Le travail décrit dans ce manuscrit présente l'utilisation de deux techniques immunoenzymatiques pour le criblage à haut débit de catalyseurs chimiques et biologiques. Ces méthodes, jusqu'à présent réservées au monde du diagnostic sont employées ici pour la découverte de nouveaux catalyseurs pour des réactions énantiosélectives ainsi que pour des réactions de couplage. Les dosages immunologiques « par compétition » faisant appel aux anticorps monoclonaux se sont avérés des excellents outils pour le criblage à haut débit de catalyseurs asymétriques pour les réactions d'insertion de carbénoïdes dans la liaison OH de l'eau tandis que celles utilisant des anticorps polyclonaux ont été utilisées avec succès pour le criblage de biocaytalyseurs pour la réaction d'ouverture énantiosélective des oxazolones par l'eau. Un autre type de méthode utilisant des anticorps monoclonaux, appelée « sandwich » nous a permis de réaliser le criblage à haut débit de catalyseurs pour la réaction de cycloaddition alcyne-azoture.

In light of the significant modulation of receptor activity previously shown by a peptide (designated L247), designed to mimic the third extracellular loop of the human P2Y2 receptor, the aim of this study was to use this peptide as an immunogen to generate and fully characterise polyclonal rabbit antibodies to the P2Y2 receptor. Other aims of this study were to characterise epithelial and endothelial cells for a thorough expression profile of P2Y receptor mRNA transcripts in order to provide a rapid screen for the molecular determinants of these receptors in these cells. These studies also aimed to confirm previously published pharmacology, thus, to set the basis for western blot studies using P2Y receptor antibodies. Bovine aortic endothelial cells that co-express P2Y1 and P2Y2 receptors; EAhy926, a human endothelial fusion cell line, that express P2Y2 receptors; and ECV304 human bladder cancer cell line, known to express P2Y2-like and P2Y11-like receptors were used in this study. The dose dependent accumulation of inositol phosphates and cAMP response to potent P2Y11 agonists and RT-PCR studies confirmed the functional expression of both P2Y2 and P2Y11 receptors in ECV304 cells. Likewise, the dose dependent accumulation of inositol phosphates in response to potent P2Y2 and P2Y6 agonists and the presence of mRNA transcripts confirmed the expression of functional P2Y2/4- like and P2Y6- like receptors in EAhy926 cells. Polyclonal antiserum raised against L247 peptide was affinity purified and the purified fractions showed strong immunoreactivity with immobilised immunogenic antigen in ELISA. In western blot analysis L247 rabbit polyclonal anti-P2Y2 antibody detected strong bands in ECV304 and EAhy926 cells. On pre-absorption with the immunogenic peptide these responses were abolished suggesting that this antibody is antigen specific. Agonist induced P2Y2 receptor desentisation studies in ECV304 cells showed that prolonged agonist incubation caused the receptor sequestration. The loss of bands caused by P2Y2 receptor desensitisation and sequestration in membrane enriched fractions of agonist incubated ECV304 cells confirmed the specificity of L247 antibody. This antibody also showed no immunoreactivity in 1321N1 human brain astrocytoma cells devoid of any P2Y receptor subtypes cells. Deglycosylation studies revealed that the P2Y2 receptors are glycosylated in ECV304 cells. The polyclonal rabbit anti-P2Y2 receptor antibodies obtained from commercial sources produced completely different immunoreactive profiles with multiple bands even in 1321N1 cells. Furthermore, in comparison to L247 anti-P2Y2 antibody the commercial antibody showed no difference between normal and agonist incubated cells suggesting that this antibody may not be recognising the P2Y2 receptors in ECV304 cells. Likewise polyclonal rabbit antibodies to other P2Y receptors either showed no response or showed strong immunoreactive profile with multiple bands even in 1321N1 cells suggesting that these antibodies may not have been extensively characterized. Furthermore, immunofluorescence studies with commercial anti-P2Y2 antibodies showed that they may be only recognising non-denatured receptors. These studies suggest that the L247 anti-P2Y2 antibody raised against peptide designed to mimic specific region in the third extracellular loop of human P2Y2 receptor is highly specific and sensitive and provides an important tool to study endogenously expressed P2Y2 receptors in both non-denatured and denatured state. These studies indicate that this strategy of generating antibodies may be used to generate highly specific antibodies to other P2Y receptor subtypes.

