O crescimento cada vez maior da industrialização e a existência de um mercado globalizado vêm exigindo dos produtores rurais à utilização de modernas tecnologias ligadas à produção animal. O baixo custo de produção e o aumento da qualidade do produto final gerado têm sido a grande meta dos pecuaristas e centros de pesquisa voltados à agropecuária. Devido à necessidade de aumento na produção, propriedades agropecuárias têm recorrido ao uso de todo e qualquer recurso disponível que possa promover melhorias na atividade produtiva.O uso indiscriminado dos antimicrobianos com a intenção de aumentar a produção animal pode resultar em concentrações residuais nos produtos como carne, ovos e leite acima das doses aceitáveis para o consumo humano. Inicialmente, os antibióticos eram usados como medida terapêutica, mas com o avanço do conhecimento e desenvolvimento de novos compostos, passaram a ser utilizados também como medida preventiva e como promotores do crescimento.O que se deve questionar não é a presença destes resíduos, mas sim o tipo e as concentrações dos mesmos. De fato, as modernas tecnologias analíticas têm permitido a detecção de partes por bilhão de substâncias químicas, componentes ou metabólitos de medicamentos de uso veterinário em alimentos e, nestas quantidades, dificilmente elas representariam perigo à saúde dos consumidores. Portanto, há necessidade de se estabelecer metodologias analíticas mais eficientes que possam auxiliar os órgãos de fiscalização da agricultura no controle do uso desses antimicrobianos na produção animal. Assim como auxiliar no controle de qualidade, principalmente em indústrias exportadoras que almejam expansão de mercado, onde essa prática é quase uma imposição no contexto do comércio internacional de produtos pecuários \"in natura\" e processados. Devido a essas necessidades, o objetivo do presente foi padronizar e desenvolver reagentes imunoenzimáticos para a pesquisa da ciprofloxacina em produtos de origem animal. Os resultados obtidos no desenvolvimento desses imunoreagentes foram positivos quando correlacionado (r2 = 0,9588) com o kit referência RIDASCREEN® Enro/Cipro da R-Biopharm. Diante disso, é possível concluir que o Brasil possui infra-estrutura adequada para padronizar esses testes rápidos, evitando com isso um grande gasto na importação desses kits / The growth each time higher of industrialization and the existence of a global market forces the rural producers to use modern technologies related to animal production. The low production cost and the increasing of obtained final product quality have been the great aim of cattle farmers and cattle farming research centers. Due to the necessity of increasing the production, rural properties have required the use of any available source able to promote better productive activities. The indiscriminated antibiotics use aiming to increase animal production may result in residual concentrations found in products such as meat, eggs and milk over acceptable doses to the human consumption. Firstly, antibiotics were used as therapeutic prescriptions although the advances on knowledge and development of new compounds were also used as a preventive way and as growth promoters. What must be questionable its not the presence of these residuals but surely what kind of residual and their concentrations. In fact, the modern analytic technologies make possible the detection of parts per billions (ppb) of chemical substances, compounds or medicine metabolites of veterinary use in food and in these quantities, hardly they would be dangerous to the consumer health. However, it\'s necessary to establish more efficient analytic methodologies to help on agriculture supervising organs in the control of these antibiotics use in the animal production. Thus, how to help in the quality control, mainly in industries that export to get the market expansion, where this activity is almost an imposition at the international trade context of farming products \"in natura\" and industrialized ones. Due to these necessities, the goal of our present work was to standard and develop immunoenzymatics reagents to the ciprofloxacin researching from animal products. The results obtained on development of these immunoreagents were positive when correlated (r2= 0,9588) to the kit RIPASCREEN® Enro/Cipro from R-Biopharm reference. At the light of these knowledge, it?s possible to conclude that Brazil has adequate proper-structure to standardize these quick tests, to avoid in this way a high cost to import these kits.

