Orientador: Sônia Maria Alves Bueno / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-17T11:27:09Z (GMT). No. of bitstreams: 1 Bresolin_IgorTadeuLazzarotto_D.pdf: 3279874 bytes, checksum: b2f9ab93ce7e5036f95f0da584a42017 (MD5) Previous issue date: 2010 / Resumo: Dentre os hemoderivados disponíveis comercialmente, as imunoglobulina do isotipo G (IgG) recebem destaque pelo seu uso em aplicações terapêuticas. Por esta razão são requeridas com elevado grau de pureza. Várias técnicas vêm sendo investigadas para a purificação de IgG a partir do soro ou plasma humano, desde a precipitação até métodos mais seletivos, como os cromatográficos. Neste trabalho, investigou-se o efeito de agentes quelantes de IMAC (cromatografia por íons metálicos imobilizados) como Tris-2(aminoetil)amina (TREN) e o-fosfoserin (OPS) como grupos ionogênicos (sem íon metálico imobilizado), além do aminoácido L-Lisina e seu polímero poli-L-Lisina (PLL) como ligantes visando a purificação de IgG a partir do soro humano. Para tanto, foram realizados experimentos de adsorção em diferentes sistemas tamponantes. A seletividade dos adsorventes foi verificada por eletroforese SDS-PAGE e análise nefelométrica. As melhores condições, para cada caso, foram utilizadas em experimentos de curvas de ruptura e isotermas de adsorção de albumina de soro humano (HSA) e IgG. No caso dos ligantes TREN e PLL, 73% e 86% da IgG alimentada foi recuperada nas frações não-retidas (cromatografia negativa) apresentado pureza superior a 90%. Quando o ligante OPS foi utilizado, por sua vez, a recuperação de IgG ocorreu nas frações retidas juntamente com IgM. Experimentos de curva de ruptura mostraram que um fator de purificação de 4.9 foi atingido, sendo a IgG recuperada com pureza de 88%. Este ligante se mostrou eficiente quando se deseja purificar IgG humana que possui pontos isoelétricos na faixa de 7,8 a 8,5. Para todos os ligantes, a recuperação de IgG a partir soro humano pode ser alcançado sob condições brandas de pH, baixa força iônica, e temperatura ambiente. De um ponto de vista de processo em larga escala, todos os ligantes apresentados neste trabalho apresentam potencial para serem usados como uma das etapas em um processo industrial de recuperação e purificação de IgG / Abstract: Among the commercially available hemoderivatives or blood products, the immunoglobulin G (IgG) is highlighted by its use in therapeutic applications, which need high purity IgG. Several techniques are being investigated aiming at the purification of IgG from human serum or plasma, usually starting with precipitation and then using more selective methods such as chromatography. In this study, we evaluated the effect of Tris-2 (aminoethyl) amine (TREN) and o-phosphoserine (OPS) - two chelating agents used in immobilized metal ion chromatography (IMAC) - as ionogenic groups (without immobilized metal ion), and the amino acid L-lysine and its polymer poly-L-lysine (PLL) as ligands aiming at the purification of IgG from human serum. For this purpose, adsorption experiments were performed using different buffering systems. The selectivity of the adsorbents was checked by SDS-PAGE and nephelometric analysis. The best conditions for each adsorbent were used in experiments of breakthrough curves and adsorption isotherms of human serum albumin (HSA) and IgG. In the case of TREN and PLL, 73% and 86% of IgG fed was recovered in the non-retained fractions (negative chromatography) with purity higher than 90%. When the ligand OPS was used, IgG was recovered in retained fractions. Experiments of breakthrough curve showed that a purification factor of 4.9 was reached, and IgG was recovered with a purity degree 88% (with IgM). This ligand is efficient when it is desired to purify human IgG with isoelectric points ranging from 7.8 to 8.5. For all ligands, the recovery of IgG from human serum was achieved under mild conditions of pH, low ionic strength and temperature. All ligands presented in this study have potential to be used in an industrial downstream processing of IgG from human serum / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutor em Engenharia Química

