Bibliography: leaves 99-106.

<p>Studies of binding reactions are of highest importance in a vast number of areas of biomedicine and biotechnology. A demand for fast and accurate small-volume measurements grows stronger, partly due to the development of therapeutic antibodies. In this report, a novel method for studies of binding reactions of antibodies is described. The use of a microfluidic platform shows promising results in determination of affinity binding constants.</p><p>Affinities between 1E-09 and 1E-11 M have been determined for four TSH antibodies. Reproducibility tests give a CV below 10%, using different Gyrolab instruments and microfluidic CD:s. The method carries the advantages of using solution-based measurements of unmodified molecules. Also an initial proof-of-concept for measurement of binding reaction rate constants shows further usage of the method. The kinetic association rate constant has been determined to 2E+06 M-1s-1 for one antibody. The possibility of using this method for screening of antibody libraries is also discussed.</p>

Three epitope-mapping procedures were used to identify B-cell epitopes on Betanodaviruses: neutralisation escape mutant sequence analysis, phage display, and pepscan. Betanodaviruses have emerged as major pathogens of marine fish. These viruses are the aetiological agents of a disease referred to as viral nervous necrosis (VNN), which affects many species of fish that are economically valuable to the aquaculture industry. The identification of betanodavirus B-cell epitopes will facilitate the rational development of vaccines to counter VNN. A panel of mouse monoclonal antibodies (MAbs) was produced using hybridoma methodology for use in each of the epitope mapping procedures. These antibodies were characterised in Western blotting, ELISA, and virus neutralisation tests. Rabbit polyclonal sera, and serum samples from nodavirus-infected fish were also used for pepscan analyses. Attempts to produce betanodavirus neutralisation escape mutants, using plaque assay or limiting dilution based methods, were not successful. Two phage libraries expressing random peptides of seven (Ph.D.7™) or twelve (Ph.D.12™) amino acids in length as fusions to the coat protein were used to identify the ligands recognised by MAbs directed against betanodavirus. Neither of these phage libraries yielded conclusive results. Phage clones containing tandem inserts were obtained after MAb selection from library Ph.D.7™. Extensive screening and nucleotide sequence analysis of MAb-selected clones from library Ph.D.12™) failed to yield a consensus sequence. Pepscan analyses were performed using the recently developed suspension array technology (SAT). This was used to map the recognition sites of MAbs and serum samples onto a panel of overlapping synthetic peptides (12mers) that mimicked the betanodavirus coat protein. The results of pepscan analyses required careful interpretation due to the binding of antibodies and serum samples to multiple peptides. However, three regions of the nodavirus coat protein were identified as containing B-cell epitopes: amino acids 1-50, 141-162, and 181-212. These results are discussed in relation to previous studies of immune responses to betanodaviruses, and to the future development of betanodavirus vaccines and diagnostic reagents.

Studies of binding reactions are of highest importance in a vast number of areas of biomedicine and biotechnology. A demand for fast and accurate small-volume measurements grows stronger, partly due to the development of therapeutic antibodies. In this report, a novel method for studies of binding reactions of antibodies is described. The use of a microfluidic platform shows promising results in determination of affinity binding constants. Affinities between 1E-09 and 1E-11 M have been determined for four TSH antibodies. Reproducibility tests give a CV below 10%, using different Gyrolab instruments and microfluidic CD:s. The method carries the advantages of using solution-based measurements of unmodified molecules. Also an initial proof-of-concept for measurement of binding reaction rate constants shows further usage of the method. The kinetic association rate constant has been determined to 2E+06 M-1s-1 for one antibody. The possibility of using this method for screening of antibody libraries is also discussed.