O presente estudo foi realizado para esclarecer o surgimento de vesículas subepidérmicas em enxertos de pele comumente descritos como áreas enxertadas. Devido à discrepância existente entre a literatura, que afirma surgirem vesículas nessas áreas, e a nossa experiência clínica, onde não observamos tal fato, decidimos investigar o problema. Para isso, estudamos a transição dermoepidérmica (TDE), em 23 pacientes submetidos à enxertia de pele, para verificar se há ou não alteração dessa estrutura que pudesse justificar a formação de vesículas. Nos 23 pacientes estudados foram feitas duas biópsias: a primeira, imediatamente antes da excisão do enxerto, na área doadora - pele sã, considerada como amostra padrão normal da TDE - Amostra Padrão AD. Após 10 dias, realizou-se uma segunda biópsia, com o mesmo vazador, próxima à área da primeira biópsia - Amostra Teste - ENX. Cada amostra foi dividida em 2 partes iguais (46 amostras) e estudadas por microscopia de luz e por imunofluorescência direta (imunomapeamento), pesquisando-se a possível alteração da zona da membrana basal (ZMB) na TDE através dos antígenos penfigóide bolhoso, laminina, colágeno IV e colágeno VII. Na microscopia de luz estudou-se, em cada biópsia, a relação entre a medida linear do relevo da trasição dermoepidérmica e a medida linear do relevo da superfície da camada granulosa, logo abaixo da camada córnea, equivalente a medida linear da superfície da pele. Nas 46 amostras as análises por microscopia de luz e de imunomapeamento para os quatro antígenos evidenciou-se a manutenção do mesmo padrão morfológico. Não houve diferença no imunomapeamento. Observou-se relações lineares das medidas com médias de 1,17 para a amostra AD e 1,44 para a amostra ENX, diferença que foi estatisticamente significativa, porém conservando a manutenção do padrão da TDE em relação à pele normal. Foi observada a manutenção do padrão do relevo da TDE no enxerto, em relação à pele sã, doadora. / SUMMARY: The present study has been done to elucidate the onset of subepidermal blisters in skin grafts, usually mistaken as grafted sites. Due to the discrepancy between literature - assigning this onset of blisters in grafts and our experience opposite we have decided to carry out this study. To do so we have studied the dermal-epidermal junction in 23 burned patients who underwent skin grafting so that we could verify whether or not there could be any alteration in the dermal-epidermal junction structure that may explain this fact. Among the 23 studied patients, two biopsies were carried out: the first one just before harvesting the skin graft from donor site healthy skin. The so called sample was regarded as an ordinary standard one of the dermal-epidermal junction STANDARD SAMPLE - DS. After graft take, by ten days, a second biopsy was performed with the same punch, close to the first biopsy TEST SAMPLE GS. Both samples were split into two equal parts (46 samples) and studied using light microscopy and direct immunofluorescence (immune mapping), searching for possible alterations in basement membrane zone in the dermal-epidermal junction through bullous pemphigoid, laminin and types IV and VII collagen antigens. On light microscopy, relation between the linear measure of dermal-epidermal junction projection and that of stratum granulosum surface, just underneath the stratum corneum, corresponding to skin surface, was studied in each biopsy. The four analyses of the antigens by light microscopy and direct immunofluorescence in the 46 samples clearly showed the keeping of the same pattern, either for STANDARD SAMPLE DS or TEST SAMPLE GS. There were no differences on the immune mapping. Regarding the relation of the linear measures it was noted a mean of 1.17 for STANDARD SAMPLE DS and a mean of 1.44 for TEST SAMPLE GS. Such difference was statistically significant. Nevertheless, it maintained the keeping of the same pattern of dermal-epidermal junction when compared to healthy skin.

