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1

García, Mark Megan Olga. "Production and validation of anti-HCV antibodies for viral neutralization." Thesis, KTH, Skolan för kemi, bioteknologi och hälsa (CBH), 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-278578.

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Hepatitis-C Virus (HCV) remains the leading cause of liver transplant in the US and the UK, and the World Health Organization (WHO) estimates that 71 million people are infected worldwide. A vaccine would drastically impact the healthcare-associated burdens that HCV causes globally. The objective of this master’s thesis project is to produce human antibody (IgG) against HCV. This project will focus on the monoclonal antibodies (mAbs) HEPC3, AR3C, HEPC74, and HCV1. These four antibodies have been isolated from patients who have successfully cleared the infection, and their sequences and structures are available in the public domain. These four antibodies have also shown to bind to E2, a glycoprotein on the surface HCV that is crucial for viral binding and entry. This interaction of the mABs with E2 has been implicated in viral neutralization, making them promising choices for this study. Overall, 3 out of 4 mAbs were successfully cloned and produced. The unsuccessful antibody, HEPC74, was discovered to have failed due to an error in the plasmid sequence. Just as the western blot to confirm secretion was ready to be run, the laboratory closed due the Covid-19 outbreak. Therefore, the data can officially declare a ¾ mAb production success, however it is safe to assume that the alternative clone for HEPC74 was also a success due to a perfect sequence match. Since the primary objective of this project was to successfully clone and produce these four antibodies, then this study is considered an overall success. Lastly, this study examined how the same protocol  could be applied the SARS-CoV-2 outbreak, by the cloning and production of anti-RBD IgG and testing them for viral neutralization.
Hepatit-C (HCV) är fortsatt den enskilt största orsaken till levertransplantationer med uppskattningsvis 71 miljoner infekterade globalt sett, enligt världshälsoorganisationen (WHO).Ett vaccin mot HCV skulle drastiskt minska trycket på global hälso- och sjukvård. Syftet med detta projekt är att producera antikroppar (igG) mot HCV. Projektet fokuserar på HEPC3, AR3C, HEPC74 och HCV1 som är monoklonala antikroppar (mAbs). Dessa antikroppsvarianter har isolerats från patienter som tillfrisknat från infektion. Både DNA-sekvenser och strukturer av antikropparna finns offentligt tillgängliga. Dessa fyra antikroppar har också visats kunna binda till E2 som är ett membranbundet glykoprotein hos HCV som är centralt för viral adhesion och fusion. Interaktionen mellan dessa mAbs och E2 har visat sig neutralisera virulens, vilket gör dem till lovande kandidater för denna studie. Tre av fyra mAbs kunde klonas och produceras framgångsrikt. Försöket med HEPC74 misslyckades på grund av ett fel i plasmidsekvensen och just som western blot skulle genomföras för att bekräfta sekretion av en alternativ klon avslutades the praktiska arbetet med anledning av Covid-19 utbrottet. Resultaten visar entydigt att tre av fyra mAb producerades framgångsrikt. Det går dock att anta att det andra försöket med HEPC74 sannolikt också lyckades pga perfekt sekventiell matchning. Då det huvudsakliga syftet med projektet var att framgångsrikt klona och producera dessa fyra antikroppar så kan studien anses vara framgångsrik. Slutligen så undersöktes huruvida samma förfarande kunde appliceras mot SARS-CoV-2 genom kloning och produktion av anti-RBD IgG och tester av viral neutralisering.
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2

Chou, Chia-Wei Thomas. "HIV neutralization through use of antibodies and pharmacokinetics of topical applications." Thesis, Boston University, 2013. https://hdl.handle.net/2144/12073.

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Thesis (M.A.)--Boston University
The Human Immunodeficiency Virus Type 1 (HIV-1), a sexually transmitted retrovirus that causes the Acquired Immunodeficiency Syndrome (AIDS), infects over two million people a year. Several methods introduced to prevent HIV-1 transmission, such as condoms, circumcision and antiretroviral drugs, have proven to be partially effective, but more effective approaches are being sought. Topical microbicides are being developed to provide a women-controlled method to prevent the transmission of HIV-1. Unfortunately, most of the candidate microbicide compounds tested to date have either elicited undesirable mucosal inflammation and epithelial lesions leading to increased seroconversions, or have been ineffective. One novel approach currently being explored is the use of monoclonal antibodies as components of topical microbicides. Monoclonal antibodies can be produced inexpensively by transfection into Nicotiana plants. We hypothesize that anti-HIV monoclonal antibodies produced in Nicotiana (MAb-N) will be effective in neutralizing HIV- 1 when used as topical microbicides at mucosal sites, and set out to test whether they retain their efficacy under physiological conditions. We tested the pharmacodynamics of anti-HIV MAb-N efficacy in Cynomolgus macaques following application of the antibodies in the vaginal compartment. We further studied the ability of MAb-N to cross through the vaginal epithelium using an EpiVaginal tissue model. To determine the pharmacodynamics of HIV neutralizing activity after the application of anti-HIV MAb-Ns to the vaginal mucosa, we used a neutralization assay based on HIV-expression in the TZM-bl cell line to test the efficacy of various doses of MAbs in a time course after they had been administered intravaginally in gel form to Cynomolgus macaques. To determine the pharmacokinetics of Mab-N transport across the vaginal epithelium, monoclonal antibodies were added to the apical surface, and a human-IgG ELISA was used to detect Mab-N that had crossed the epithelium into the basal supernatant. Immunohistology was used to confirm and validate ELISA data for evidence of transfer of Mabs across the epithelial layer. Our results show that anti-HIV MAb-Ns were effective in neutralizing cell-free HIV in TZM-bl neutralization assays. We found that MAb-Ns retained their anti-viral efficacy in monkeys after 4-hours. However, neutralizing activity was decreased after 24- hours and 72-hours, with wide variability in effectiveness between individual macaques. Mab-ns tested in the EpiVaginal tissue model showed minimal transfer of antibodies across the epithelium, ranging from 0.005% to 0.09%. Immunohistological data showed that antibodies applied apically to tissue models concentrated only in the superficial layers of the stratum corneum and did not penetrate the epithelium. Our data indicate that anti-HIV MAb-Ns are effective in neutralizing HIV-1 following vaginal application for at least 4 hours, and that they do not pass through the vaginal epithelium in significant amounts. Our data support their further development as vaginal microbicides.
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3

