Quenched Molecular Dynamics (QMD) used to explore molecular conformations was developed to operate in Insight II platform for two simulation engines: CHARMm and Discover. Two scripts and procedures were written for molecular minimization, dynamics, minimization of each of several hundred conformers, and cut off. Experience with Insight II/Discover versus Quanta/CHARMm, and between Insight II/CHARMm versus Quanta/CHARMm has taught that the forcefield is the key factor in QMD studies. Protein A has been used for the purification of commercial antibodies, but it is expensive. Seven peptidomimetics of protein A were designed based on the hot-spots located at the helix-loop-helix region of protein A, and synthesized via solid phase using the Fmoc approach. These peptidomimetics were characterized by MS and NMR. The conformations of four peptidomimetics were studied by NMR and CD in water/hexafluoroisopropanol (pH 4). The CD and NMR data show that addition of hexafluoroisopropanol stabilizes their a-helical conformations. The structures of these peptidomimetics in solution were generated with Quanta/CHARMm using NMR data as limits for the QMD technique. Protein G has also been used to purify antibodies, but it is expensive too. A number of protein G mimics were designed as trivalent molecules. An efficient preparation of trivalent molecules having a useful primary amine arm has been developed through solid phase synthesis. The cheap, commercially available poly(propylene imine) dendrimers were used as scaffolds which allow multimerization of functionalized compounds. A small library of trivalent compounds were synthesized using this approach. A portion of compounds in this library were tested by Amersham Biosciences. The seven amino acid modified DAB-Am-4 exhibits strong binding to the IgG/Fab, and is a potential ligand for IgG purification. The interactions between neurotrophins (ie NGF and NT-3) and their receptors are typical drug targets. Fourteen second-generation peptidomimetics showing NGF-like or NT3-like activities in a preliminary bioassay, were resynthesized and tested again. Preliminary and retested data were compared. To access a direct binding assay, five fluorescently labeled peptidomimetics 41a-e were synthesized for a fluorescence activated cell sorting (FACScan) assay. Six monomeric precursors 42 and 43 were prepared on large scales for the library of bivalent turn analogs

<p>A capture step with protein A is the most common purification step in the downstream purification process of monoclonal antibodies. It is therefore of great importance to increase the knowledge of the interactions involved in this purification technique. The purpose of this master thesis project was to determine the affinity of protein A domains to IgG subclasses by surface plasmon resonance (SPR).</p><p>Besides the five homologous IgG-binding protein A domains (E, D, A, B, and C) an engineered domain, similar to domain B and used in the protein A media MabSelect Sure™ (GE Healthcare) was included in the study. The domains were expressed in E.coli, affinity purified and immobilized onto sensor chip surfaces by amine coupling. The antibodies used in the interaction analyses were of the human IgG subclasses 1, 2, 3, and 4. Affinity determination was performed by kinetic analyses with the SPR-biosensor Biacore™ 2000.</p><p>All human IgG subclasses except IgG3 were shown to bind to all protein A domains including the monomer of the SuRe ligand. The equilibrium constants, KD-values, obtained were all in the low nanomolar range. For IgG1 and IgG4, no significantly differences in the affinity to any of the protein A domains were found, except for domain E where there might be quality issues of the prepared domain. Furthermore, a detected quality issue with the commercial IgG2 made it impossible to determine the KD-values for this subclass with any reliability.</p>

A capture step with protein A is the most common purification step in the downstream purification process of monoclonal antibodies. It is therefore of great importance to increase the knowledge of the interactions involved in this purification technique. The purpose of this master thesis project was to determine the affinity of protein A domains to IgG subclasses by surface plasmon resonance (SPR). Besides the five homologous IgG-binding protein A domains (E, D, A, B, and C) an engineered domain, similar to domain B and used in the protein A media MabSelect Sure™ (GE Healthcare) was included in the study. The domains were expressed in E.coli, affinity purified and immobilized onto sensor chip surfaces by amine coupling. The antibodies used in the interaction analyses were of the human IgG subclasses 1, 2, 3, and 4. Affinity determination was performed by kinetic analyses with the SPR-biosensor Biacore™ 2000. All human IgG subclasses except IgG3 were shown to bind to all protein A domains including the monomer of the SuRe ligand. The equilibrium constants, KD-values, obtained were all in the low nanomolar range. For IgG1 and IgG4, no significantly differences in the affinity to any of the protein A domains were found, except for domain E where there might be quality issues of the prepared domain. Furthermore, a detected quality issue with the commercial IgG2 made it impossible to determine the KD-values for this subclass with any reliability.