No presente trabalho, a técnica de Espectroscopia de Correlação Angular Perturbada (CAP) foi usada para medir a interação de quadrupolo elétrico em amostras de DNA de diferentes linhagens de camundongos (A/J, C57BL/6, B6AF1, BXA1 e BXA2), amostras de antiimunoglobulinas pertencentes as subclasses (IgG1, IgG2a e IgG2b) e de porções ativas de imunoglobulinas fracionadas ou inteiras, correspondentes a parte diversificada da imunoglobulina, responsável pelo padrão de resposta imunológica apresentado pelos organismos. Também foram realizadas medidas da interação de quadrupolo elétrico em amostras de bases nitrogenadas do DNA (adenina, citosina, guanina e timina). As medidas CAP foram realizadas utilizando os núcleos de prova 111In111Cd; 111mCd111Cd; 111Ag111Cd; e 181Hf181Ta, nas temperaturas ambiente e do nitrogênio líquido para investigar as interações dinâmicas e estáticas, respectivamente. As biomoléculas foram marcadas por meio direto, onde os átomos dos núcleos de prova devem se ligar a um determinado sítio da biomolécula. Os materiais biológicos e os núcleos de prova foram escolhidos com o objetivo de verificar, em todos os seus aspectos, a possibilidade de aplicação da espectroscopia de CAP na investigação dos parâmetros hiperfinos em um núcleo de prova de um átomo metálico ligado às biomoléculas (incluindo o uso de diferentes núcleos de prova, resultantes do decaimento do núcleo pai de quatro diferentes metais) e estudar, por meio dos parâmetros hiperfinos medidos, o comportamento dessas diferentes moléculas. Os resultados obtidos mostram as diferenças entre a interação das biomoléculas estudadas com os núcleos de prova Estas diferenças foram observadas por meio de variações nos parâmetros hiperfinos medidos, que depende do tipo de molécula, mostrando que o núcleo de prova em alguns casos ligou-se as moléculas e em outros não houve esta ligação. / In the present work perturbed angular correlation (PAC) spectroscopy was used to measured electric quadrupole interactions in DNA biomolecules of different mice lineages (A/J, C57BL/6, B6AF1, BXA1 e BXA2), samples of different isotypes of immunoglobulin G (IgG1, IgG2a e IgG2b) and active portions of complete and fragmented immunoglobulin responsible by the immune response. Electric quadrupole interactions were also measured in DNA nitrogenous bases (adenine, cytosine, guanine, thymine). PAC measurements were performed using 111In111Cd; 111mCd111Cd; 111Ag111Cd; e 181Hf181Ta as probe nuclei, and carried out at room temperature and liquid nitrogen temperature, in order to investigate dynamic and static hyperfine interactions, respectively. The biomolecule samples were directly marked with the radioactive parent nuclei, whose atom link to a certain site in the biomolecules. The biological materials as well as the probe nuclei were chosen to investigate the possibility to use PAC spectroscopy to measure hyperfine parameters at nuclei from metallic elements bound to biomolecules (including the use of different probe nuclei produced in the decay of parent nuclei of four different metals) and also to study the behavior of different biomolecules by means of the measured hyperfine parameters. Results show differences in the hyperfine interactions of probe nuclei bound to the studied biomolecules. Such differences were observed by variations in the hyperfine parameters, which depend on the type of biomolecule and the results also show that the probe nuclei atom bound to the molecule in some cases and in others do not.