Purpose: Control of micrometastatic ovarian cancer in the peritoneal cavity remains a major objective in post-surgical treatment. The purpose of this project was to investigate the efficacy and toxicity of targeted alpha therapy (TAT) for ovarian cancer in vitro and in vivo in animal models and to select the optimal targeting vector for an ovarian cancer clinical trial. Animal models of ovarian, breast and prostate cancer were developed and for further TAT; a phase I melanoma clinical trial was supported, paving the way for an ovarian cancer clinical trial. Methods: The expression of the turnor-associated antigens (Her2, MUC1, uPAfuPAR) on cancer cell line, animal model xenografts and human ovarian cancer tissue was tested by immunostaining. MTS and TUNEL assays were used to evaluate cell killing of alpha conjugates in monolayer and spheroids. Toxicity and maximum tolerance doses for different vectors were tested and determined in vivo. Pharmacokinetics was studied for different time points and different parameters. The antiproliferative effect of 213Bi-C595 and 213Bi-PAI2 was tested at 9 days post-peritoneal cell inoculation of the ovarian cancer cell line OVCAR3. The treatment efficacy of 213Bi-Herceptin was tested at a 2 days post-subcutaneous breast cancer cell BT474 inoculation. Mice were injected (i.p) with various concentrations of alpha conjugates (AC). Changes in cancer progression were assessed by girth size and tumor size. Results: uPA/uPAR and MUCI are expressed on ovarian cancer cell lines and more than 45% ovarian cancer tissue, while HER2 was only positive in one cell line and was positive in less than 15% of ovarian cancer tissues. The ACs can target and kill cancer cells in vitro in a dose dependent fashion. TUNEL positive cells were found after incubation with the different ACs. PAI2 and C595 vectors were selected for in vivo ascites model study of OVCARJ cell with high expression. Delayed and acute toxicity in animal models showed that radiation nephropathy was the cause of body weight loss. Biodistribution studies showed that kidney was the major uptake organ. L-lysine can reduce kidney uptake for 213Bi-PAI2, but no significant differences were found. A single ip injection of 213Bi-C595 or 213Bi-PAI2 can inhibit ascites growth, whereas, 213Bi-Herceptin can inhibit breast cancer growth in a nude mice model. Conclusion: 213Bi labelled targeting vectors can specifically target ovarian cancer cells in vitro and inhibit tumor growth in vivo. These ACs may be useful agents for the treatment of ovarian cancer at the minimum residual disease stage.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 209-236). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-06-22Bitstream added on 2014-06-13T18:44:27Z : No. of bitstreams: 1 caetano_ag_dr_jabo.pdf: 1343619 bytes, checksum: 387f2f16ea3939c118f65585ece520a6 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O vírus da Bronquite infecciosa (VBI) é um Coronavírus aviário que infecta aves domésticas de corte e postura, ocasionando grandes perdas econômicas na indústria avícola. Dada a natureza altamente contagiosa e aguda da doença, há uma grande necessidade do desenvolvimento de métodos diagnósticos que possam ajudar na detecção e/ou caracterização de estirpes variantes do VBI. Sendo assim, para auxiliar no diagnostico laboratorial da infecção, foi construída uma biblioteca de fragmentos de anticorpos monoclonais pela técnica de Phage-display. Para tanto, após a imunização de galinhas com a estirpe vacinal H120, foi extraído o RNA total do baço das aves imunizadas e amplificadas as cadeias variáveis leve e pesada que foram unidas por linker, originando o fragmento gênico de cadeia única scFv. Após a realização de três ciclos de seleção foram obtidos 400 clones que foram avaliados em ensaios de ELISA e Western blotting para averiguação da especificidade dos mesmos frente às proteínas da estirpe H120. Após realização dos testes foram selecionados dois clones, um que apresentou grande reatividade para com a proteína de nucleocapsídeo (N) (scFv-N) e o outro com reatividade para com a subunidade 1 da glicoproteína de superfície (S) (scFv-S1). O anticorpo scFv-S1 quando utilizado em ensaio de vírus-neutralização em ovos embrionados mostrou titulo significativo de proteção. Já em testes de ELISA utilizando estirpes de referência e isolados brasileiros de campo do VBI, o anticorpo scFv-N foi capaz de detectar todas as estirpes (H120, M41, Arkansas, IBVPR05, IBVPR02, IBVPR01, IBVSC01), enquanto que o scFv-S1 pode discriminar as estirpes pertencentes ao sorotipo Massachusetts (H120, M41 e IBVSC01) das demais estirpes variantes avaliadas. Os fragmentos de anticorpos scFv-N e scFv-S1 também mostraram bons resultados quando utilizados na técnica... / Infectious bronchitis virus (IBV), the coronavirus of the chicken, is one of the main causes of economic loss within the poultry industry, affecting the performance of meattype and egg-laying domestic fowls. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect and/or characterize IBV strains. In order to improve the laboratory diagnosis of IBV infection, phage-displayed recombinant antibody library derived from splenic mRNA of chickens immunized with H120 vaccine strain of infectious bronchitis virus (IBV) was constructed as single chain variable fragments (scFv) by overlap extension polymerase chain reaction (PCR) of the individual heavy (VH) and light (VL) chain variable gene segments. After three rounds of panning selection, ten scFv phage display antibodies of 400 randomly chosen clones were demonstrated to react with IBV antigens by ELISA. The western blot analysis selected two scFv antibodies reacting strongly with nucleocapsid (N) (scFv-N) protein or subunit 1 of spike glycoprotein (S1) (scFv-S1) of IBV. The anti-S1 scFv antibody showed a significant neutralization titre in embryonating chicken egg test. In ELISA analysis using reference IBV strains and Brazilian field isolates, the anti-N scFv antibody was able to detect all strains (H120, M41, ARKANSAS, IBVPR05, IBVPR02, IBVPR01, IBVSC01), while the anti-S1 could discriminate Massachusetts serotype (H120, M41 and IBVSC01) between variant strains. A scFv-based indirect immunoperoxidase (IP) procedure was also applied to detect infectious bronchitis virus (IBV) antigens in formalin-fixed tracheal tissue sections. Thus, the results showed that scFv-N and scFv-S1 antibodies can be used for the detection and differentiation of IBV strains.