Orientador: Maria Esther de Carvalho / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T09:30:13Z (GMT). No. of bitstreams: 1 Santos_AndersonPachecodos_M.pdf: 3380754 bytes, checksum: 9736aa6f219de1691e4d55d474a7d293 (MD5) Previous issue date: 2008 / Resumo: As zonas endêmicas de leishmanioses e doença de Chagas, duas grandes endemias causadas por cinetoplastídeos, sobrepõe-se na América do Sul, especialmente na região Sudeste do Brasil, o que pode gerar casos de co-infecção entre Leishmania spp. e Trypanosoma cruzi. Ocorrem reações cruzadas, mesmo com baixos títulos, em inquéritos sorológicos de pacientes infectados com os parasitas, o que leva a falsas estimativas de prevalência de ambas as infecções. Este trabalho tem o objetivo de observar o efeito de um diluente de amostra proposto para reduzir a ocorrência de reações cruzadas em testes sorológicos para doenças causadas por cinetoplastídeos e outros parasitas. Foram selecionadas 585 amostras de sangue provenientes de quatro regiões do Estado de São Paulo, entre litoral e planalto, colhidas no período compreendido entre 1996 a 2003, durante atividades do Programa de Controle da Doença de Chagas. As análises das amostras foram feitas através das Reações de Imunofluorescência (RIFI) e do ensaio imunoenzimático (ELISA). Com as amostras reagentes (onze no total), para doença de Chagas e leishmaniose em ambas as técnicas, foi realizado o teste ELISA alta avidez, que utiliza um diluente com caotrópico, que demonstrou redução nos títulos da reação positiva falsa, restando apenas uma amostra sem redução significativa do seu título, que sugeriu ser um caso de co-infecção de doença de Chagas e leishmaniose, epidemiologicamente factível dada a existência de ambas as infecções na área estudada. O uso deste diluente em estudos clínicos e epidemiológicos pode aprimorar o diagnóstico para a identificação de soropositivos em regiões de sobreposição de endemias causadas por cinetoplastídeos / Abstract: Leishmaniases and Chagas disease are endemic in extensive areas of the American Continent. These parasitic diseases can occur together in South America, particularly in southwestern regions of Brazil, which poses a serious epidemiological problem due to the observed serological cross-reactivity between Leishmania spp. and Trypanosoma cruzi, even at low titers. As false prevalence estimates of these agents are critically misleading, we designed an experiment aiming at analyzing the effect of a diluent intended to reduce the chances of false positive tests resulting from serological cross-reactivity between these kinetoplastids and other parasites. As a part of the activities of the Program for the Control of Chagas disease, we selected 585 blood samples collected, during the period from 1996 to 2003, from residents in four regions situated between the seacoast and the plateau of the State of São Paulo. The serological tests used (IFAT and ELISA) produced a total of eleven positive reactions. Lower false positive titers resulted from the use of high avidity ELISA plus a chaotropic diluent. The only serological result in which titers corresponding to both infections were not significantly reduced was suggestive of mixed infection, on the basis of the fact that it was connected to a patient evidently exposed to both agents. This experiment suggests that the use of a chaotropic diluent can be a useful tool to reduce the proportion of such false parasitic mixed infection results as those investigated by us / Mestrado / Mestre em Parasitologia

Neste trabalho, anticorpos anti-IgGh foram conjugados às nanopartículas de prata (NPAg) para detectar imunoglobulina G humana (IgGh). Um imunoensaio colorimétrico baseado na diminuição da agregação devido ao aumento da repulsão eletrostática após a interação ligante-alvo. A agregação é induzida pela variação da força iônica e uma mudança da coloração da suspensão coloidal de amarelo para vermelho pode ser observada. Na presença de IgGh, a agregação é inibida e a coloração da suspensão coloidal não se altera. As nanopartículas foram obtidas por meio de cinco procedimentos diferentes e caracterizadas por espectroscopia UV-Vis, espalhamento dinâmico de luz, difração de raios-X e microscopia