As many genome sequencing efforts today are completed, we are now provided with the genetic maps for several organisms, including man. With these maps at hand, the scientific focus is now shifting towards investigations of the functionality of proteins. This task is even more challenging than the genomic field since proteins, in contrast to DNA, do not allow themselves to be specifically probed or amplified by easy and generic methods. However, to achieve knowledge regarding protein function, useful information includes where, when and how much certain proteins are expressed in an organism. Such information can be obtained if protein-specific binding molecules are available as tools. One such class of target specific binders are the antibody molecules, traditionally employed in a broad variety of biotechnical applications, including protein localization studies on both cellular and sub cellular levels. In a first serie of studies, new methodology for recombinant production and purification of antigens for generation of antibodies via immunization routes were investigated. Parallel affinity gene fusion-based expression systems were used for evaluation of different concepts for production of antigen and post-immunization antibody purification. Carefully designed protein antigens from different organisms were produced and used to raise antisera which were affinity purified on their respective antigens to obtain highly specific polyclonal antibodies (monospecific antibodies). One of the constructed expression systems includes an affinity handle, ZSPA-1, previously selected from a combinatorial protein library for its capacity to selectively bind protein A. This allows for convenient, non IgG-dependent, affinity purification of proteins on conventional protein A resins. A strategy where highly target specific antibody preparations could be affinity purified in a more streamlined setup is also presented. By this strategy it was possible to fractionate antibodies showing reactivity to different parts of the antigen into separate fractions. This resulted in affinity purified antibodies showing monospecific but still multi-epitope reactivity. Purified monospecific antibodies were used in different studies including Western blot immunofluorescence and recovery applications. For affinity purification of endogenous target from its native surrounding a selective elution strategy where the recombinant antigen was used to competitively elute the captured target was developed. / QC 20100824

Made available in DSpace on 2014-06-11T19:30:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-12-08Bitstream added on 2014-06-13T20:00:43Z : No. of bitstreams: 1 cangiani_ee_dr_arafcf.pdf: 879107 bytes, checksum: bd2b88a537298b5f3ad944c986ece870 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Yersinia enterocolitica é um enteropatógeno que pode levar ao desenvolvimento de artrite reativa. Um dos mecanismos imunomoduladores usados pelos patógenos artritogênicos é a ativação policlonal de linfócitos. O objetivo deste estudo foi verificar se ocorre ativação policlonal de linfócitos B e comparar o padrão isotípico de imunoglobulinas produzidas durante a infecção com Y. enterocolitica O:8 em linhagens de camundongos suscetíveis (BALB/c) e resistentes (C57BL/6), bem como analisar o padrão de secreção de citocinas pró-inflamatórias e regulatórias pelas populações de células T CD4+ e CD8+. / Yersinia enterocolitica is an enteropathogen that can lead to the development of reactive arthritis. One of the immunomodulating mechanisms used by arthritogenic pathogens is the polyclonal activation of lymphocytes. The objective of this study was to verify if the polyclonal activation of B-lymphocytes occur and to compare the different immunoglobulin (Ig) isotypes produced during the infection with Y. enterocolitica O:8 in susceptible (BALB/c) and resistant (C57BL/6) mice strains. We also analyzed the production of pro-inflammatory and regulatory cytokines in T CD4+ and T CD8+ lymphocytes populations.