While Rhodococcus equi (R. equi) remains the most common cause of subacute or chronic granulomatous bronchopneumonia in foals, development of a relevant model to study this bacterium has proven difficult. As a result, the reasons for the underlying foal’s susceptibility to this disease are not well understood. Furthermore, data regarding the immune response of foals to R. equi infection remains controversial. We hypothesized that foals are susceptible to R. equi early in life and that this susceptibility decreases with age. Also, we hypothesized that specific subclasses of IgG antibodies to the virulence-associated protein of R. equi, VapA, predict the outcome of exposure. The objectives of this study were: (1) to develop an R. equi challenge model that resulted in slow progressive disease in some foals as well as spontaneous regression of lesions in others, (2) using the developed model, to investigate the age-related susceptibility of young foals to R. equi, (3) to describe the humoral immune response of foals following experimental challenge and natural infection. The use of a low dose of R. equi to challenge neonatal foals resulted in slow, progressive disease characterized by pulmonary abscessation and spontaneous regression in approximately 50% of the foals. When this low dose was used in 1, 2 or 3-week-old foals, a marked decrease in disease susceptibility was observed as the foals aged. The immunological responses seen after experimental challenge reflect those observed after natural infection. While there was a significant increase of VapA-specific IgG and IgG subclasses over time in both pneumonic and healthy foals, use of VapA-specific IgG(T) showed good sensitivity and specificity when used as a diagnostic tool for R. equi pneumonia. In summary, this study shows that foal susceptibility to R. equi occurs early in life and decreases with age. Whereas all foals developed VapA-specific IgG antibodies post-exposure, IgG(T) appeared to be predictive of infection.

Thesis (M.Sc. in Microbiology) -- Mahidol University, 1976.

Dissertation (Ph.D.)--University of Toledo, 2010. / "Submitted to the Graduate Faculty In partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Science." Title from title page of PDF document. "A Dissertation entitled"--at head of title. Non-Latin script record Bibliography: p. 68-101.

The purpose of diploma thesis was evaluate the effect of milk production an the season on the quality colostrum Czech fleckvieh cattle in a selected breeding. Between monitored parameters were included solids, active acidity, density, imunoglobulins content and refraction. Literary review was processed to the given problem. Viewed samples came from the farm Agrodružstvo Tištín. Samples were taken during the whole year (from 17. 4. 2014 to 18. 1. 2015). In this period has been taken 39 samples of colostrum, from selected cows. These cows were all on the second lactation. The measured values were statistically processed. The results indicate, that cows with high milk production have better quality of colostrum. Seasons affected the quality of colostrum. The cows, who gave birth in the autumn and winter, had colostrum quality. We found, that healthy cows had in colostrum more immunoglobulins and solids, during the assessing the health condition.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Recombinant proteins from Toxoplasma gondii have been used in several experimental models, as well as for serodiagnosis of human toxoplasmosis, particularly to differentiate acute from chronic phases of the infection. In the present study, we evaluated the kinetics of IgM, IgA, and IgG isotypes, in addition to IgG1 and IgG3 subclasses, by testing sequential serum samples from patients with acute toxoplasmosis. It was carried out immunoassays by using SAG2A recombinant antigen and soluble antigen of Toxoplasma (STAg). The avidity of IgG1 antibody was assessed using slot blot assay. Additionally, a ratio between IgG1 and IgG3 subclasses (IgG3:IgG1) was determined and evaluated its degree of association with levels of IgM and IgA specific for STAg and the avidity index of IgG1 specific for SAG2A. The results showed a decreasing kinetic profile for SAG2A and STAg for IgM and IgA. The kinetic profile for the IgG antibody was increasing for both antigens. Compared to the avidity for IgG1, it was observed that sera from an early stage showed a low average avidity of IgG1 for SAG2A while the same samples showed intermediate mean avidity of IgG1 when STAg was used as antigen. In a later phase, the average avidity observed was high for STAg and intermediate for SAG2A in the same tested sera suggesting that SAG2A may be a promising tool for the detection of avidity. Associations between IgG3/IgG1 and specific IgM and IgA levels for STAg and the avidity index of specific IgG1 for SAG2A were found, and together these parameters could be used as valuable tools in the diagnosis of human toxoplasmosis, especially in situations when the determination of different phases is critical. / Proteínas recombinantes de Toxoplasma gondii têm sido utilizadas em diversos modelos experimentais, assim como para o diagnóstico sorológico da infecção humana por este parasito, principalmente com o intuito de diferenciar as fases aguda e crônica da toxoplasmose. Neste estudo, foi avaliada a cinética dos anticorpos IgM, IgA ,IgG e subclasses (IgG1 e IgG3) através de imunoensaios realizados em amostras seqüenciais de soros humanos, provenientes de pacientes com toxoplasmose aguda. Estas amostras foram testadas frennte ao antígeno recombinante SAG2A, utilizando-se como paradigma de comparação o antígeno solúvel total de Toxoplasma (STAg). A avidez do anticorpo IgG1 foi avaliada utilizando a metodologia slot-blot. Adicionalmente, a razão entre as subclasses IgG3 e IgG1 (IgG3:IgG1) foi determinada e avaliada quanto ao grau de associação com os níveis de IgM e IgA específicos para STAg e aos índices avidez de IgG1 específicos para SAG2A. Os resultados demonstraram a presença de níveis decrescentes de IgM e IgA para ambos os antígenos utilizados, enquanto que para o isotipo IgG o perfil cinético demonstrou níveis crescentes para ambas preparações antígênicas. Em relação aos índices de avidez para IgG1, foi observado que amostras de soros de uma fase inicial apresentaram baixa avidez média de anticorpos IgG1 dirigidos para SAG2A, enquanto que as mesmas amostras demonstraram avidez média intermediária de IgG1 quando STAg foi utilizado como antígeno. Já em uma fase mais tardia, a avidez média observada foi alta para STAg e intermediária com SAG2A. A razão entre IgG3:IgG1 obtida no primeiro bimestre foi significantemente maior para SAG2A em comparação com STAg. Tomados em conjunto, os resultados obtidos no presente estudo indicam que a proteína recombinantes SAG2A pode se constituir em uma ferramenta efetiva na diferenciação das fases da infecção humana por T. gondii. / Mestre em Imunologia e Parasitologia Aplicadas