eletrônica. Glicose e borohidreto de sódio foram utilizados como agentes redutores, enquanto CTAB e β-ciclodextrina foram utilizados como estabilizantes. Citrato de sódio foi utilizado como agente redutor e/ou estabilizante. Nanoesferas de carbono foram obtidas por tratamento hidrotérmico de uma solução aquosa de glicose e também foram utilizadas no preparo das nanopartículas. As nanopartículas foram funcionalizadas com ácido mercaptossuccínico e a conjugação ocorreu devido à interação entre grupos aminas e grupos carboxílicos ionizados, presentes no anticorpo e agente de acoplamento, respectivamente. A estabilidade dos conjugados e o efeito da adição de IgGh foram avaliados para todos os sistemas preparados. As nanopartículas de prata preparadas com borohidreto de sódio e citrato de sódio foram selecionadas para serem aplicadas no desenvolvimento do imunoensaio e as condições experimentais foram avaliadas. Em condições ótimas, observou-se uma correlação linear entre a diminuição da agregação do sistema (NPAg-anti-IgGh) e a concentração de IgGh (0 a 200 ng mL-1). O limite de detecção foi estimado em 25 ng mL-1. O método colorimétrico apresentou boa seletividade para a detecção de IgGh. Além disso, foi obtido um resultado satisfatório ao aplicar o método para determinação do fator IX de coagulação. Foi desenvolvido também um método para determinação de ATP baseado na agregação de nanopartículas de ouro. Aptâmeros foram utilizados como elemento de reconhecimento. Em princípio, o método pode ser aplicável à determinação de outros analitos, por meio da substituição do aptâmero utilizado neste trabalho pelo oligonucleotídeo específico para o alvo de interesse. / In this work, antibodies to human immunoglobulin G (anti-IgGh) were used in combination with silver nanoparticle (NPAg) to detect IgGh. A colorimetric immunoassay based on the decrease of aggregation due to increased electrostatic repulsion upon ligand-target interaction. Aggregation was induced by varying the ionic strength and the solution of NPAg-anti-IgGh shows obvious visible color change from yellow to red. In the presence of IgGh aggregation the nanoparticle is inhibited and coloration of the colloidal solution does not change. The nanoparticles were obtained using five different procedures and they were characterized by UV-Vis spectroscopy, dynamic light scattering, X-ray diffraction and electron microscopy. Glucose and sodium borohydride were used as reducing agent, as CTAB and β-cyclodextrin reagents were used as stabilizers. Sodium citrate was used as reducing and stabilizing agent. Carbon nanospheres were prepared by hydrothermal treatment of glucose and used in the preparation of NPAg. The nanoparticles were functionalized with dimercaptosuccinic acid and their conjugation occurred due to the interaction of positively charged amine groups and anionic groups (-COO-) present on the antibody and coupling agent. The stability of conjugates and the variation of aggregation in the presence of IgGh were evaluated for all systems. The NPAg prepared by sodium borohydride were selected for use in the immunoassay and the optimum conditions of the assay were investigated. Under the optimal conditions, the ration between the absorbance at 396 nm and 564 nm was linearly proportional to the IgGh concentration on a range from 0 to 200 ng mL-1, with a detection limit of 25 ng mL-1. The colorimetric method showed good selectivity for IgGh detection. It was possible to adapt the method to the determination of other proteins, such as factor IX. In another approach, anti-aptamer ATP was used to develop a colorimetric method for the determination of ATP based on stabilization of gold nanoparticles provided by strands of DNA.The strategy was based on stabilization of nanoparticles due to interaction with single strands of DNA, and the change of the stability of the nanoparticles provided by the conformational change of the aptamer following recognition. This method could in principle be used to detect analytes by substituting the aptamer used in this study by the specific aptamer for the target of interest