The present research focused on the development of a new methodology to assess the strength of the interaction between vaccine antigens and elicited polyclonal antibodies through SPR biosensors. Quantifying the binding strength of polyclonal antibodies is of first importance to evaluate the quality of the vaccine as well as to increase the scientific knowledge of immune protection mechanisms. To now the development of such tool has been complicated by the non-specific binding caused by high protein abundance in the blood and serum samples but also by the way of interpreting the data resulting from multi-interaction events measured at the same time. At first, we unsuccessfully tried to segregate the individual affinity contribution of each antibody population by measuring the signal as the sum of singular interactions. Differentiation of the singular contribution would have needed the fulfillment of the “additivity” hypothesis, meaning that each antibody bind identically alone or in mixture with other antibody. This hypothesis was not met and mathematical assessment by the sum of singular contribution led to fitting results that did not reflect the biological reality. It was therefore decided to switch the analysis method and to measure the end association binding level reached by the different samples injected at the same specific antibody content. The dissociation behavior was interpreted by the percentage of binding after long and fixed dissociation time. In a first application, we compared the antibodies elicited by two different commercially available vaccines and we showed that the binding interaction was not concentration dependent as, highly different levels were reached when injecting identical antibody concentration. No statistical significant difference was observed between both vaccines. Research firstly focused on the decrease of the non-specific binding and we found that ionic strength was a key parameter, increasing the buffer salt concentration reduced the non-specific binding without diminishing the binding strength. The sample composition was also a key parameter and purifying the IgG allowed to decrease dramatically the undesired binding events. A second application aimed at showing the equivalence between two different antigen constructions for two antibodies population. Even if identical antigen level immobilization is a challenge, the methodology is completely suitable to perform a 2-dimensional comparison (ligand and analyte). A last application was dedicated to the comparison between D and Q-pan Flu vaccines, and results showed that there was no statistical evidence of significant differences between both vaccines. End association level correlated well with haemagglutination inhibition assay at least when serum samples were not diluted at the same antibody content. This last application also showed that throughput may be extended to more than 50 samples per 80 hours / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Made available in DSpace on 2014-06-11T19:27:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-13Bitstream added on 2014-06-13T19:55:43Z : No. of bitstreams: 1 teixeira_wfp_me_araca.pdf: 257034 bytes, checksum: 74c2e0dcd5f52341aac3382521691e4d (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste estudo foi padronizar a reação de Imunofluorescência direta (IFD) para detecção de oocistos de Cryptosporidium parvum em amostras fecais de bezerros. Os anticorpos policlonais anti-C. parvum foram produzidos em dois coelhos adultos da raça Nova Zelândia, e titulados por meio do ensaio imuno-enzimático indireto (ELISA). A fração de imunoglobulina G (IgG) foi purificada a partir do soro do coelho imunizado, utilizando-se diálise em sulfato de amônio seguida de cromatografia em coluna de DEAE celulose e conjugação com isotiocianato de fluoresceína. Por meio da IFD padronizada, foi testada a reatividade cruzada do conjugado produzido contra outras espécies de Cryptosporidium (C. andersoni e C. serpentis) e com outros agentes etiológicos presentes em fezes de bovinos (Escherichia coli, Eimeria sp. e Candida sp.). A IFD foi comparada à microscopia com contraste de fase em solução de Sheather (MCF), avaliando a capacidade de detecção de oocistos de Cryptosporidium sp. em alíquotas de fezes de bezerro inoculadas com oocistos de C. parvum, e em amostras fecais de bezerros naturalmente infectados provenientes de 37 propriedades leiteiras da região de Araçatuba, SP. Nas amostras comprovadas como positivas, foi ainda feita uma análise semi-quantitativa de oocistos de Cryptosporidium sp., visualizados por campo de microscopia em ambas as técnicas. O conjugado anti-C. parvum apresentou reatividade com C. andersoni e C. serpentis. A IFD foi utilizada com sucesso para detecção de oocistos de C. parvum em fezes de bezerros, apresentando sensibilidade para detecção de até 104 oocistos em 3 g de fezes. Entre as 300 amostras fecais de bezerros naturalmente infectados, 19,67% (59/300) foram positivas para a presença de oocistos de Cryptosporidium sp. pela