detect the analytes by means of the strong interactions between antibodies and antigens, which provide concentration-dependent signals. For the herein highlighted voltammetric immunosensors, the analytical measurements are due to changes in the electrical signals on the surface of the transducers. The possibility of using disposable and miniaturized immunoassays is a very interesting alternative for voltammetric analyses, mainly, when associated with screen-printing technologies (screen-printed electrodes, SPEs), and microfluidic platforms. The aim of this paper is to discuss a carefully selected literature about different examples of SPEs-based immunosensors associated with microfluidic technologies for diseases, food, agricultural and environmental analysis. Technological aspects of the development of the voltammetric immunoassays such as the signal amplification, construction of paper-based microfluidic platforms and the utilization of microfluidic devices for point-of-care testing will be presented as well.


Assuntos
Antígenos/isolamento & purificação , Técnicas Biossensoriais/métodos , Imunoensaio/métodos , Técnicas Analíticas Microfluídicas/métodos , Agricultura , Anticorpos Imobilizados/imunologia , Antígenos/imunologia , Análise de Alimentos/métodos , Humanos
11.
Sensors (Basel) ; 18(11)2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30445792

RESUMO

A simple approach was proposed to decrease the detection limit of sandwich lateral flow immunoassay (LFIA) by changing the conditions for binding between a polyvalent antigen and a conjugate of gold nanoparticles (GNPs) with antibodies. In this study, the potato virus Y (PVY) was used as the polyvalent antigen, which affects economically important plants in the Solanaceae family. The obtained polyclonal antibodies that are specific to PVY were characterized using a sandwich enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). For LFIA, the antibodies were conjugated with GNPs with a diameter of 17.4 ± 1.0 nm. We conducted LFIAs using GNP conjugates in a dried state on the test strip and after pre-incubation with a sample. Pre-incubating the GNP conjugates and sample for 30 s was found to decrease the detection limit by 60-fold from 330 ng∙mL-1 to 5.4 ng∙mL-1 in comparison with conventional LFIA. The developed method was successfully tested for its ability to detect PVY in infected and uninfected potato leaves. The quantitative results of the proposed LFIA with pre-incubation were confirmed by ELISA, and resulted in a correlation coefficient of 0.891. The proposed approach is rapid, simple, and preserves the main advantages of LFIA as a non-laboratory diagnostic method.


Assuntos
Anticorpos/imunologia , Antígenos/isolamento & purificação , Técnicas Biossensoriais , Potyvirus/isolamento & purificação , Anticorpos/química , Antígenos/imunologia , Ensaio de Imunoadsorção Enzimática , Imunoconjugados/química , Limite de Detecção , Nanopartículas Metálicas/química , Potyvirus/patogenicidade , Solanum tuberosum/virologia , Ressonância de Plasmônio de Superfície
12.
Methods Mol Biol ; 1827: 165-178, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30196497

RESUMO

The isolation of antibody fragments targeting proteins implicated in cancers and other diseases remains a crucial issue on targeted therapy or diagnostic tool development. In many case, the protein of interest, or a relevant portion of this protein such as its extracellular domain, is available as purified protein. In such cases, phage display on purified antigen is an easy and fast way to select antibody fragment able to efficiently bind this antigen. However the output of phage selection can vary significantly depending on the way to immobilize the purified antigen during selection. The following protocols describe the selection of phage antibody on purified antigen adsorbed on plastic, i.e., panning, or a selection in solution, using a biotinylated antigen as well as the corresponding screening produces, and give hints on the advantage and drawbacks of each approach.


Assuntos
Antígenos/isolamento & purificação , Técnicas de Visualização da Superfície Celular/métodos , Anticorpos/metabolismo , Bacteriófagos/metabolismo , Biotinilação , Precipitação Química , Ensaio de Imunoadsorção Enzimática , Biblioteca de Peptídeos , Solubilidade
13.
PLoS One ; 13(5): e0197179, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29742170

RESUMO

Citrinin (CIT) is a hepato-nephrotoxic fungal metabolite produced by the genera Penicillium, Aspergillus and Monascu. There is an increasing demand for rapid and economical methods for detection CIT residues in fruit. In this study, we developed an immunochromatographic strip (ICS) for detection of citrinin (CIT) residues in fruit for the first time. Anti-CIT monoclonal antibody (McAb) 2B9 was prepared, with a binding affinity of 9.39 × 108 L/moL. Conjugates CIT-BSA and McAb 2B9 were used to develop the ICS which could be completed in 5 min, with the detection limit of 50 ng/mL and no cross reactivity with other mycotoxins. Analysis of CIT in 64 fruit samples revealed that data obtained from the ICS test were in good agreement with indirect competitive enzyme-linked immunosorbent assays (ic-ELISAs) and high performance liquid chromatography (HPLC). This result demonstrated that the ICS test could be used as a rapid, reliable, cost-effective and user-friendly qualitative tool for detection of CIT residues on-site.