Depuis leur découverte en 1975 par Köhler et Milstein, le monde des Ac monoclonaux a beaucoup évolué. Ils occupent actuellement une place prépondérante dans la prise en charge de nombreux cancers. De nos jours, les Ac monoclonaux, ayant une AMM ou en essai clinique, sont tous de classe IgG voire IgG1. Cette classe d’Ac a cependant montré des limites à son utilisation, et l’étude d’autres isotypes d’Ac, comme les IgA, pourrait être intéressante. Les IgA, isotype d’Ac particulier en raison notamment de leur hétérogénéité dans les formes moléculaires, demeurent peu étudiées à l’instar des IgG. Dans ce travail, nous proposons un radiomarquage des IgA monomériques, polymériques et sécrétoires, avec le 99mTc par une méthode indirecte impliquant le 2-iminothiolane et le cœur tricarbonyl. Par le biais de ce radiomarquage, la biodistribution des IgA monomériques et polymériques après administration i.v. a été évaluée chez l’animal sain et chez l’animal porteur de tumeur à localisation muqueuse. Ces études nous ont permis d’entrevoir le potentiel diagnostique des IgA, mais aussi leur intérêt en thérapie ciblée de tumeurs à localisation muqueuse. D’autre part, grâce à leur résistance enzymatique et au phénomène de retranscytose, une nouvelle voie d’administration des Ac monoclonaux pourrait être développée. Dans cette optique, des IgA sécrétoires ont été administrées par voie orale lors d’études préliminaires de biodistribution. / Since their discovery in 1975, by Köhler and Milstein, monoclonal antibodies (mAbs) world has significantly evolved and they currently hold a prominent place in cancers care. Today, the mAbs, having a marketing authorization or in clinical trial, are all IgG class (IgG1). However, this Ab class showed limitations on its use, and the study of other isotypes, such as IgA, could be interesting. Unlike IgG, IgA, original isotype particularly because of their heterogeneity in molecular forms, remains understudied. In this work, we propose a radiolabeling of monomeric, polymeric and secretory IgA with 99mTc by an indirect method, involving 2-iminothiolane and tricarbonyl core. Biodistribution of radiolabeled monomeric and polymeric IgA was evaluated, after intravenous administration, in healthy animals and in mucosal tumor-bearing animals. These studies have allowed us to glimpse the IgA diagnostic potential, but also their interest in targeted therapy of tumors with mucosal localization. Moreover, thanks to their enzymatic strength and retranscytosis, a new administration route of mAbs could be developed. In this context, secretory IgA were administered orally in preliminary biodistribution studies.

Orientador: Mateus José Rodrigues Paranhos da Costa / Resumo: Objetivou-se avaliar a eficácia do uso do colostro comercial em pó como substituto do colostro de vaca na primeira mamada de bezerras. Foram utilizadas 31 bezerras mestiças Holandês (H) x Gir (G), provenientes dos grupos genéticos 3/4HG, 5/8HG, 7/8HG e LA, divididas em dois grupos experimentais: G1 (n=15) 2L de colostro de vaca e G2 (n=16) 470g de colostro comercial em pó (SCCL, Saskatoon, SK, Canadá) diluídos em 1,5L de água morna, ambos fornecidos nas primeiras três horas de vida, e posteriormente recebendo mais 2L de colostro de vaca em até 12 horas após o nascimento. As concentrações de IgG presentes (p=0,006) foram maiores no colostro comercial em pó, enquanto as médias da β- caseína (p<0,0001), haptoglobina (p<0,0001) e α1-glicoproteína ácida (p=0,002) foram maiores nos colostros de vaca. As concentrações séricas de PT (G1 = 7,70 ± 1,00g/dL e G2 = 6,73 ± 0,65g/dL; p=0,003) e IgG (G1 = 2110,25 ± 595,03mg/dL e G2 = 1567,6 ± 418,25mg/dL; p=0,004) demonstraram diferenças estatísticas entre os grupos de manejo, com maiores médias para o G1. Houve diferenças significativas para a concentração de albumina sérica (p=0,005), com maiores médias para o G1 (4660,7 ± 384,61mg/dL). Não ocorreu diferença significativa para a haptoglobina sérica (p=0,29), porém suas concentrações médias se apresentaram bem mais altas em ambos os grupos de manejo (G1 = 33,86 ± 4,90 e G2 = 29,31 ± 5,63mg/dL). As enfermidades com maior ocorrência foram diarreia e pneumonia, sendo registrados também casos ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The purpose