IFD, apresentando diferença estatisticamente significante (p<0.05) em relação às amostras positivas pela MCF / The objective of this experiment was to standardize the direct immunofluorescence assay (DIF) for detection of Cryptosporidium parvum oocysts in fecal samples of calves. The anti-C. parvum polyclonal antibodies were produced in two adult rabbits of New Zealand breed, and titrated by an indirect enzyme-linked immunosorbent (ELISA). The immunoglobulin G (IgG) was purified from the serum of immunized rabbits by dialysis in ammonium sulfate followed by column chromatography on DEAE cellulose, and conjugated with fluorescein isothiocyanate. Using DIF the anti-C. parvum conjugate was tested for the presence of cross-reactivity against other species of Cryptosporidium (C. andersoni and C. serpentis), and other infectious agents present in cattle fecal samples (Escherichia coli, Eimeria sp. and Candida sp.). A comparison between the DIF and phase contrast microscopy in Sheather solution (MCF) was accomplished for evaluation of the efficiency of both techniques for detection of Cryptosporidium sp. oocysts in fecal samples of calves seeded with C. parvum oocysts, and in fecal samples from naturally infected calves from 37 dairy farms in the region of Araçatuba, SP. A semi-quantitative analysis of Cryptosporidium sp. oocysts per microscopic field was accomplished for both techniques. The anti-C. parvum conjugate showed cross-reactivity with C. andersoni and C. serpentis oocysts. The DIF has been used successfully in the detection of C. parvum oocysts in fecal samples of calves, with sensitivity for detecting 104 oocysts in 3 g of fecal samples. Among the 300 fecal samples from naturally infected calves, 19.67% (59/300) were positive for Cryptosporidium oocysts by DIF, with significant statistical difference (p <0.05) when compared to the number of positive samples by MCF

Resumo: Yersinia enterocolitica é um enteropatógeno que pode levar ao desenvolvimento de artrite reativa. Um dos mecanismos imunomoduladores usados pelos patógenos artritogênicos é a ativação policlonal de linfócitos. O objetivo deste estudo foi verificar se ocorre ativação policlonal de linfócitos B e comparar o padrão isotípico de imunoglobulinas produzidas durante a infecção com Y. enterocolitica O:8 em linhagens de camundongos suscetíveis (BALB/c) e resistentes (C57BL/6), bem como analisar o padrão de secreção de citocinas pró-inflamatórias e regulatórias pelas populações de células T CD4+ e CD8+. / Abstract: Yersinia enterocolitica is an enteropathogen that can lead to the development of reactive arthritis. One of the immunomodulating mechanisms used by arthritogenic pathogens is the polyclonal activation of lymphocytes. The objective of this study was to verify if the polyclonal activation of B-lymphocytes occur and to compare the different immunoglobulin (Ig) isotypes produced during the infection with Y. enterocolitica O:8 in susceptible (BALB/c) and resistant (C57BL/6) mice strains. We also analyzed the production of pro-inflammatory and regulatory cytokines in T CD4+ and T CD8+ lymphocytes populations. / Orientador: Beatriz Maria Machado de Medeiros / Coorientador: Iracilda Zeppone Carlos / Banca: Maria Regina D'Império Lima / Banca: Phileno Pinge / Banca: Ângela Maria Victoriano de Campos Soares / Banca: Deise Pasetto Falcão / Doutor

Orientador: José Osmar Gaspar / Banca: Hugo Kuniyuki / Banca: Fátima Pereira de Souza / Resumo: O lenho estriado de rupestris ou cascudo (Rupestris stem pitting - RSP), um dos componentes do Complexo do lenho rugoso ("Rugose wood" - RW), é considerado uma das doenças de videira transmitidas por enxertia de grande relevância econômica para a viticultura. O Rupestris stem pitting associated virus - RSPaV foi associado com a doença do lenho estriado ou cascudo, sendo classificado como espécie do gênero Foveavirus, pertencente a família Flexiviridae. No presente trabalho, descrevem-se o sequenciamento do gene da proteína capsidial (CP) de um isolado brasileiro do RSPaV (RSPaV-SP), sua expressão em Escherichia coli, purificação da proteína capsidial recombinante e a produção de anti-soro policlonal em coelho. O sequenciamento do gene resultou em uma seqüência de 780 nucleotídeos e 259 aminoácidos deduzidos com massa molecular estimada de 28 kDa. A análise filogenética, entre a seqüência correspondente à CP do RSPaV-SP e outras variantes do mesmo vírus, evidenciou a formação de 4 grupos distintos, sendo o isolado brasileiro incluído no grupo da variante BS do RSPaV. A proteína capsidial recombinante foi purificada em coluna de afinidade e apresentou massa molecular estimada de 32kDa (4kDa da seqüência do vetor e 28kD da CP do RSPaV-SP). O anti-soro produzido apresentou-se específico na detecção da proteína capsidial recombinante purificada por "Western-blot", sem reação com proteína heteróloga a partir da diluição 1:4000. Nesta diluição, o anti-soro foi efetivo na detecção do vírus em extratos de plantas infectadas, sendo que nenhuma reação foi observada com extratos de plantas