Martin, Nicole C. Couillard Nicole. "Differential dengue tropism & neutralization : potential mechanisms of pathogenesis /." Download the dissertation in PDF, 2006. http://www.lrc.usuhs.mil/dissertations/pdf/Martin2006.pdf.

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4

Gias, Edna Lyn Michael. "Mechanism of human respiratory syncytial virus (hRSV) resistance to neutralization by anti-fusion glycoprotein antibodies." Thesis, University of Newcastle Upon Tyne, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435566.

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5

Yari, Fayezeh. "Expression of recombinant neutralizing anti-HIV-1 antibodies in bacteria and eukaryotic cells /." Stockholm : Karolisnska institutet, 2007. http://diss.kib.ki.se/2007/978-91-7357-079-4/.

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6

Johnson, Jacklyn. "Properties of HIV-1 env and human seminal fluid that determine virus inhibition by antibodies and microbicides." Diss., University of Iowa, 2019. https://ir.uiowa.edu/etd/6966.

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Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection that leads to acquired immunodeficiency syndrome (AIDS). Approximately 36 million people worldwide are living with HIV-1, which is commonly acquired through sexual contact. Antiviral therapies control disease progression, but do not eliminate this virus from the host. Thus, global efforts are focused on developing vaccines that prevent HIV-1 transmission. Such vaccines are based on eliciting the production of protective antibodies that target the envelope glycoproteins (Envs) of this virus. Unfortunately, HIV-1 immunization trials have shown limited efficacy. A better understanding of the antibody-mediated inactivation process is needed to improve vaccine strategies. In this work we describe two novel factors that contribute to HIV-1 inactivation. First, we show that structural stability of the Env protein determines its sensitivity to vaccine-elicited antibodies. Different interactions within Env contribute to its stability. Perturbation of the Env-stabilizing interactions by physical and chemical treatments enhances sensitivity of HIV-1 to antibodies. Second, we found that the chemical composition of the transmission medium affects Env inhibition by antibodies and other inhibitory agents. Semen is the most common vehicle for HIV-1 transmission. This medium contains high concentrations of the sugar fructose. We found that semen fructose competitively blocks binding of antiviral agents that target sugar residues on Env. Together, this work advances our understanding of the mechanism that underlies HIV-1 inactivation by vaccine-elicited antibodies and provides novel strategies to enhance their potency.
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7

Ubillus, Borja Elizabeth Noelia. "Titulación de anticuerpos al virus Chikungunya mediante la técnica de neutralización por reducción de placas." Bachelor's thesis, Universidad Ricardo Palma, 2016. http://cybertesis.urp.edu.pe/handle/urp/845.

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El presente trabajo de tesis tuvo como objetivo titular anticuerpos neutralizantes contra una cepa endémica del virus Chikungunya que circula en la costa norte peruana. Para desarrollar esta investigación, se empleó la prueba de neutralización por reducción en placas (PRNT), la cual se realizó en el Instituto Nacional de Salud (INS)– Laboratorio de Aislamiento y Cultivo Celular perteneciente al área de Metaxénicas Virales. La justificación de la investigación se basa en la necesidad de comprobar que los anticuerpos detectados por la prueba de ELISA son neutralizantes y logran inhibir la dispersión del CHIKV en el cuerpo humano. Además, la prueba es necesaria para evaluar drogas antivirales y futuras vacunas que lleguen al Perú. El diseño metodológico utilizado fue analítico y experimental. La cepa y las muestras fueron proporcionadas por el INS y provinieron del departamento de Tumbes. Las muestras positivas fueron previamente diagnosticadas con ELISA utilizando IgM e IgG y las muestras negativas fueron de individuos sanos sin contacto previo con arbovirus. La prueba de PRNT se realizó en 24 horas usando la línea celular VERO CCL-81 en monocapa. La valoración de la prueba se realizó al 50% de neutralización. Se obtuvo como resultado títulos de anticuerpos en el rango de 1/8 y 1/16. Ésta variación responde a una relación inversa con el inicio de los días de síntomas. Por lo tanto, se concluye que la población de la costa norte del Perú sí está desarrollando anticuerpos neutralizantes para contrarrestar el virus Chikungunya endémico en la región.The present thesis aims to titrate neutralizing antibodies against an endemic strain of the Chikungunya virus that circulates in the northern coast of Peru. In order to develop this research, the plaque reduction neutralization test (PRNT) has been used, which has been carried out at the National Institutes of Health (INS) - Isolation and Cell Culture Laboratory belonging to the Viral Metaxenics area. The justification of the investigation is based on the need to verify that the antibodies detected by the ELISA test are neutralizing and manage to inhibit the dispersion of CHIKV in the human body. In addition, the test is necessary to evaluate antiviral drugs and future vaccines that arrive in Peru. The methodological design used was analytical and experimental. The strain and samples were provided by the INS. The strain is endemic to the department of Tumbes. The positive samples were previously diagnosed with ELISA using IgM and IgG and the negative samples were from individuals that had not had contact with any arbovirus. The PRNT test was performed in 24 hours using the VERO CCL-81 cell line in monolayer. The titration of the test was performed at 50% neutralization. Antibody titres were obtained in the range of 1/8 and 1/16. This variation responds to an inverse relationship with the onset of symptom days. Therefore, it is concluded that the population of the northern coast of Peru is developing neutralizing antibodies to counteract the endemic Chikungunya virus in the region.
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8