Assuntos
Antígenos/isolamento & purificação , Cromatografia de Afinidade/métodos , Citrinina/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , China , Citrinina/imunologia , Reações Cruzadas , Contaminação de Alimentos , Frutas/química , Humanos , Limite de Detecção
14.
Ticks Tick Borne Dis ; 9(5): 1158-1172, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29728336

RESUMO

The African argasid tick Ornithodoros moubata transmits two important pathogens, the African swine fever virus and the spirochete Borrelia duttoni, the cause of human relapsing fever. To date, only conventional control measures such as widespread application of acaricides, strict control measures, and animal movement restrictions have been implemented to confine these diseases. Vaccines against tick infestations have the potential to be among the most efficacious interventions for the management of these diseases. Plasma membrane-associated proteins upregulated in tick midgut cells in response to blood feeding and digestion are thought to play vital functions in tick physiology and in the transmission of tick-borne pathogens. In addition, their antigenic extracellular regions are easily accessible to antibodies synthesised by immunised hosts, which makes them interesting targets for tick vaccine design. The mialomes (midgut transcriptomes and proteomes) of unfed O. moubata females and of engorged females at 48 h post-feeding have recently been obtained, providing a wealth of predicted midgut protein sequences. In the current study, these mialomes were screened using in silico tools to select predicted antigenic transmembrane proteins that were upregulated after feeding (516 proteins). The functionally annotatable proteins from this list (396 proteins) were then manually inspected following additional criteria in order to select a finite and easy-manageable number of candidate antigens for tick vaccine design. The extracellular antigenic regions of five of these candidates were obtained either as truncated recombinant proteins or as KLH-conjugated synthetic peptides, formulated in Freund's adjuvant, and individually administered to rabbits to assess their immunogenicity and protective potential against infestations by O. moubata and the Iberian species Ornithodoros erraticus. All candidates were highly immunogenic, but provided low protection against the O. moubata infestations (ranging from 7% to 39%). Interestingly, all candidates except one also protected against infestations by O. erraticus, achieving higher efficacies against this species (from 20% to 66%). According to their protective potential, three of the five antigens tested (Om17, Om86 and OM99) were considered little suitable for use in tick vaccines, while the other two (OM85 and OM03) were considered useful antigens for tick vaccine development, deserving further studies.


Assuntos
Antígenos/imunologia , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Ornithodoros/genética , Proteoma , Infestações por Carrapato/prevenção & controle , Algoritmos , Animais , Antígenos/administração & dosagem , Antígenos/genética , Antígenos/isolamento & purificação , Proteínas de Artrópodes/isolamento & purificação , Biologia Computacional/métodos , Simulação por Computador , Comportamento Alimentar , Feminino , Imunogenicidade da Vacina , Ornithodoros/anatomia & histologia , Ornithodoros/imunologia , Ornithodoros/fisiologia , Peptídeos/síntese química , Peptídeos/imunologia , Proteômica/métodos , Coelhos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Regulação para Cima , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia
15.
Analyst ; 143(4): 865-873, 2018 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-29327741