of this study was value the effectiveness in commercial colostrum powder using as a cow colostrum substitute in the first calf suckling. Twenty-one Holstein (H) x Gir (G) crossbred calves from 3/4HG, 5/8HG, 7/8HG and LA genetic groups were divided into two experimental groups: G1 (n = 15) 2L of cow colostrum and G2 (n = 16) 470g of commercial powdered colostrum (SCCL, Saskatoon, SK, Canada) diluted in 1.5L of warm water, both supplied within the first three hours of life, and subsequently receiving an additional 2L of cow colostrum in up to 12 hours after birth. Present IgG concentrations (p = 0.006) were higher in commercial powdered colostrum, while mean β-casein (p < 0.0001), haptoglobin (p <0.0001) and α1-acid glycoprotein (p = 0.002) were higher in cow colostrums. Serum concentrations of PT (G1 = 7.70 ± 1.00 g/dL and G2 = 6.73 ± 0.65 g/dL; p = 0.003) and IgG (G1 = 2110.25 ± 595.03 mg/dL and G2 = 1567.6 ± 418.25 mg/dL; p = 0.004) demonstrated statistical differences between management groups, with higher means for G1. There were significant differences in serum albumin concentration (p = 0.005), with higher means for G1 (4660.7 ± 384.61mg / dL). There was no significant difference for serum haptoglobin (p = 0.29), but its mean concentrations were much higher in both management groups (G1 = 33.86 ± 4.90 and G2 = 29.31 ± 5.63 mg/dL). The most common diseases were diarrhea and pneumonia, with cases of parasitic sadness and omphalitis, and two deaths in G2. Althou... (Complete abstract click electronic access below) / Mestre

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20282号 / 医博第4241号 / 新制||医||1021(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 鈴木 茂彦, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

During the first days after parturition, mammalian milk (colostrum) is specifically formulated to nourish newborns. Immunoglobulins are a particularly important component for newborn New World camelids, as their immune system is almost totally dependent on the intestinal transfer of colostral immunoglobulins to acquire passive immunity. In this study, colostrum samples were collected from 20 alpaca mares in the first four days after parturition and analyzed for their immunoglobulin concentration. Sampling started on the day of parturition. The associations of immunoglobulins with other components were determined. The immunoglobulin G (IgG) concentrations decreased significantly within the first four days after parturition. The correlation coefficients between IgG content and the content of various minerals were significant but variable. The correlation between IgG content and fat and lactose content was negative but between IgG content and protein content was highly positive. This strong association could be used for a brief estimation of the IgG content of the colostrum based on the measured protein concentration. The results of the present study can be used for the development of colostrum replacers where motherless rearing is required. Abstract Colostrum provides the newborn with nutrients and immunoglobulins. Immunoglobulins and their intestinal transfer play a major role in the immune system of neonates since they are born agammaglobulinemic. In this study immunoglobulin G (IgG) content was determined in alpaca colostrum and the correlations of the IgG concentration by fat, protein, lactose and minerals were calculated. Colostrum samples were collected daily from 20 multiparous alpaca mares during the first four days after parturition. The IgG concentrations were determined by radial immunodiffusion using a Camelid IgG Test Kit. The IgG concentration decreased significantly from 26,319 mg/dL on day 1 to 3848.8 mg/dL on day 4. There were significant correlations between IgG concentration and the other components of the colostrum. While the correlations between IgG and fat (r = −0.69, p ≤ 0.001) and lactose (r = −0.64, p ≤ 0.001) were negative, the correlations with protein (r = 0.91, p ≤ 0.001), magnesium (r = 0.86, p ≤ 0.001) and cobalt (r = 0.87, p ≤ 0.001) were strongly positive. Due to the strong association, the colostrum protein concentration could be used for a brief estimation of the IgG content.