SANTOS, e. SILVA ED C. "Uso de venenos de serpentes australianas como potencial alternativa para a produção de soro anti-elapídico." reponame:Repositório Institucional do IPEN, 2015. http://repositorio.ipen.br:8080/xmlui/handle/123456789/25666.

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Dissertação (Mestrado em Tecnologia Nuclear)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
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9

Gagneux-Brunon, Amandine. "Rôle du réservoir viral et de l'immunité humorale du sperme dans la transmission sexuelle de HIV." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSES050/document.

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HIV est principalement transmis par voie sexuelle. Bien que les stratégies de prévention basées sur les antirétroviraux (TasP et PrEP) soient efficaces, d'autres approches (en particulier vaccinales) restent d' actualité.Dans la première partie de ce travail, nous avons caractérisé le réservoir séminal de HIV chez des patients chroniquement infectés et sous trithérapie antirétrovirale (TARV). Dans le sperme, HIV est présent sous forme libre et/ou associée aux NSMC. Ces deux formes peuvent être transmises. Le TARV réduit la quantité de virus libre dans le sperme, sans éliminer le virus associé aux NSMC. A heure du TasP, comme meilleur outil de prévention de la transmission sexuelle, mieux caractériser l'état de ce virus résiduel (latent, réplicatif, défectif ?) est nécessaire. Dans notre travail, le virus associé aux NSMC n'a pas été réactivé, laissant supposer que le virus résiduel dans les NSMC de patients sous TARV au long cours, peut être défectif. Ces données restent à confirmer dans de plus grandes cohortes.Dans la deuxième partie du travail, nous avons caractérisé sur le plan isotypique et fonctionnel, les anticorps dirigés contre HIV du sperme. L'objectif est de mettre en évidence des anticorps « large spectre » neutralisant HIV, et/ou inhibant la transcytose, et/ou capables d'ADCC et ciblant les souches virales potentiellement transmissibles par voie sexuelle. Nous avons identifié dans le plasma séminal de certains patients des anticorps neutralisant des souches X4 tropiques, alors que les souches majoritairement transmises par voie sexuelle sont R5 tropiques. Nous avons observé la capacité d' anticorps dirigés contre HIV du plasma séminal à traverser un épithélium monocouche. Le rôle protecteur ou facilitateur est à préciser. Des anticorps protecteurs pourraient être utilisés dans des formulations de type microbicides ou en immunothérapie et contribuer également à un développement de vaccin après identification des épitopes cibles
HIV new infections are mostly related to sexual transmission. Semen contains HIV free particles and cell-associated HIV. Both forms are sexually transmitted. Although PrEP and TasP are efficient to prevent HIV sexual transmission, other strategies like a Vaccine remain needed.In a first part, we studies HIV reservoir in HIV-infected patients receiving combined antiretroviral treatment (cART) in semen. cART reduced HIV viral load in semen, but had a no activity against cell-associated HIV. We tried to reactivate HIV cell reservoir in semen to better characterize its role. We did not observe any reactivation; HIV reservoir in semen might be mostly latent, or defective. This observation should be confirmed in larger cohort of patients.In a second part, we aimed to study the prevalence of anti-HIV antibodies in semen and their role in sexual transmission. Semen of HIV-infected men contains anti-HIV immunoglobulins (Igs), mostly IgG. We observed that unspecific Igs (IgG, IgAl, IgA2) and anti-HIV IgG and IgA were able to transmigrate across an epithelium monolayer mimicking endocervix. This transmigration property may facilitate HIV transcytosis. We also observed a neutralizing activity by Igs purified from semen of 2 chronically infected patients against a X4-tropic viral strain. Seminal anti-HIV may exhibit a dual role (facilitating or limiting) HIV sexual transmission. Protective antibodies purified from semen might be used in preventive strategies like microbicides or serotherapy, and may help to develop vaccine after identification of their epitopes
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10

Prachanronarong, Kristina L. "Understanding Drug Resistance and Antibody Neutralization Escape in Antivirals: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/840.