O ovo é o alimento naturalmente mais completo, uma vez que possui todos os nutrientes necessários, como vitaminas, aminoácidos e minerais essenciais para manter uma vida. Porém, em contra partida, possui várias proteínas promotoras de alergias em considerável parcela da população mundial. Para determinar as proteínas dos alimentos alergênicos, um dos testes mais utilizados é o imunoensaios tais como ELISA (ensaio imunoenzimático - enzyme linked immunosorbent assay), onde o anticorpo reconhece o antígeno e essa conexão é mostrada por um sistema enzimático, em outras palavras, a densidade óptica. O objetivo deste estudo foi determinar a eficiência do anticorpo policlonal, produzido em laboratório, para identificar a presença do antígeno ovomucóide em ovos tratados por irradiação gama para a sua desativação. Para avaliar os tratamentos, o anticorpo policlonal foi produzido em quatro (04) coelhos da raça Nova Zelândia, do sexo feminino, com 45 dias de vida, imunizadas com ovomucóide bioconjugado. Foi utilizado o adjuvante de Freund completo na primeira imunização e a solução tampão PBS, foram realizadas, posteriormente, quatro imunizações a cada quinze dias, mais um reforço 48 horas antes da retirada do plasma sanguíneo. O soro sangüíneo foi titulado por PTA-ELISA (Plate trapped antigen). Todos os procedimentos foram aprovados pelo Comitê de Ética e Experimentação Animal do Instituto de Ciência Animal e Pastagens (IZ) e precedida de acordo com as normas europeias para o bem-estar e ética animal. Foram utilizados ovos comerciais in natura, fornecidos pelo Departamento de Genética da Universidade de Agricultura \"Luiz de Queiroz\" - ESALQ / USP. As amostras foram submetidas à radiação gama proveniente de uma fonte de Co60, do tipo Multipropósito no Instituto de Pesquisas Energéticas e Nucleares (IPEN), sob uma taxa de dose de 19,4 e 31.8Gy/hora, nas doses: 0 (controle); 10kGy; 20KGy e 30KGy, em todas as taxas. Pelo teste de ELISA, foi encontrado o alérgeno ovomucóide das amostras ovo e, pelo resultado apresentados, constatou-se que o tratamento da radiação não mostrou alterações significativas, quando avaliado por anticorpos policlonais. Assim, podemos concluir que o anticorpo produzido é capaz de identificar a proteína alergênica ovomucóide e, a irradiação gama em tais taxas não apresenta mudanças na estrutura da proteína, por esta forma de avaliação. Porém, apresentou algumas alterações na cor e viscosidade visual das amostras de ovos / The egg is the most complete natural food; it has all the necessary nutrients such as vitamins, aminoacids and essential minerals to maintain a life. However, although, has several proteins that promote allergies in considerable part of the world population. To determine allergenic food proteins, one of the most used tests is the immunoassays such as ELISA (enzyme linked immunosorbent assay), where the antibody recognizes the antigen and this connection is showed by an enzymatic system, in other words, optical density. The aim of this study was to determine the polyclonal antibody efficiency, produced in laboratory, to identify the presence the ovomucoid antigen in treated eggs by gamma irradiation for its inactivation. To evaluate the treatments, polyclonal antibody was produced in four New Zealand female rabbits, at 45 days old, immunized with bioconjugated ovomucoid. Was used Freund Complete Adjuvant at first immunization and PBS Buffer at four subsequently immunizations every fifteen days, plus a booster 48 hours before the blood retreated. The blood serum was tittered by PTAELISA (Plate trapped antigen). All procedures were approved by Institute of Animal Science and Pastures (IZ)´s Committee of Ethical and Animal Experimentation and preceded according to European Norms for ethical and animal welfare. It was used, in nature, commercial laying eggs, from the Genetic Department of Agricultural University Luiz de Queiroz ESALQ/USP. So the samples were submitted to the gamma radiation coming from a source of Co60, type Multipurpose at the Energetically Researches and Nuclear Institute (IPEN), under a dose rate of 19.4 and 31.8Gy/hour, in the doses: 0 (control); 10KGy; 20KGy and 30KGy, in all rates. By the ELISAs test we can find the egg allergen ovomucoid and the radiation treatment do not showed considerable changes. So we can concluded that the antibody produced is capable of identify the ovomucoid allergenic protein and the gamma irradiation in such rates does not shows changes in that protein, therefore showed some changes in the color and visual viscosity of the egg samples

A leptospirose está entre as principais zoonoses causadoras de morbidade e morte em humanos. Neste tipo de infecção, a resposta imune inata e humoral são essenciais para o seu controle. Nós avaliamos a importância de C3 na fagocitose Leptospira interrogans sorovar Kennewicki estirpe Pomona Fromm por macrófagos obtidos de camundongos selvagens e deficientes da proteína C3 do Sistema Complemento. Nossos resultados demonstram que as leptospiras são refratárias à ação dos macrófagos e que a internalização dessas bactérias, só pôde ser melhorada após adição de soro, como fonte de C3b e iC3b. Além disso, nós também investigamos a participação de C3 na produção de anticorpos. Para tanto, nós imunizamos camundongos selvagens e deficientes em C3 com a bactéria. Os camundongos deficientes em C3 produziram baixos níveis de anticorpos em comparação com os camundongos selvagens. É importante destacar que a produção de anticorpos envolve a participação do fragmento C3d(g), reconhecido como importante adjuvante na imunidade humoral. / Leptospirosis is amongst the main bacterial zoonosis that cause morbidity and death in human beings. In this type of infection, the innate and humoral immune response is essential for its control. We evaluated the importance of C3 in the phagocytosis of Leptospira interrogans serovar Kennewicki strain Pomona Fromm by macrophages obtained from wild and deficient mice of the Complement System C3 protein. Our results demonstrate that leptospires are refractory to the action of macrophages and that the internalization of these bacteria could only be improved after addition of serum as the source of C3b and iC3b. In addition, we also investigated the role of C3 in the production of antibodies. To do so, we immunized wild and C3 deficient mice with the bacterium. C3 deficient mice produced low levels of antibodies compared to wild mice. It is important to note that the production of antibodies involves the participation of the fragment C3d (g), recognized as an important adjuvant in humoral immunity.

