Selecção e caracterização de mini-anticorpos recombinantes anti-ciprofloxacina

Mestrado em Microbiologia MolecularEste trabalho teve como objectivo central a obtenção de mini-anticorpos recombinantes com especificidade para o antibiótico ciprofloxacina (CPFX). A utilização em saúde humana, animal e vegetal deste antibiótico tornou-o, à semelhança de outros, um problema de contaminação ambiental. Actualmente, o doseamento de CPFX em amostras é feito, na maioria das vezes, por cromatografia líquida de alta resolução (HPLC), o que é um método bastante moroso. Assim, há grande interesse em desenvolver métodos alternativos, rápidos e sensíveis para detecção daquele composto. Imunoensaios com recurso a anticorpos recombinantes, ou fragmentos de anticorpos, é uma via interessante e esta constitui o objectivo último desta investigação. Para tal, neste trabalho escolheu-se efectuar uma selecção numa biblioteca de fagos (Griffin.1) que expressam mini-anticorpos de cadeia única (scFv`s) à sua superfície (denominados phAbs), para isolar alguns com afinidade para CPFX, através da técnica de phage display. O processo exigiu, num primeiro passo, a imobilização da CPFX num suporte sólido, tendo-se optado por esferas magnéticas. Efectuou-se a ligação covalente entre o grupo carboxílico da CPFX e os grupos amina à superfície das esferas por reacção com carbodiimida. Conseguiu-se uma aparente saturação dos grupos amina com CPFX. Para quantificação de CPFX nas soluções, foi previamente desenvolvido e validado um método de HPLC que se distingue de outros métodos reportados. Seguidamente, efectuou-se um protocolo de selecção sobre a biblioteca de minianticorpos acima referida utilizando as esferas derivatizadas com CPFX. Em vários ciclos sucessivos de selecção, a eluição das esferas de phAbs (específicos) foi efectuada com quantidades decrescentes de CPFX livre em solução, e com tempos de eluição também decrescentes, de modo a promover uma pressão selectiva cada vez mais apertada. Desenvolveu-se também um método para separar phAbs eluídos da CPFX em solução, baseado em cromatografia de exclusão molecular. Para testar a afinidade e especificidade de phAbs, desenvolveu-se com sucesso métodos de ELISA que empregam as esferas magnéticas derivatizadas com CPFX e os próprios phAbs (em vez dos mini-anticorpos separados dos fagos). Após quatro ciclos de selecção, analisaram-se por ELISA 32 clones, tendo-se detectado cinco clones com afinidade por CPFX. Procedeu-se, então, à sequenciação dos genes dos fragmentos daqueles cinco clones, mas só três deles tiveram resultados conclusivos. Curiosamente, todos três correspondem a fragmentos VL, e não a scFv`s, sendo que dois são da mesma família (VKII). Produziram-se os phAbs dos três clones e analisaram-se em ELISA competitiva, a fim de identificar aqueles que ligam CPFX em solução, e não apenas CPFX imobilizada nas esferas. Apenas dois dos phAbs satisfizeram este critério, mas só um (correspondente ao clone 406) mostrou um comportamento regular na ELISA. Este ensaio mostrou um limite de detecção de CPFX de 9,3 nM. Analisou-se também por ELISA competitiva a especificidade deste phAb, verificando-se que ele possuía reactividade cruzada com outras três fluoroquinolonas, mas não com outros dois antibióticos não-quinolonas. Os fragmentos VL, separados dos fagos, do clone 406 foram produzidos com sucesso por cultura de E. coli em pequena escala, conforme confirmado por análise dot blot. Este conjunto de resultados sugere como trabalho futuro a produção em maior escala de fragmentos isolados, bem como o desenvolvimento e a validação de imunoensaios para detecção e quantificação de CPFX em amostras ambientais. ABSTRACT: The main objective of this research was to obtain recombinant mini-antibodies with specificity for the antibiotic ciprofloxacin (CPFX). The use of this drug in human, animal and plant health turned it an environmental problem, like many other antibiotics. Currently, quantification of CPFX is carried out most often by high performance liquid chromatography (HPLC), which is a time demanding method. Therefore, it is of interest to develop rapid, sensitive, alternative methods for detection of that compound. Immunoassays using recombinant antibodies, or their fragments, constitute an interesting route and this is the ultimate goal of this research. For that purpose, the strategy followed was to carry out a selection procedure in a library of phage particles (Griffin.1) that express single-chain antibodies (scFv`s) at their surface (named phAbs), in order to isolate a few with affinity for CPFX. This process required, as a preliminary step, the immobilization of CPFX on to a solid support. Magnetic beads were chosen for this purpose. The covalent linkage between CPFX molecules and amine groups at the surface of the beads was carried out by a carbodiimidemediated reaction. An apparent saturation of the amine groups in the beads was attained. In order to quantify CPFX in the solutions, a HPLC protocol, different from others previously reported in the literature, was developed and validated. Next, a selection procedure was executed on the above mentioned library and using the CPFX-derivatized beads. In the various cycles of selection, the elution of (specific) phAbs from the beads was attained with decreasing quantities of free CPFX in solution, and with decreasing contact times, in order to provid an increasing selective pressure. A method to separate the eluted phAbs from CPFX in solution based on size-exclusion chromatography was also developed. In order to test the affinity and specificity of phAbs, ELISA methods employing the magnetic beads-CPFX and the phAbs themselves (instead of the mini-antibodies separated from the phages) were successfully developed. After four rounds of selection, 32 clones were analysed by ELISA. Of these, five showed affinity for CPFX. The sequencing of the genes of the antibody-fragments in those five clones was then carried out, but with conclusive results only for three of them. Curiously enough, all three correspond to VL fragments, and not to scFv`s. Two of them belong to the same family (VKII). The phAbs of the three clones were produced and then tested in competitive ELISA, in order to identify those that bind CPFX in solution, besides CPFX bound to the beads. Only two of the phAbs fulfilled this criterion, but only one (corresponding to clone 406) showed a regular pattern in ELISA. This assay showed a limit of detection of CPFX of 9.3 nM. The specificity of this phAb was also studied by competitive ELISA. It was observed that it has cross-reactivity with three other fluoroquinolones, but not with two other antibiotics non-quinolones. VL fragments, separated from the phage, from the clone 406 were successfully produced by E. coli culture in small scale, as confirmed by dot blot analysis. This set of results suggest as future work the production in larger scale of isolated antibody fragments, followed by the development and validation of immunoassays for detecting and quantifying CPFX in environmental samples