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Antiviral drug resistance is a major problem in the treatment of viral infections, including influenza and hepatitis C virus (HCV). Influenza neuraminidase (NA) is a viral sialidase on the surface of the influenza virion and a primary antiviral target in influenza. Two subtypes of NA predominate in humans, N1 and N2, but different patterns of drug resistance have emerged in each subtype. To provide a framework for understanding the structural basis of subtype specific drug resistance mutations in NA, we used molecular dynamics simulations to define dynamic substrate envelopes for NA to determine how different patterns of drug resistance have emerged in N1 and N2 NA. Furthermore, we used the substrate envelope to analyze HCV NS3/4A protease inhibitors in clinical development. In addition, influenza hemagglutinin (HA) is a primary target of neutralizing antibodies against influenza. Novel broadly neutralizing antibodies (BnAbs) against the stem region of HA have been described and inhibit several influenza viral subtypes, but antibody neutralization escape mutations have emerged. We identified potential escape mutations in broadly neutralizing antibody F10 that may impact protein dynamics in HA that are critical for function. We also solved crystal structures of antibody fragments that are important for understanding the structural basis of antibody binding for influenza BnAbs. These studies can inform the design of improved therapeutic strategies against viruses by incorporating an understanding of structural elements that are critical for function, such as substrate processing and protein dynamics, into the development of novel therapeutics that are robust against resistance.
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11

Miyazaki, Yasuyuki. "Analyses of sensitivity to antibody-mediated neutralization and induction of neutralizing antibodies to HIV-1 Env by simian/human immunodeficiency virus-macaque model." Kyoto University, 2002. http://hdl.handle.net/2433/149868.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(人間・環境学)
甲第9660号
人博第144号
13||129(吉田南総合図書館)
新制||人||35(附属図書館)
UT51-2002-G418
京都大学大学院人間・環境学研究科人間・環境学専攻
(主査)教授 速水 正憲, 教授 津田 謹輔, 教授 松村 道一, 助教授 三浦 智行
学位規則第4条第1項該当
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12

Otterstrom, Jason John. "Visualizing Influenza Virus Membrane Fusion: Inhibition and Kinetics." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11251.

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The influenza virus hemagglutinin (HA) surface protein is a primary antigenic target for neutralization of viral infection. HA also mediates membrane fusion between the virus and a cell, which is the first critical step during infection. Traditional techniques to study infection neutralization by antibodies or the membrane fusion process rely on ensemble measurements, confounding the precise mechanism of infection neutralization and obscuring transient conformational intermediates. This dissertation describes advances made in a fluorescence microscopy-based single-particle fusion assay to overcome the limitations of ensemble measurements in these types of studies. Virus particles are labeled to visualize lipid mixing between a virus and a target membrane formed upon a glass or polymer support. Optionally, the viral lumen can be labeled to visualize the subsequent release of viral contents.
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13

Miranda, Emily Hime. "Padronização e validação do teste de neutralização por redução de placas de Lise em placa de 96 poços para avaliar a imunogenicidade do componente caxumba da vacina MMR." reponame:Repositório Institucional da FIOCRUZ, 2015. https://www.arca.fiocruz.br/handle/icict/13006.

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Fundação Oswaldo Cruz. Instituto de Tecnologia em Fármacos/Farmanguinhos. Rio de Janeiro, RJ, Brasil.
A caxumba é uma doença infecto-contagiosa imunoprevinível por vacinação. A imunogenicidade vacinal é avaliada através de testes sorológicos que detectam anticorpos neutralizantes, que são considerados correlatos de proteção. O Teste de Neutralização (PRNT) apresenta vantagens frente a outros testes por ser mais específico na detecção desses anticorpos neutralizantes. O presente estudo visou padronizar e validar a técnica. Durante a padronização foram definidos o M.O.I de 0,001 e o melhor dia de pico de vírus infeciosos (3 dias), para definir um protocolo de produção viral. A partir do vírus produzido foram avaliados qualitativamente o fenótipo da placa de lise e a diluição viral em diferentes condições analíticas. Os seguintes parâmetros foram definidos: diluição viral de 1:1600 para obter 30 placas de lise/poço, bicarbonato de sódio como tampão no meio de cultura, meio semi-sólido com carboximetilcelulose (CMC) 1,5%, tempo de adsorção de 3 horas e tempo de incubação final de 4 dias. Para avaliar o impacto do tempo de neutralização na potência viral e no de título de anticorpos neutralizantes, dois intervalos de tempo (1 e 2 horas) foram testados e a neutralização por 2 horas se demonstrou mais adequada ao ensaio. Posteriormente, foi definido um ponto de corte de 23, utilizando um painel sorológico contendo 126 soros pré e pós-vacinais de crianças imunizadas com a vacina tríplice viral. O ponto de corte foi determinado como a área sob a curva ROC usando os resultados de ELISA previamente avaliados em comparados com os resultados de PRNT. Dentre os resultados analisados, 35% foram concordantes na positividade dos resultados em ambos os testes. Valores preditivos positivos de 95,373 e valores negativos 97,680 determinam a real presença ou não da doença. Critérios de aceitação para o teste foram estabelecidos como: faixas de variação do end point do vírus (13 a 22) e faixas de variação dos controles (baixo, médio e alto). Também foi estabelecido como as amostras indeterminadas devem ser tratadas. Posteriormente, o PRNT foi submetido ao processo de validação, avaliando os parâmetros de linearidade, especificidade, exatidão e precisão, conforme preconizado pela RDC 27 da ANVISA. Nas análises de precisão foram obtidos coeficientes de variação a 15% para o limite inferior de quantificação do método (LIQ) e 20% para as demais concentrações. O mesmo ocorreu nas análises de exatidão. Quanto a seletividade, não foi detectada reação cruzada nas amostras quando desafiadas com o vírus de sarampo.
Mumps is an infectious disease preventable by an available vaccine. Vaccine immunogenicity is evaluated by serological tests for detection of neutralizing antibodies, which are considered the correlate of protection. Plaque Reduction Neutralization Test (PRNT) shows advantages for neutralizing antibodies’ dosage because of its better specificity when compared to other tests. The present study aims to standardize and validate this technique. During the standardization process, it was defined the multiplicity of infection of 0,001 and the viral peak production on the third day, for defining a viral production protocol. From the virus lot produced, it were qualitatively evaluated the plaque phenotypes and viral dilutions under different analytical conditions. The following parameters were defined: viral dilution of 1:1600 to achieve 30 plaques per well; sodium bicarbonate as buffer of the culture media, semisolid overlay media content of 1,5% of carboxymethylcellulose (CMC); adsorption time of 3 hours and final incubation of 4 days. In order to evaluate the impact of incubation time for neutralization step on viral potency and neutralizing antibodies’ titers, two intervals (1 and 2 hours) were evaluated and the 2-hour neutralization time was considered more appropriate. After that, a cutoff value of 23 was defined using a serological panel containing 126 pre and post- vaccination sera of children immunized with MMR vaccine. The cutoff value was defined from the area under the ROC curve using ELISA data previously analyzed in comparison with PRNT results. Among the results, there were 35% of positive results in agreement in both tests. Positive predictive value of 95.373 and negative predictive value of 97.680 determine the actual presence or absence of disease. Acceptance criteria for the test were established such as: viral endpoint range (13 to 22) and titer variation ranges for control samples (low, medium and high titer). It was also determined how indeterminate samples should be treated. Finally, PRNT were submitted to validation regarding linearity, specificity, accuracy and precision as recommended by RDC 27 of ANVISA. Regarding the precision analyses, coefficient of variations lower than 15% were achieved for the sample with the lower limit of quantification, and lower than 20% for the other sample concentrations tested. The same occurred in the accuracy analyses. Regarding specificity, no cross reaction was detected in the samples when challenged with measles virus.
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Morgand, Marion. "Contribution à la recherche d'une stratégie d'immunisation visant à induire une réponse anti-VIH-1 largement neutralisante." Thesis, Tours, 2017. http://www.theses.fr/2017TOUR3804/document.