Orientador: Claudio Lucio Rossi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T14:37:04Z (GMT). No. of bitstreams: 1 Nascimento_FernandaSantos_M.pdf: 1195114 bytes, checksum: 8f7292945661258264c33482af428d55 (MD5) Previous issue date: 2008 / Resumo: A toxoplasmose, uma zoonose com ampla distribuição mundial, causada pelo parasita intracelular obrigatório Toxoplasma gondii, é geralmente adquirida por meio da ingestão de cistos ou oocistos viáveis do parasita, presentes, respectivamente, em carne crua ou mal cozida e no solo, alimento ou água contaminados com fezes de gatos infectados. A toxoplasmose pode ser altamente debilitante, e ocasionalmente fatal, em crianças infectadas no útero e em receptores de transplante. O diagnóstico de infecção aguda primária em mulheres grávidas é geralmente baseado em testes sorológicos, visto que, na grande maioria dos casos, a toxoplasmose não é reconhecida clinicamente. A longa persistência dos anticorpos IgM em algumas pessoas e a dificuldade para demonstrar soroconversão ou aumento significativo da concentração de anticorpos específicos, têm complicado a interpretação dos testes sorológicos, quando se suspeita de infecção aguda. Com relação à infecção toxoplámica em pacientes transplantados, em muitos casos o status sorológico do doador não é conhecido e a pesquisa periódica de anticorpos anti-T. gondii no receptor raramente é realizada. O objetivo do primeiro estudo foi determinar o valor da demonstração dos anticorpos IgA anti-T.gondii para o diagnóstico da fase aguda da infecção toxoplásmica. Nossos resultados mostraram que os anticorpos IgA são detectados com alta freqüência em amostras de soros obtidas de mulheres com evidência clínica e/ou sorológica de infecção toxoplásmica aguda. Entretanto, em 19% das mulheres apresentando persistência de anticorpos IgM e alto índice de avidez dos anticorpos IgG, anticorpos IgA anti-T. gondii foram detectados em amostras de soros coletadas mais de 9 meses após o início da infecção, indicando que esses anticorpos não podem ser considerados marcadores confiáveis de infecção aguda primária. No segundo estudo, nós relatamos o diagnóstico de infecção toxoplásmica primária em um paciente com mieloma múltiplo submetido a transplante alogênico não-mieloablativo de células hematopoiéticas, provenientes de doador com sorologia negativa para toxoplasmose. A resposta primária contra o T. gondii foi baseada na soroconversão dos anticorpos IgM, IgG e IgA. O paciente foi prontamente tratado e nenhuma complicação relacionada à toxoplasmose foi observada nos meses subseqüentes. Esse caso ressalta a necessidade da detecção dos anticorpos anti-T. gondii no doador e no receptor antes do transplante e a importância do monitoramento sorológico do receptor durante o seguimento pós-transplante / Abstract: Toxoplasmosis, a cosmopolitan zoonotic disease caused by the intracellular parasite Toxoplasma gondii, is usually acquired through the ingestion of viable parasite cysts or oocysts, present, respectively, in raw or undercooked meat and in soil, food or water contaminated with feces of infected cats. Toxoplasmosis can be highly debilitating and occasionally fatal in children infected in utero and in transplant recipients. The diagnosis of acute primary infection in pregnant women is usually based on serology, because in the great majority of cases primary infection is not recognized clinically. The sustained persistence, in some persons, of specific IgM antibodies and the difficulty in demonstrating seroconversion or a significant increase in specific antibody concentrations, have complicated the interpretation of serological tests when acute infection is suspected. With regard to toxoplasmic