In this thesis I develop and implement “antibody landscapes”, a method to profile immunity against a pathogen as a function of antigenic differences between a range of strains. Theoretically applicable to any antigenically variable pathogen and measurement of immunity, the work here focusses on antibody-mediated immunity against the A/H3N2 influenza subtype. Applying the methodology to study annual serum samples from individuals monitored for influenza infection over a period of six years, patterns of influenza immunity were found to be remarkably distinct and maintained almost unchanged over time in the absence of influenza exposure. Upon infection, the initial response is strikingly antigenically broad, including responses against viruses far beyond the extent of cross-reactivity observed after a primary infection. Analysis of two vaccination cohorts, one receiving an antigenically advanced vaccine strain and one a more typical vaccine strain choice, revealed many of the same patterns of response as seen with infection. Antigenically advanced vaccination generated greater responses against later strains but surprisingly, due to equivalent boosting of prior immunity, this came at no cost to responses generated against contemporary or older strains. Exploring in more detail the development of immunity over time, analysis of a cohort of children demonstrated that - in contrast to adults with diverse exposure histories - antibody responses to a first infections were remarkably similar in pattern and magnitude. Interestingly, for second infections, although post-infection antibody titres against circulating strains were comparable to those after first infections, overall cross-reactivity of the response against future antigenic variants appeared to be diminished. The findings here underline the significant role prior-immunity plays in affecting the response to new exposures and the importance of understanding it. An important conclusion is that by failing to account for it, current approaches to influenza vaccine strain selection may be suboptimal and pre-emptive vaccine strain updates may improve overall vaccine efficacy where immunity to current strains already exists in the population. Building on the work presented here should help to optimise strain choice and vaccine efficacy even further.

Background: Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease characterised by autoantibody production and variable clinical features, ranging from mild to severe disease. Patients with SLE are at increased risk of developing accelerated atherosclerosis. Biomarkers have potential utility in SLE as markers of disease or predictors of future clinical events and mortality. Objective The aim of this thesis was to identify serological biomarkers predictive for erosive arthritis (EA), cardiovascular events (CVEs), mortality and subclinical atherosclerosis in SLE. Methods: In chapters 2 to 4, study subjects were SLE patients from Bath. Anti-cyclic citrullinated peptide antibodies (ACPA) and HLA-DR and -DQ were studied for markers of EA, and anticardiolipin (aCL) and lipoprotein profiles for markers of CVEs and mortality. In chapters 5 and 6, study subjects were women with SLE from Manchester. B-mode ultrasound scans of subjects' carotid arteries were performed at baseline and follow-up time-points to detect atherosclerotic plaque. Baseline IgG and IgM antiphospholipid (aPL) antibodies and CV risk factors were studied for markers of subclinical atherosclerosis. Clinical data collected for all studies included SLE features and auto-antibody profiles. Results: ACPA was identified as a marker of a SLE phenotype with EA - "rhupus". Patients with major erosive arthritis were HLA-DQB1*0302 carriers. Increased aCL GPL levels and total cholesterol : high density lipoprotein-C (TC : HDL-C) ratio were markers for future CVEs, and increased TC : HDL ratio, aCL GPL and lipoprotein(a) concentrations were markers for increased mortality. Lower HDL-C concentrations and anti-annexin A5 (anti-AnxA5) GPL were markers of carotid plaque progression. Conclusion: This thesis identified new markers for EA, subclinical atherosclerosis and future CVE and mortality risk in SLE. Strategies to incorporate these new CV markers into clinical CV risk assessments may assist in distinguishing the subset of SLE patients most at risk of developing accelerated atherosclerosis.