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La difficulté à induire des anticorps capables de neutraliser (anticorps neutralisants, AcN) la très grande diversité des isolats circulants du VIH-1 reste à l’heure actuelle un obstacle majeur au développement d'un vaccin préventif contre le VIH-1. L’objectif du projet a été de déterminer quels étaient les épitopes neutralisants les plus conservés au sein des 4 groupes M, N, O et P de VIH-1 puis de concevoir un immunogène qui serait capable d’induire la production d’AcN anti-VIH-1. Nous avons montré que l’épitope N160-glycane dépendant de la région V1/V2 de l’enveloppe virale est le plus conservé au sein des 4 groupes du VIH-1 (Morgand et al., JAIDS 2016). Nous avons ensuite montré la faisabilité d’obtenir, en système d’expression transitoire, des particules chimères constituées de la protéine d’enveloppe HBs du virus de l’hépatite B exprimant à leur surface les glycoprotéines d’enveloppe de différents groupes et sous-type du VIH-1. Malgré la présence de certains épitopes neutralisants (supersite N332-V3, site de liaison au CD4, région MPER- membrane proximal external region-), les épitopes d’intérêt de la région V1/V2 ne sont pas exposés sur ces particules chimères
The difficulty to induce antibodies able to neutralize (neutralizing antibodies, NAb) the large diversity of HIV- 1 isolates remains a major hurdle toward the development of an anti-HIV-1 vaccine. The aim of our study was first, to identify which epitopes are the most conserved within the 4 HIV-1 groups (M, N, O, P) and then, to design an immunogen that would be able to induce NAb against HIV-1. We showed that the V1/V2 N160- glycan epitope is the most conserved within the 4 HIV-1 groups (Morgand et al., JAIDS 2016). Subsequently, we showed the feasibility to generate chimeric particles based on the HBs envelope protein exposing the envelope glycoproteins of different groups and subtypes of HIV-1 at their surface. Although we demonstrated the presence of several neutralizing epitopes on these chimeric particles (N332-V3 supersite, CD4 binding site, membrane proximal external region), none of them exposed the V1/V2 epitopes of interest
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15

Feitosa, Ana Luisa Pereira. "Padronização de uma nova técnica para detecção de anticorpos neutralizantes anti-dengue baseada na RT-PCR em tempo real." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/17/17138/tde-06012016-154117/.

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Por representar a mais importante arbovirose em nível mundial, as infecções causadas pelos vírus da dengue são de grande importância em nosso país, apresentando uma ampla variedade de sintomas clínicos que vão desde infecção assintomática até formas mais graves da doença. O título de anticorpos neutralizantes produzidos frente à infecção por dengue parece ser determinante na forma de apresentação da doença no paciente. Atualmente, a forma com que o teste de neutralização é realizado demanda tempo para sua realização. O objetivo deste trabalho foi padronizar um ensaio de neutralização viral por RT-PCR em tempo real em cepas virais dos quatro sorotipos dengue para, posteriormente, ser empregada na detecção rápida e em grande escala de anticorpos em soro de pacientes e de candidatos vacinais. Para isso, foram construídas curvas padrão, para cada sorotipo viral, por meio da transcrição in vitro do RNA viral. O ensaio de neutralização padronizado nesse estudo reduziu o número de dias de detecção da neutralização em 48 horas quando comparada com a técnica de neutralização tradicional (PRNT), além de utilizar técnicas moleculares sensíveis e específicas para detecção como a RT-PCR em tempo real que garantem maior aplicabilidade do teste.
Because Dengue virus is the most important arboviral disease worldwide, infections caused by this pathogen are of great importance in Brazil, producing a wide variety of clinical symptoms ranging from asymptomatic infection to more serious forms of the disease. The title of neutralizing antibodies produced against the dengue infection appears to be determinant in the outcome of the disease. Currently, neutralization tests that have been performed take time for its execution. The aim of this study was to standardize a viral neutralization assay by real-time RT-PCR of viral strains of the four Dengue serotypes to subsequently be used for rapid detection and large-scale using antibodies of patients and for vaccine candidates. For this matter, standard curves were constructed for each Dengue serotype, by in vitro transcription of viral RNA. The neutralization assay in this study reduced the period of neutralization to 48 hours, compared to traditional neutralization test (PRNT), and it uses a more sensitive and specific molecular technique for detection of neutralizing antibodies, such as real time RT-PCR, to ensure greater applicability of the test
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16