RESUMO

This study investigated the fabrication and proof of concept design demonstrating rapid and highly sensitive antigen capture utilizing electrospun polystyrene (PS) microfiber mat substrates paired with vacuum pump pressurization to induce bulk flow. In comparison with conventional flat PS surfaces used for immunoassay purposes, this system optimizes the increased surface area of the electrospun polystyrene (ESPS) fiber mat substrates and the accelerated propagation of the antigen through the detection platform by using a vacuum pump to enable efficient and rapid antigen capture. The novelty of this work was demonstrated through a parametric study detailing how a fiber substrate can capture antigen sensitively and at high speeds. In terms of sensitivity, the current system is comparable to the conventionally used flat PS substrates. Additionally, the amount of antigen captured on a flat PS substrate in 60 minutes was surpassed in under 5 seconds when utilizing the ESPS-vacuum system. Three-dimensional ESPS fiber mats were then noted as a comparison between Damkohler numbers and between flat PS and ESPS-vacuum systems. The bulk flow of the ESPS-vacuum system allows for a Damkohler number of 0.37 indicating a balance between the flow rate and the reaction rate as opposed to a PS flat platform of 5.80 × 104 which illustrates a diffusion rate limited system. Finally, the overall ESPS-vacuum system was tested for its immunoassay capability. A sandwich fluorescence-based immunoassay was performed on both PS flat-diffusion and ESPS-vacuum systems. The ESPS-vacuum system indicated a wider detection range capability from 5 to 1000 ng mL-1 in comparison with the PS flat-diffusion system at 5 to 100 ng mL-1.


Assuntos
Antígenos/isolamento & purificação , Imunoensaio , Poliestirenos , Anticorpos Imobilizados , Fluorescência
16.
Biosens Bioelectron ; 102: 456-463, 2018 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-29182928

RESUMO

In this work, we report the development of a general strategy for enhancing the efficiency of target capture in immunoassays, using a bifunctional fusion protein construct which incorporates a substrate-anchoring moiety for the high-abundance immobilization of an antigen-binding domain. This approach was informed by the development of a pseudo first-order rate constant model, and tested in a paper-based assay format using a fusion construct consisting of an rcSso7d binding module and a cellulose-binding domain. These rcSso7d-CBD fusion proteins were solubly expressed and purified from bacteria in high molar yields, and enable oriented, high-density adsorption of the rcSso7d binding species to unmodified cellulose within a 30-second incubation period. These findings were validated using two distinct, antigen-specific rcSso7d variants, which were isolated from a yeast surface display library via flow cytometry. Up to 1.6 micromoles of rcSso7d-CBD was found to adsorb per gram of cellulose, yielding a volume-averaged binder concentration of up to 760µM within the resulting active material. At this molar abundance, the target antigen is captured from solution with nearly 100% efficiency, maximizing the attainable sensitivity for any given diagnostic system.


Assuntos
Antígenos/isolamento & purificação , Técnicas Biossensoriais/métodos , Patologia Molecular/métodos , Proteínas Recombinantes de Fusão/química , Antígenos/química , Sítios de Ligação , Celulose/química , Enzimas Imobilizadas/química , Citometria de Fluxo , Humanos , Ligação Proteica
17.
Biosens Bioelectron ; 102: 470-478, 2018 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-29182930

RESUMO

Immunosensors are biosensors based on interactions between an antibody and antigen on a transducer surface. Either antibody or antigen can be the species immobilized on the transducer to detect antigen or antibody, respectively. Because of the strong binding forces between these biomolecules, immunosensors present high selectivity and very high sensitivity, making them very attractive for many applications in different science fields. Electrochemical immunosensors explore measurements of an electrical signal produced on an electrochemical transductor. This signal can be voltammetric, potentiometric, conductometric or impedimetric. Immunosensors utilizing electrochemical detection have been explored in several analyses since they are specific, simple, portable, and generally disposable and can carry out in situ or automated detection. This review addresses the potential of immunosensors destined for application in food and environmental analysis, and cancer biomarker diagnosis. Emphasis is given to the approaches that have been used for construction of electrochemical immunosensors. Additionally, the fundamentals of immunosensors, technology of transducers and nanomaterials and a general overview of the possible applications of electrochemical immunosensors to the food, environmental and diseases analysis fields are described.


Assuntos
Antígenos/isolamento & purificação , Técnicas Biossensoriais/tendências , Técnicas Eletroquímicas/tendências , Imunoensaio/tendências , Anticorpos/química , Anticorpos/imunologia , Antígenos/imunologia , Biomarcadores/química , Nanoestruturas/química
18.
Biosens Bioelectron ; 102: 267-275, 2018 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-29153948