sadias. Considerando-se que este vírus apresenta variações de concentração na planta durante as estações do ano, e que, os testes sorológicos foram realizados durante a estação de baixa concentração do vírus, os resultados ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Rupestris stem pitting (RSP), a component of the rugose wood (RW) complex, is one of the most graft-transmissible grapevine virus diseases with great economic importance for viticulture . Rupestris stem pitting-associated virus (RSPaV), genus Foveavirus within the family Flexiviridae, has been associated with this disease. This work reports the sequencing of the coat protein (CP) gene of a brazilian an isolate of RSPaV (RSPaV-SP), its expression in Escherichia coli, purification of the recombinant coat protein and production of a polyclonal antiserum in rabbit. CP gene was found to be 780nt long, with a 256 deduced amino acid sequence encoding a predicted protein of 28 kDa. In filogenetic analysis, with RSPaV-SP and other variants of the virus, four groups were found and the sequence of RSPaV-SP showed the highest identity with the variant RSPaV-BS. The recombinant coat protein was purified by affinity chromatography and showed a molecular weight of 32kDa (4 kDa from a small vector sequence plus 28 kDa for the CP of RSPaV-SP). The antiserum proved specific for detection of the recombinant protein by Western Blot, and did not react with heterologous proteins starting at a dilution of 1:4000. At this dilution, the antiserum was effective in the virus detection of leaf extracts of infected plants and no reaction was observed with extracts from healthy grapevines. Considering that the virus is found at low concentrations in the plants during the seasons of the year, the results obtained so far were highly satisfactory for RSPaV detection. Serological methods have advantages over the biological indexing method, since they are cheaper and can be used in large-scale tests such as ELISA. Experiments using the ELISA technique were not successful. Purification of the native recombinant protein would be an alternative more efective to detect the virus using these technique. / Mestre

Resumo: O objetivo deste estudo foi padronizar a reação de Imunofluorescência direta (IFD) para detecção de oocistos de Cryptosporidium parvum em amostras fecais de bezerros. Os anticorpos policlonais anti-C. parvum foram produzidos em dois coelhos adultos da raça Nova Zelândia, e titulados por meio do ensaio imuno-enzimático indireto (ELISA). A fração de imunoglobulina G (IgG) foi purificada a partir do soro do coelho imunizado, utilizando-se diálise em sulfato de amônio seguida de cromatografia em coluna de DEAE celulose e conjugação com isotiocianato de fluoresceína. Por meio da IFD padronizada, foi testada a reatividade cruzada do conjugado produzido contra outras espécies de Cryptosporidium (C. andersoni e C. serpentis) e com outros agentes etiológicos presentes em fezes de bovinos (Escherichia coli, Eimeria sp. e Candida sp.). A IFD foi comparada à microscopia com contraste de fase em solução de Sheather (MCF), avaliando a capacidade de detecção de oocistos de Cryptosporidium sp. em alíquotas de fezes de bezerro inoculadas com oocistos de C. parvum, e em amostras fecais de bezerros naturalmente infectados provenientes de 37 propriedades leiteiras da região de Araçatuba, SP. Nas amostras comprovadas como positivas, foi ainda feita uma análise semi-quantitativa de oocistos de Cryptosporidium sp., visualizados por campo de microscopia em ambas as técnicas. O conjugado anti-C. parvum apresentou reatividade com C. andersoni e C. serpentis. A IFD foi utilizada com sucesso para detecção de oocistos de C. parvum em fezes de bezerros, apresentando sensibilidade para detecção de até 104 oocistos em 3 g de fezes. Entre as 300 amostras fecais de bezerros naturalmente infectados, 19,67% (59/300) foram positivas para a presença de oocistos de Cryptosporidium sp. pela IFD, apresentando diferença estatisticamente significante (p<0.05) em relação às amostras positivas pela MCF / Abstract: The objective of this experiment was to standardize the direct immunofluorescence assay (DIF) for detection of Cryptosporidium parvum oocysts in fecal samples of calves. The anti-C. parvum polyclonal antibodies were produced in two adult rabbits of New Zealand breed, and titrated by an indirect enzyme-linked immunosorbent (ELISA). The immunoglobulin G (IgG) was purified from the serum of immunized rabbits by dialysis in ammonium sulfate followed by column chromatography on DEAE cellulose, and conjugated with fluorescein isothiocyanate. Using DIF the anti-C. parvum conjugate was tested for the presence of cross-reactivity against other species of Cryptosporidium (C. andersoni and C. serpentis), and other infectious agents present in cattle fecal samples (Escherichia coli, Eimeria sp. and Candida sp.). A comparison between the DIF and phase contrast microscopy in Sheather solution (MCF) was accomplished for evaluation of the efficiency of both techniques for detection