THOMAZI, GABRIELA O. C. "Resposta imunológica em modelos animais imunizados contra o muco nativo ou irradiado por raios gama de 60 Co da raia de água doce Paratrygon aiereba." reponame:Repositório Institucional do IPEN, 2016. http://repositorio.ipen.br:8080/xmlui/handle/123456789/27130.

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As raias são peixes peçonhentos e estão frequentemente associadas a acidentes em seres humanos, principalmente na região Norte do Brasil, favorecidos pelo hábito desses peixes de permanecerem no fundo de águas rasas e pela contínua utilização humana dos rios. Os ferrões das raias causam lesões dolorosas, edema, necrose, e o muco que recobre toda a extensão do corpo desses peixes pode aumentar a gravidade desses ferimentos. O objetivo deste trabalho foi avaliar a resposta imunológica induzida pelo muco de Paratrygon aiereba nativo ou irradiado por raios gama de 60Co em modelos animais. Foram realizados ensaios imunoenzimáticos e Western blotting para verificar a resposta humoral e reatividade cruzada dos soros provenientes de camundongos Swiss e coelhos New Zealand previamente imunizados contra o veneno, muco nativo ou irradiado. A indução da produção de anticorpos in vitro, as subclasses de IgG e a quantificação de citocinas foram analisados. Além de realizados ensaios de soroneutralização da atividade edematogênica in vitro e in vivo e de viabilidade celular. Os dados foram analisados estatisticamente por meio de análise de variância. O protocolo de imunização possibilitou a obtenção de soros com títulos satisfatórios de anticorpos policlonais. O muco e veneno de P. aiereba são imunogênicos e apresentam reatividade antigênica. O muco nativo ou irradiado induziu a produção de anticorpos IgG e esses reconheceram antígenos presentes no muco de outras espécies de raias. Células esplênicas de animais imunizados contra o muco irradiado produziram IFN-γ, TNF- α e IL-10 e também foi observada a produção sérica de TNF-α (grupo imunizado contra o muco irradiado) e de IL-6 e IL-17 (grupo imunizado contra o muco nativo). O soro anti-muco irradiado reduziu a atividade edematogênica in vitro, ao contrária da in vivo que não foi neutralizada. Os resultados corroboram o uso da radiação ionizante, com produção de anticorpos altamente responsivos e melhor resposta imune, além de comprovar que o muco de Paratrygon aiereba foi capaz de estimular resposta imune adaptativa celular e humoral.
Tese (Doutorado em Tecnologia Nuclear )
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
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17

Leippe, Donna Marie. "Stoichiometry of picornavirus neutralization by murine monoclonal antibodies." 1991. http://catalog.hathitrust.org/api/volumes/oclc/25611248.html.

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Thesis (Ph. D.)--University of Wisconsin--Madison, 1991.
Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 133-135).
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18

Szydlik, Agnieszka. "Expression and neutralization capacity of single domain HIV antibody fragments." Thesis, 2018. https://hdl.handle.net/10539/25256.

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A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine in the specialty of Virology, June 2018
The discovery of broad and potent neutralizing HIV antibodies (bNAbs) has opened up new opportunities of passive immunization for HIV-1 prevention. In this study, we have engineered CAP256-VRC26.25, a V1V2 bNAb that neutralizes 70% of clade C viruses, as a single domain antibody (sdAb). These small antigen binding entities are derived from naturally occurring heavy chain only antibodies present in members of the dromedary families, and are characterized by the absence of a light chain, long complementarity-determining regions (CDR) heavy (H) chain 3 and high stability. Since CAP256.25 contains a highly charged and protruding CDR-H3 that binds mainly through its heavy chain, we hypothesized that it may function well as an sdAb. Multiple camelization approaches to engineer CAP256.25 as a sdAb were tested in silico utilising structural modelling software. Parameters such as germline sequence homology, hydrophobicity and solubility, folding energy, torsion angles and native conformation of CAP256.25 in complex with its binding epitope were major factors considered during the modelling process. Four CAP256.25 sdAb derivatives were generated from parental antibody, the mut_0 or a wild type (WT), which was used as a base line for downstream optimization. CAP256.25 mut_4 in which residues involved in LC interactions were replaced with residues strongly conserved in camel sdAbs, which minimize hydrophobic interface of the sdAb. Mut_8 variant, which included four additional substitutions to increase solubility and mut_9 contained a single additional mutation at the base of CDR-H3 to improve the energetic landscape of sdAb. All genes were synthesized and sub-cloned into a mammalian expression vector and recombinant proteins expressed in HEK293T cell line, and purified by Immobilized Metal Ion Affinity Chromatography (IMAC) and Fast Protein Liquid Chromatography (FPLC). CAP256.25_mut0 expression was below the detectable level and whilst mut_4 expressed at low levels, it showed no neutralization activity. CAP256.25 sdAb mut_8 and mut_9 expressed at significantly lower levels compared to m36, a previously described sdAb used a positive control. Nevertheless CAP256.25mut_8 sdAb showed neutralization capability although it lost significant potency in comparison to the parental antibody, yet still within the therapeutic window of the VRC01 bNAb. Importantly, CAP256.25 sdAb was unable to neutralize the K169E mutant confirming that it retained specificity for the V2 epitope. These data suggest that camelization of human antibodies is possible although further engineering is required to increase expression and improve stability. As such, sdAb engineering could be an encouraging step for the generation of small antigen binding fragments for future therapeutic purposes including topical delivery at mucosal surfaces, to interrupt or block sexual transmission of HIV.
XL2018
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19

Chiang, Chen-Yi, and 江貞儀. "Application of Monoclonal Antibodies in Detection and Neutralization of Porcine Endogenous Retroviruses." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/45833872926833162751.