An effective antibody response is essential for immunity against influenza virus infection and is the primary goal for vaccine development. In this study, codon optimized and wild type DNA vaccines expressing hemagglutinin (HA) antigens of human flu viruses A/H1N1/NewCal/20/99 (H1 serotype) were compared to test the antigenic differences of the constructs in mammalian systems. Furthermore, to determine if a prime-boost immunization strategy was more effective in eliciting a greater immune response, a codon optimized HA vaccine was administered as a prime in conjunction with the trivalent inactivated vaccine (TIV), Fluzone, as a boost and immune responses were measured. We found that protein expression and antibody response levels of HA antigens were increased with the codon optimized construct when compared to the wild type HA gene construct. Prime-boost vaccination of NZW rabbits was able to elicit a greater immune response when compared to TIV alone as measured by enzyme-linked immunosorbent assay (ELISA), hemagglutinin inhibition (HI) and neutralizing antibody (NAb) assays. Together, these studies indicate that optimal HA DNA vaccine formulations should be codon optimized and can be used as part of a prime-boost vaccination strategy.

Orientador: Elenice Deffune / Resumo: A Hemorragia feto-materna (HFM) se dá pela transferência do sangue fetal para o compartimento intravascular materno, devido à ruptura na membrana vásculo-sincicial da placenta. A HFM pode ser responsável pela alosensibilização do sistema imune materno, levando a morbidade e mortalidade de gravidez corrente e/ou de futura, assim como também constitui a base da etiopatogenia de várias afecções, como se verifica na doença hemolítica perinatal que expõe a complicações fetais como, hidropsia, danos cerebrais hipóxicos e morte fetal. Detectar e quantificar hemácias fetais ajuda a prevenir as consequências devida a ocorrência da HFM. Entre os testes diagnósticos utilizados na detecção e quantificação têm - se os mais sensíveis o teste quantitativo de Kleihauer e Betke e o de Citometria de fluxo, sendo o primeiro considerado padrão ouro. Tendo em vista a importância do diagnóstico laboratorial da HFM foi desenvolvida a proposta da construção de imunossensor para detecção de amostras de sangue fetal na corrente sanguínea materna. Foi realizada a produção de anticorpo monoclonal com objetivo de produzir anticorpos contra antígenos de superfícies de hemácias fetais humana e também foram realizadas a construção de unidades sensoriais para detecção de sinal biológico de imunoafinidade entre anticorpos que detectam hemácias fetais e a hemoglobina fetal presente nestas células, a fim de se diagnosticar a ocorrência da HFM. Foram obtidos como resultados a produção de anticorpo monoclonal que... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Fetomaternal hemorrhage (FMH) occurs through the transfer of fetal blood into the maternal intravascular compartment, due to rupture in the placenta-syncytial membrane. FMH may be responsible for the alosensitization of the maternal immune system, leading to morbidity and mortality of current and / or future pregnancies, as well as being the basis of the etiopathogenesis of various conditions, as in perinatal haemolytic disease that exposes to fetal complications such as hydrops, hypoxic brain damage, and fetal death. Detecting and quantifying fetal erythrocytes helps to prevent the consequences due to the occurrence of FMH. Among the diagnostic tests used in the detection and quantification are the most sensitive the quantitative test of Kleihauer and Betke and the one of Flow cytometry, being the first considered gold standard. Considering the importance of the laboratory diagnosis of FMH, the proposal of the construction of immunosensor for the detection of fetal blood samples in the maternal blood was developed. The production of a monoclonal antibody was carried out with the objective of producing antibodies against antigens on human fetal red blood cells and also the construction of sensorial units for the detection of biological signal of immunoaffinity between antibodies that detect fetal red blood cells and the fetal hemoglobin present in these cells, in order to diagnose the occurrence of FMH. The results obtained are the production of monoclonal antibody that detec... (Complete abstract click electronic access below) / Mestre