of Cryptosporidium sp. oocysts in fecal samples of calves seeded with C. parvum oocysts, and in fecal samples from naturally infected calves from 37 dairy farms in the region of Araçatuba, SP. A semi-quantitative analysis of Cryptosporidium sp. oocysts per microscopic field was accomplished for both techniques. The anti-C. parvum conjugate showed cross-reactivity with C. andersoni and C. serpentis oocysts. The DIF has been used successfully in the detection of C. parvum oocysts in fecal samples of calves, with sensitivity for detecting 104 oocysts in 3 g of fecal samples. Among the 300 fecal samples from naturally infected calves, 19.67% (59/300) were positive for Cryptosporidium oocysts by DIF, with significant statistical difference (p <0.05) when compared to the number of positive samples by MCF / Orientador: Marcelo Vasconcelos Meireles / Coorientador: Cáris Maroni Nunes / Banca: Ricardo Velludo Gomes de Soutello / Banca: Luzia Helena Queiroz / Mestre

Made available in DSpace on 2014-08-20T13:32:45Z (GMT). No. of bitstreams: 1 dissertacao_juliana_alcoforado_diniz.pdf: 1026827 bytes, checksum: f7e6dae0a1eabce2f815ea02d01d8e6a (MD5) Previous issue date: 2012-09-12 / Leptospirosis is a zoonosis caused by bacteria of the Leptospira genus. In recent years, efforts to identify immunogenic components of leptospires have resulted in the characterization of several outer membrane lipoproteins that are expressed during infection. Previously, our group had produced polyclonal IgY antibodies against whole-cell leptospires strain Fiocruz L1-130, which were used in different forms of capture ELISAs. In this study we produced and characterized IgY antibodies against LipL32 in recombinant form (rLipL32), the most abundant lipoprotein in the proteome of pathogenic leptospires, in order to use they in diagnostic assays and prophylaxis of leptospirosis. For this, two 22-week-old hens were immunized on day 0 with rLipL32 and oil adjuvant by intramuscular route. Three booster immunizations on days 14, 28 and 42 were held, and the eggs were collected daily from day 45. The yolks were processed and the antibodies purified with polyethylene glycol, and monitored by SDS-PAGE. Specificity of purified antibodies was assessed through indirect ELISA and Western blot (WB) using both rLipL32 and whole-cell Fiocruz L1-130. After standardization of the first batch of IgY, antibodies were conjugated with horseradish peroxidase and fluorescein (FITC). In addition, we conducted a passive immunoprotection assay in hamster model, using homologous and heterologous challenge. SDS-PAGE confirmed the successful purification of IgY and a batch with a concentration of 13μg.μL-1 was stored at -20ºC until use. Both IgY and IgY conjugated with horseradish peroxidase recognized the recombinant and native protein in ELISA and WB. In this study we produced and characterized successfully IgY against rLipL32. Although these antibodies have not been tested with clinical samples from humans or animals, they have antigen detecting potential for use in the diagnosis of leptospirosis. / A leptospirose é uma zoonose causada por bactérias do gênero Leptospira. Nos últimos anos, esforços para a identificação de componentes imunogênicos nas leptospiras resultaram na caracterização de várias lipoproteínas de membrana externa que são expressas durante a infecção. Anteriormente, nosso grupo havia produzido IgY contra Leptospira interrogans cepa Fiocruz L1-130 inteira, a qual foi usada em diferentes formatos de ELISA de captura. No presente estudo, objetivamos produzir e caracterizar anticorpos IgY contra a proteína LipL32 na forma recombinante (rLipL32), a lipoproteína mais abundante no proteoma das leptospiras patogênicas, com intuito de utilizá-los posteriormente, no diagnóstico e na imunoprofilaxia da leptospirose. Para isso, duas galinhas com 22 semanas de idade foram imunizadas no dia 0 com rLipL32 e adjuvante oleoso, através da via intramuscular. Três outras imunizações foram realizadas nos dias 14, 28 e 42 e os ovos coletados diariamente a partir do dia 45. Os anticorpos foram obitidos através do processamento das gemas, purificados com polietilenoglicol e monitorados por SDS-PAGE. A especificidade dos anticorpos foi avaliada através de ELISA indireto e Western blot (WB), ambos utilizando rLipL32 e Fiocruz L1-130 inteira. Após a padronização do primeiro lote de IgY, os anticorpos foram conjugados com peroxidase e fluoresceína (FITC), e avaliados em um ensaio de imunização passiva em hamsters, com desafio homólogo e heterólogo. O SDS-PAGE confirmou o sucesso de purificação, e um lote de IgY com a concentração de 13μg.μL-1 foi estocado a -20ºC até o uso. A IgY pura e conjugada com peroxidase reagiram com as proteínas recombinante e nativa no ELISA e no WB. Nesse estudo, produzimos e caracterizamos com sucesso IgY contra rLipL32. Embora esses anticorpos não tenham sido testados com amostras clínicas de humanos e animais, eles possuem potencial para compor ensaios que visem a detecção do antígeno no diagnóstico da leptospirose.