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博士
國立清華大學
生命科學系
93
Porcine endogenous retrovirus (PERV) is ubiquitous in pigs. The virus has drawn much attention recently because of the widespread use of pig organs and tissues in xenotransplantion. The virus has been found to be capable of infecting a bread range of cells including those of human origins in vitro and thus possesses a potential threat to xeno-transplant recipients who are generally immunosuppressed. Nevertheless, it remains unclear whether PERVs infection can cause human diseases. Therefore, the development of a highly sensitive and specific immunoassay for clinical surveillance is imperative in patients receiving xenotransplantation. We describe here the generation of monoclonal antibodies (mAbs) named A-11, 8E10, and 7C4 that specifically recognize either Gag or Env protein of PERV. In immunoblotting assay, the A-11 mAb was found to be able to detect all three classes of PERV: 8E10 mAb recognizes PERV-A and -B, whereas 7C4 mAb recognized only PERV-A. A-11 mAb can be used in detection of PERV in cultured cells, especially human kidney epithelial cells, larynx epithelial cells, hepatoma epithelial cells, and muscle spindle cells by immunochemistry. No cross-reaction with Gag and Env proteins of murine leukemia virus and human immunodeficiency virus-1,2 was observed, indicating that it was highly specific to PERV. In addition, the region recognized by mAb A-11, 8E10, and 7C4 was localized to amino acid positions 313 to 322 on the Gag protein, 427 to 434 and 517 to 537 on the Env protein, respectively. The synthetic peptides Gag313-322, Env417-434, recombinant Env397-481, and Env460-539 proteins effectively competed the binding of the mAb with recombinant Gag and Env proteins, respectively. The recombinant protein of Env was able to neutralize PERV infection. Furthermore, A-11 mAb was used in tissue tropism of PERV infection for different human cell lines by flow cytometry. Higher efficiency of PERV infection and higher expression of viral proteins were observed in human kidney cell line than others human cell lines. Therefore, these mAbs can be used as tools for the identification of viral proteins in basic research as well as clinical studies and application in neutralization of virus infection.
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20

Prasad, Nijaguna B. "Neutralization of toxic property Russell’s viper (Viper Russelli) Venom by polyclonal antibodies prepared against selected toxins." Thesis, 1996. http://hdl.handle.net/2009/1936.

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21

Registre, Ludy. "Examination of the role of envelope directed antibodies on co-receptor usage in HIV-1B infection." Thesis, 2018. https://hdl.handle.net/2144/29958.

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HIV-1 primarily utilizes the CCR5 receptor as a co-receptor, but over time, viruses can evolve to use the CXCR4 protein. Changes in the viral envelope V3 loop mediate this switch. The emergence of CXCR4-utilizing viruses has been presumed to occur as a consequence of decreased humoral immunity. We show that exclusively CXCR4-using (X4) viruses contain a 2 to 3 amino acid insertion in the V3 loop. Structural modeling revealed that this insertion caused a protrusion in the V3 loop, which impacts CCR5 receptor interaction. These genotypic and structural motifs affected neutralization susceptibility because X4, as compared to co-circulating CCR5-utilizing (R5) viruses, were less neutralization sensitive to autologous contemporaneous and heterologous plasma. Individuals with co-circulating X4 and R5, as compared to those with only R5, viruses had similar neutralization breadth and potency indicating that the emergence of X4 viruses is not associated with decreased humoral immunity. These results suggest that X4 viruses are neutralization escape variants and arise due to humoral selective pressure. This work has implications for future antibody-based therapeutics. Along with providing a framework for developing an HIV-1 vaccine, broadly neutralizing antibodies (bnAbs) are also being investigated as a potential therapeutic. BnAbs target a limited number of conserved HIV-1 envelope structures, including glycans in and around the V1/V2 and V3 domains. Along with the V3 loop, changes in V1/V2 are also known to impact co-receptor usage. We show that viruses that exclusively use the CXCR4 co-receptor, as compared to variants that only utilize CCR5, were less neutralization sensitive to V1/V2 and V3 directed bnAbs. In contrast, R5 and X4 viruses did not demonstrate neutralization differences to bnAbs that target non-V1/V2 and V3 envelope regions, such as the CD4 binding site and the membrane proximal external region. Structural modeling revealed that the predicted orientation of the V1/V2 loop among diverse HIV-1 variants predicts susceptibility to V3 loop directed bnAbs. In aggregate, our results suggest that viruses with different co-receptor usage have differing bnAb susceptibility. Furthermore, structural modeling may be used as a tool to predict neutralization susceptibility to bnAbs against regions associated with co-receptor usage.
2020-06-12T00:00:00Z
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22

Lin, Hong-En, and 林宏恩. "Study of the epitopes on dengue virus envelop protein recognized by different antibodies and the relationship to neutralization potency, specificity and maturation status." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/57756384855617318367.