RESUMO

The application of Surface Plasmon Resonance Imaging (SPRi) for the detection of transmembrane antigen of the Rhesus (Rh) blood group system is demonstrated. Clinically significant Rh blood group system antigens, including D, C, E, c, and e, can be simultaneously identified via solid phase immobilization assay, which offers significant time savings and assay simplification. Red blood cells (RBCs) flowed through the micro-channel, where a suitable condition for Rh blood group detection was an RBC dilution of 1:10 with a stop-flow condition. Stop flow showed an improvement in specific binding compared to continuous flow. Rh antigens required a longer incubation time to react with the immobilized antibody than A and B antigens due to the difference in antigen type and their location on the RBC. The interaction between the immobilized antibodies and their specific antigenic counterpart on the RBC showed a significant difference in RBC removal behavior using shear flow, measured from the decay of the SPR signal. The strength of the interaction between the immobilized antibody and RBC antigen was determined from the minimum wall shear stress required to start the decay process in the SPR signal. For a given range of immobilized antibody surface densities, the Rh antigen possesses a stronger interaction than A, B, and AB antigens. Identification of 82 samples of ABO and Rh blood groups using SPRi showed good agreement with the standard micro-column agglutination technique. A wider coverage of antigenic recognition for RBC when using the solid phase immobilization assay was demonstrated for the RBC with the antigenic site located on the transmembrane protein of the clinically significant Rh antigen. Given the level of accuracy and precision, the technique showed potential for the detection of the Rh minor blood group system.


Assuntos
Sistema ABO de Grupos Sanguíneos/sangue , Antígenos/isolamento & purificação , Técnicas Biossensoriais , Sistema do Grupo Sanguíneo Rh-Hr/sangue , Sistema ABO de Grupos Sanguíneos/genética , Sistema ABO de Grupos Sanguíneos/isolamento & purificação , Anticorpos/química , Anticorpos/imunologia , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Antígenos/sangue , Eritrócitos , Humanos , Sistema do Grupo Sanguíneo Rh-Hr/genética , Sistema do Grupo Sanguíneo Rh-Hr/isolamento & purificação , Ressonância de Plasmônio de Superfície
19.
Cold Spring Harb Protoc ; 2017(12): pdb.prot098640, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29196601

RESUMO

This immunoprecipitation protocol details individual steps for the enrichment and purification process of specific proteins from a complex cell lysate using an antibody bound to a solid matrix. Purified antigen(s) can be eluted by various methods, and the resultant protein target can be analyzed and/or identified by numerous assays, including the enzyme-linked immunosorbent assay (ELISA), western blotting, or mass spectrometry.


Assuntos
Antígenos/isolamento & purificação , Imunoprecipitação/métodos , Proteínas/isolamento & purificação
20.
Eur J Immunol ; 47(8): 1377-1385, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28654217

RESUMO

Antibody conjugates applicable in both conventional flow and mass cytometry would offer interesting options for cross-platform comparison, as well as the enrichment of rare target cells by conventional flow cytometry (FC) sorting prior to deep phenotyping by mass cytometry (MC). Here, we introduce a simple method to generate dual fluorochrome/metal-labelled antibodies by consecutive orthogonal labelling. First, we compared different fluorochrome-conjugated antibodies specific for CD4, such as FITC, Vio667, VioGreen or VioBlue for their compatibility with the conventional secondary MAXPAR® labelling protocol. After labelling with 141 Pr, the fluorescence emission spectra of all fluorochromes investigated retained their characteristics, and CD4 dual conjugates (DCs) provided consistent results in immune phenotyping assays performed by FC and MC. The phenotypical composition of CD4+ T-cells was maintained after enrichment by FC sorting using different CD4 DCs. Finally, magnetic cell depletion was combined with FC sorting using CD19-VioBlue-142 Nd, CD20-VioGreen-147 Sm, CD27-Cy5-167 Er and CD38-Alexa488-143 Nd DC to enrich rare human plasmablasts to purities >80%, which allowed a subsequent deep phenotyping by MC. In conclusion, DCs have been successfully established for direct assay comparison between FC and MC, and help to minimise MC data acquisition time for deep phenotyping of rare cell subsets.


Assuntos
Anticorpos Monoclonais/química , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Plasmócitos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Antígenos/isolamento & purificação , Antígenos CD4/imunologia , Antígenos CD4/isolamento & purificação , Citometria de Fluxo/instrumentação , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Humanos , Imunofenotipagem/instrumentação , Plasmócitos/química , Coloração e Rotulagem/instrumentação , Coloração e Rotulagem/métodos , Linfócitos T/imunologia
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