infection in transplant patients, in many cases the serological status of the donor is not known and the periodic research of anti-T. gondii antibodies in the receptor is rarely performed. In the first study, we investigated the usefulness of detecting anti-T. gondii IgA for the diagnosis of an acute acquired Toxoplasma infection. Our results showed that anti-T. gondii IgA antibodies are detected with a high frequency in serum samples obtained from women with clinical and/or serologic evidence of acute acquired Toxoplasma infection. However, in 19% of the women presenting a sustained IgM antibody response and a high IgG avidity index, anti-T. gondii IgA antibodies were detected in serum samples collected more than nine months after the beginning of infection, indicating that IgA cannot be considered a dependable marker for acute primary infection. In the second study, we report the diagnosis of a primary toxoplasmic infection in a patient with multiple myeloma following a non-myeloablative allogeneic transplant with hematopoietic stem cells from a donor with negative serology for toxoplasmosis. The primary response to T. gondii was supported by IgM, IgG and IgA seroconversion. The patient was promptly treated and there were no complications related to toxoplasmosis in the subsequent months. This case stresses the importance of detecting anti-T. gondii antibodies in the donor and in the recipient before transplantation, and of serologically monitoring the recipient during long-term follow-u / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Made available in DSpace on 2014-06-12T15:52:08Z (GMT). No. of bitstreams: 2 arquivo5003_1.pdf: 1484239 bytes, checksum: 04b84269045a9fccab4418903a3a27bc (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2002 / A lectina de folhas de Bauhinia monandra (BmoLL) foi previamente purificada por fracionamento com sulfato de amônio (F 0-60%) seguido por cromatografia de afinidade com gel de Guar. Para o estudo da BmoLL foi desenvolvido um anticorpo policlonal em coelho contra esta lectina; anti-BmoLL IgG foi purificada em coluna de Sepharose-Proteína A. Em imunodifusão, anti-BmoLL IgG apresentou reações cruzadas com lectinas de Bauhinia purpurea e Ulex europaeus I. Lectinas de Bandeiraea simplicilofia II e Triticum vulgaris reagiram inespecificamente com substâncias presentes no antissoro mas ausentes na preparação obtida de IgG. Precipitações de anti-BmoLL IgG com extratos de outros tecidos de B. monandra, revelaram outras formas moleculares da lectina. Anti-BmoLL IgG foi conjugada com peroxidase e aplicada em ELISA, confirmando os resultados de imunodifusão. Para a sua aplicação no biossensor piezoelétrico, o rendimento do método de imobilização foi testado imobilizando anti-BmoLL IgG em placas de ouro (4 x 4 x 0.5 mm3) em quantidades de 0, 110 e 220 mg em 100 ml utilizando dextrana tratado com bromocianeto. As concentrações de IgG não imobilizada foram medidas pela quantificação de proteína após a imobilização e lavagens com PBS. As quantidades de 110 e 220 mg de anti-BmoLL IgG apresentaram respectivamente, rendimentos de imobilização de 46% e 33%. Anti-BmoLL IgG (110 mg/ml) foi imobilizada na superfície de ouro de Microbalança de Cristal de Quartzo (do inglês Quartz Crystal Microbalance, QCM). Na execução de ensaio de piezoelétrico, a QCM foi incubada com solução de BmoLL (91 mg/ml) seguido de uma redução de freqüência com 5 kHz, mostrando um grande reconhecimento da lectina pela IgG imobilizada. No potenciometro e no medidor de turbidez, anti-BmoLL IgG não provocou nenhuma alteração na ligação de galactose por BmoLL, indicando que a IgG purificada não tem afinidade pelos sítios de ligação de carboidrato de BmoLL