Though antibodies as therapeutics are limited to extracellular targets, their repertoire of molecular interactions has particular relevance to the many intracellular cellular proteins for which small molecule screening has reached impasse. For such proteins there is little recourse to theory, since molecular recognition is, in practical terms, still not well understood. Here I apply antibody discovery to the lysine demthylases KDM4D and PHF8, two proteins difficult to inhibit selectively due to the similarity of their binding pockets to those of the larger family. With a selective, picomolar affinity antibody, dependent on residues distal to the KDM4D active site, I present what is likely the first example of allosteric inhibition of a KDM4 lysine demethylase, demonstrating that there is opportunity outside active sites oversubscribed with pan inhibitors. Antibody discovery for PHF8, however, was plagued by a familiar problem: antibodies that bound when their antigen was immobilised directly to a surface, but barely bound at all when it was free in solution. The common explanation is that the partial denaturation that accompanies immobilisation reveals epitopes unavailable in solution, but examining the problem in detail for the Plant Homeodomain of PHF8 revealed a connection to its rarely sampled conformations. The prominence these antibodies in the immune responses to PHF8, and to some extent KDM4D, motivates two hypotheses on their origin: either the states are very immunogenic or there is a connection between states of irreversible damage and those sampled reversibly, but rarely, by a protein in solution.

Made available in DSpace on 2014-06-11T19:24:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-27Bitstream added on 2014-06-13T19:11:25Z : No. of bitstreams: 1 souza_avg_me_botfm.pdf: 5208360 bytes, checksum: d0caf95e74fc05f32950a4bbcec4f765 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O Vírus Sincicial Respiratório humano (VSR) é o principal agente causal das Infecções Respiratórias Agudas (IRAs) em lactantes e pré-escolares. Apresenta dois subgrupos – A e B – sendo o primeiro responsável por quadros clínicos mais graves. Os RNA mensageiros virais codificam 11 proteínas conhecidas, das quais a proteína F é responsável pela fusão viral à célula e se apresenta na forma de F0 com peso molecular de 67kDa, podendo ser clivada em duas unidades: F2 de 20kDa e a F1 de 47kDa. A proteína F recombinante (Fr), expressa em E.coli (BL21A no vetor pET28a), foi cedida por pesquisadores da UNESP de São José do Rio Preto. Com intuito de produzir anticorpos monoclonais contra a proteína Fr para fins de diagnóstico laboratorial, inoculamo-la em camundongos isogênicos Balb/c. Durante o desenvolvimento do monoclonal, foi necessário a eliminação da contaminação de cultura por Mycoplasma ssp. Após ajuste de dose de inoculação e descontaminação do antígeno (hipótese: contaminação com endotoxinas provenientes de E.coli), seis clones secretores de anticorpos monoclonais foram produzidos (5 do tipo IgM e 1 do tipo IgG2a) e testados, simultâneamente, contra toxina bacteriana e proteína Fr (40μg/mL) por técnica de ELISA indireto. Paralelamente, testes por citometria de fluxo foram realizados, incubando-se os clones obtidos com E.coli (bactéria com membrana permeabilizada e in natura). Os clones (VIRSV2-87A74 e VIRSV2-87A80) apresentam um reconhecimento inespecífico de proteínas da E. coli e não foi possível caracterizar os clones obtidos em função da dificuldade de se obter novas amostras de proteína Fr. Para o futuro, os anticorpos obtidos deverão ser marcados com isotiocinato de fluoroceína e comporem um amplo teste epidemiológico em paralelo com o kit disponível em mercado respeitando a sazonalidade da doença. Novos ensaios para caracterização... / The Human Respiratory Syncytial Virus (HRSV) is the main cause of Acute Respiratory Insufficiency in suckling child and children below 5 years old. It has 2 subgroups – A and B – being the first one responsible for more dangerous clinical situations. The viral messenger RNA codifies 11 known proteins, which the F protein is responsible for the viral fusion and it is presented as F0 with molecular weight of 67kDa, but once is broke it generates two units: F2 with 20kDa and F1 with 47kDa. The recombinant F (Fr) protein were expressed on E.coli (BL21A, on vector pET28a), and it was given to us by researches from UNESP of São José do Rio Preto. Wishing to produce monoclonal antibodies (Mab) against Fr protein to laboratorial diagnosis, mice (Balb/c) were inoculated with de antigen (Fr protein). During the development of the Mab, it was necessary to eliminate, from the culture, Mycoplasma spp. After we adjusted the necessary amount to inoculate on the mice and eradicated the contamination of the antigen (hypotheses: there were endotoxins from the E.coli), six clones that secrete Mab were produced (5 of the kind IgM, and 1 of the kind IgG2a) and they were tested, simultaneously, against bacterium toxin and Fr protein (40μg/mL) using ELISA technique. Together, flow cytometry test were performed when we incubated the clones with E. coli (bacterium with permeable membrane and in natura). The clones (VIRSV2-87A74 and VIRSV2-87A80) presented an unspecific recognition for the proteins from E.coli and it was not possible to characterize the cells because we couldn´t get more samples of Fr protein. For the future, the Mab should be conjugated with FITC and then, they must be part of a wide epidemiologic test side by side from the commercial kit, always respecting the seasonal disease. New assays to characterize antibodies (like Western blotting and imunepreciptation) are already being done... (Complete abstract click electronic access below)