The antibody Immunoglobulin G (IgG) main function is to protect and prevent the body from infections, and it is normally found in human serum. This study is about IgG glycosylation, which is associated with different types of diseases such as neurological diseases, cancers and immunodeficiency etc. This study attempts to optimize IgG glycopeptide enrichment in a 100 μL micropipette tip set up, and to separate the enriched glycopeptides using capillary electrophoresis (CE). Matrix-assisted laser desorption/ionization – mass spectrometry (MALDI-MS) was used for data acquisition and glycopeptide profiling.  In this study, loading solutions with different combinations of acetonitrile (ACN) and trifluoroacetic acid (TFA), together with various precondition and sample preparation procedures were evaluated on IgG digest samples. Best enrichment performance, particularly regarding the selectivity, was achieved using the parameters as follows: loading solution of 83% ACN/16% H2O/1% TFA, sample solution in H2O containing 83% ACN, using a 100 μL micropipette tip packed with 1 mg cotton wool. A re-enrichment step was carried out on enriched glycopeptide samples, and improved selectivity of glycopeptides could be observed. Enriched glycopeptides could be separated into three major groups by CE using an acidic background electrolyte of 50 mM formic acid and 50 mM acetic acid, pH 2.5. / Huvudfunktionen för antikroppen Immunoglobulin G (IgG) är att skydda kroppen och förhindra infektioner och det finns normalt i mänskligt serum. Denna studie handlar om glykosylering, som är kopplad till olika typer av sjukdomar såsom neurologiska sjukdomar, cancer och immunbrist etc, och är en potentiell biomarkör för sjukdomar. Denna studie försöker optimera en IgG glycopeptidanrikningsmetod i en 100 μL mikropipettspets och separera de anrikade och intakta glycopeptiderna med hjälp av kapillärelektrofores (CE). Matrisassisterad laserdesorption/jonisering – masspektrometri (MALDI-MS) användes för datainsamling och glykopeptidprofilering  I denna studie utvärderades lösningar med olika kombinationer av acetonitril (ACN) och trifluororättiksyra (TFA) tillsammans med olika föberedelseförfaranden och provberedningsprocedurer på IgG prover. Anrikningsprestanda, särskilt selektiviteten, uppnåddes bäst med användning av följande parametrar: lösningen av 83% ACN / 16% H2O / 1% TFA, provlösning i H2O innehållande 83% ACN, med användning av en 100 mikroliter mikropipettspets fylld med 1 mg bomull. Återreningssteget genomfördes på anrikade glykopeptidprover och förbättrad selektivitet för glykopeptider kunde observeras. Anrikade glykopeptider kunde separeras i tre huvudgrupper med CE med användning av sur bakgrundselektrolyt med 50 mM myrsyra och 50 mM ättiksyra, pH 2,5.

Immunoglobulins are key mediators of humoral immunity and can be appreciated in an isotype-dependent manner in autoimmune diseases, and to an extent, immune-mediated metabolic diseases. The most common isotype is Immunoglobulin G (IgG), yet there is little understanding of the role IgG plays in the pathogenesis of macrophage-driven metabolic disorders like atherosclerosis, metabolic dysfunction-associated steatotic liver disease (MASLD), and metabolic dysfunction-associated steatohepatitis (MASH). The contents of this dissertation introduce IgG as an activator of the macrophage inflammasome and its deposition in atherosclerotic plaques and fatty livers as a driver of disease progression. The presence of IgG in these depots and its accumulation is dictated by the neonatal Fc receptor (FcRn) in macrophages. IgG levels are known to be determined by recycling, especially in macrophages, rather than by production, and FcRn, encoded by the gene Fcgrt, is the sole receptor responsible. Interestingly, we uncover a myriad of roles for FcRn involved in regulating the biological properties of macrophages, such as their transcriptional and secretory profiles and their polarization amidst an immune response. Taken together, we identify FcRn as a hitherto unknown contender in the manipulation of macrophage function and regulation of IgG in the development of macrophage-associated cardiometabolic diseases using a multitude of methodologies. These findings highlight the importance of macrophage IgG recycling in metabolism and may warrant the potential to explore this phenomenon for therapeutic pursuits.

Serum cortisol and immunoglobulin G (IgG) concentrations were measured to investigate the relationship between vitamin A status and immune function in lambs. Twenty-four crossbred ewe lambs were each fed 900 g·d-1 of a carotene—deficient diet composed of 95.5% whole oats, 3% molasses, .5% trace mineral salt and 1% limestone. All lambs were injected monthly with vitamins D and E and with selenium. The 12 control lambs also received a 100,000 IU oral dose of vitamin A palmitate in capsule form every 2 wk. All lambs were challenged by injecting them with 1 mg ovalbumin in 1 ml of Freund’s complete adjuvant. At the time of challenge, serum vitamin A levels for the control and A-deficient (A—def) lambs were 33.3 and 3.1 ug·dl-1 respectively. Blood was collected prior to and 6, 13, 20 and 34 d post—challenge. The lambs were then reschallenged using the same antigen and blood was obtained 1, 2, 6 and 22 d post—challenge. Lambs were sacrificed at the end of the second challenge period. Spleen weights were obtained and gross post—mortem observations were made at this time. / Master of Science