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博士
國立臺灣大學
微生物學研究所
101
The four serotypes of dengue virus (DENV) are the leading cause of arboviral diseases in humans; currently here is no licensed dengue vaccine. The envelope (E) protein of DENV is the major target of neutralizing antibodies and vaccine development. The long-term goal of this study is to provide important information for the development of dengue vaccine. The objective of this study is to investigate the epitopes on dengue virus envelop protein recognized by different antibodies and the relationship of epitopes to specificity, maturation status of particles and neutralization potency of different antibodies. The first specific aim is to establish a high-throughput method of epitope mapping. We used a panel of 67 alanine mutants of surface exposed E residues to rapidly identify the epitopes of anti-E monoclonal antibodies (mAbs) and the predominant epitope of anti-E antibodies in polyclonal sera, followed by verification with capture-ELISA using virus-like particles (VLPs). Of the 12 mouse anti-E mAbs, three recognized a novel epitope in the E protein domain II central interface, and three recognized an epitope involving domain III and lateral ridge of domain II. This interdomain epitope can be recognized by this method. Using the same approach, we found the predominant epitope of anti-E antibodies in polyclonal sera of dengue patients consisted of the fusion loop of domain II and surrounding residues. The second aim is to determine the conservation index of each epitope residue based on the degree of conservation among different serocomplexes of flaviviruses and calculate the conservation scores of each epitope. We found a good correlation between the conservation scores of epitope and the specificity of antibodies. Conservation scores of epitope can explain the specificity of different antibodies. The third aim is to establish a method to produce mature DENV VLPs and study the effect of maturation status of VLPs to the binding of different anti-E mAbs. We found most group-reactive (GR) anti-E mAbs bound immature VLPs better than mature VLPs, suggesting that these antibodies are primarily induced by immature particles. In summary, we establish a high-throughput method to identify the epitopes on DENV E protein, including several interdomain epitopes. The conservation scores of epitopes analyzed in this study can explain the specificity of different antibodies. Moreover, we found the maturation status of VLPs affected the binding of different categories of anti-E mAbs recognizing different epitopes. These provide important information for the future development of dengue vaccine.
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Sekavová, Alžběta. "Příprava polyomavirových nanostruktur pro diagnostiku BK virových infekcí." Master's thesis, 2017. http://www.nusl.cz/ntk/nusl-355719.

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24

Batraville, Laurie-Anne. "Étude des immunoglobulines G dirigées contre l’enveloppe du VIH-1 dans des spécimens vaginaux et sanguins de travailleuses du sexe béninoises." Thèse, 2014. http://hdl.handle.net/1866/12123.

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Objectif: La caractérisation des facteurs immunitaires associés à la protection contre l’infection au VIH est cruciale pour le développement de stratégies de prévention. Cette étude évalue les IgGs dirigées contre l’enveloppe du VIH-1 dans le sérum et les liquides cervicovaginaux (LCV) de travailleuses du sexe béninoises. Méthode : Notre étude porte sur 23 travailleuses du sexe séropositives (TS+) et 20 travailleuses du sexe séronégatives (TS-). Le potentiel de neutralisation a été évalué par un essai de neutralisation. La détection d’IgGs a été effectuée par un ELISA sur base cellulaire. La capacité d’induire une réponse ADCC a été est évaluée par l’élimination de cellules cibles recouvertes de gp120 par le mécanisme d’ADCC. Résultats : Malgré que nous n’ayons pas détecté d’IgG dirigées contre l’enveloppe du VIH-1, ni d’activité de neutralisation ou d’élimination de cellules cibles par ADCC chez les TS-, nous avons facilement détecté ces activités de neutralisation et d’élimination de cellules cibles par ADCC chez les TS+ dans le sérum et les LCV qui reconnaissent mieux la forme de l’enveloppe liée à CD4. Ces IgGs pourraient être impliquées dans l’élimination par ADCC des cellules cibles présentant les glycoprotéines de l’enveloppe à leur surface, et ce à la muqueuse vaginale, le premier site de transmission virale. Conclusion : Ces résultats permettent pour la première fois de montrer la conformation de l’enveloppe préférentiellement reconnue par les IgGs présentes au site de transmission par voie sexuelle.
Objective: Characterization of the immune correlates of protection against HIV infection is crucial for the development of preventive strategies. This study examined HIV-1 envelope glycoproteins (Env) specific IgG in systemic and mucosal compartments of female Beninese commercial sex workers (CSWs). Design: 23 HIV-1-positive and 20 highly-exposed HIV-1-seronegative (HESN) CSWs were studied. Methods: HIV-1-Env specific IgG detection in sera and cervico-vaginal lavages (CVLs) from the study population was done by cell-based ELISA. The HIV neutralizing activity was evaluated with a neutralization assay. HIV-1 specific antibody-dependent cellular cytotoxicity (ADCC) response of the cohort was measured with a FACS-based assay evaluating the ADCC-mediated elimination of gp120-coated target cells. Results: No anti-HIV-1-Env-specific IgG, neutralizing or ADCC activities were detected in samples from HESN CSWs. Samples from HIV-1-infected CSWs presented ADCC activity in both sera and CVLs. Anti-Env IgG from sera and CVLs from HIV-1-infected CSWs preferentially recognized the Env in its CD4-bound conformation. Conclusion: Theses results demonstrate for the first time that HIV-1-infected CSWs have ADCC-mediating IgG that preferentially recognize Env in its CD4-bound conformation at the mucosal site.
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