La glycoprotéine VI (GP VI) des plaquettes sanguines humaines est le récepteur principal du collagène, composé le plus thrombogénique d'une paroi vasculaire lésée. Ainsi, GPVI est souvent considérée comme une cible de premier plan pour développer des tests diagnostiques ou des stratégies thérapeutiques innovantes, efficaces et sûres afin d'améliorer encore la prise en charge des accidents ischémiques. Les anticorps monoclonaux et leurs fragments actifs produits par ingénierie moléculaire constituent aujourd'hui une nouvelle classe de biomolécules en plein essor avec des propriétés bien adaptées à des applications thérapeutiques et diagnostiques. Notre groupe a produit plusieurs anticorps monoclonaux anti-GPVI par immunisation génique de souris. Ces anticorps ont une affinité élevé pour leur cible. Ils de distinguent les uns des autres par leur spécificité épitopique ainsi que par les effets engendrés par leur liaison à GPVI. Parmi ces anticorps, l'un présente un fort potentiel diagnostique parce qu'il reconnait les formes mono- et dimériques de GPVI, mais sa liaison aux plaquettes peut induire une activation ou la perte de GPVI. Un autre anticorps présente un fort potentiel thérapeutique parce que ses fragments actifs monovalents obtenus par papaïnolyse neutralisent l'interaction entre les plaquettes et le collagène, sans activer les plaquette. Cependant, l'origine xénogénique de cet anticorps est responsable d'une forte immunogénicité qui en interdit des applications en médecine humaine. Dans cette étude, nous avons conçus un fragments variable d'anticorps simple chaine (scFv) utile pour quantifier l'expression de la GPVI à la surface des plaquettes sanguines. Ce scFv a été reformaté de façon à lui insérer un motif de reconnaissance de la Protéine L (PpL) qui facilite sa détection et sa purification sans avoir recours à un peptide "drapeau". Nous avons également humanisé et créé plusieurs fragments d'anticorps recombinants monovalents inhibiteurs de l'interaction GPVI / collagène. Ces fragments d'anticorps présentent un potentiel thérapeutique élevé. / Human platelets glycoprotein VI (GPVI) is evidenced to be a platelet receptor of major importance in the occurrence of arterial thrombosis. Thus, it can be considered to be of great interest in diagnosis and therapeutic of atheriosclerotic diseases. Antibodies are powerful molecules which can be used in both diagnostic as well as for therapeutic purposes due to their unique characteristics. Monoclonal and recombinant antibodies have antigen restricted specificity, high affinity and can be used in various assays. Moreover, the good knowledge of their structure and molecular engineering facilities now allows the antibody modulation according to desired properties.Our group has already produced several monoclonal antibodies to human GPVI by gene gun immunization against the immunoadhesin hGPVI-Fc, which differ in fine epitopespecificity, affinity and other functional properties (Lecut et al. 2003). One, 3J24, with diagnostic potential while the other, 9O12, has a therapeutic potential because it blocks the binding of GPVI to collagen. Its Fab fragment has been extensively characterized in vitro,ex vivo and in vivo for its antithrombotic properties.Here, we designed and reshaped a single-chain antibody fragment (scFv) based on 3J24variable domains for the quantification of GPVI with diagnostic potential. We were also involved in the design, production and functional evaluation of humanized anti-GPVI recombinant antibody fragments (scFvs and Fabs) with therapeutic properties.