Development of an immunoassay for ciprofloxacin based on phage-displayed antibody fragments

The widespread use of ciprofloxacin in human, animal and plant health has raised an environmental problem, paralleled by several other antibiotics. The aim of this work is the development of a rapid and sensitive ELISA assay for ciprofloxacin, which can constitute an alternative to time-consuming HPLC methods. For this purpose, we worked with antibody fragments, instead of whole antibodies, and used magnetic beads as solid support. Ciprofloxacin was successfully immobilized onto this support with a carbodiimide-mediated reaction. A library of phage particles that express human single-chain antibodies at their surface was then screened with an optimized protocol. Several positive fragments were isolated and identified as being V L fragments. These were then fully characterized. A reproducible competitive ELISA was developed using the magnetic beads -ciprofloxacin as support and the phages displaying the V L fragment as recognition entity. This assay showed limits of detection and quantification of 9.3 nM and 33 nM, respectively. Also, competitive ELISAs with ciprofloxacin homologues and other molecules showed cross-reactivities lower than 12%

Development of an immunoassay for ciprofloxacin based on phage-displayed antibody fragments

The widespread use of ciprofloxacin in human, animal and plant health has raised an environmental problem, paralleled by several other antibiotics. The aim of this work is the development of a rapid and sensitive ELISA assay for ciprofloxacin, which can constitute an alternative to time-consuming HPLC methods. For this purpose, we worked with antibody fragments, instead of whole antibodies, and used magnetic beads as solid support. Ciprofloxacin was successfully immobilized onto this support with a carbodiimide-mediated reaction. A library of phage particles that express human single-chain antibodies at their surface was then screened with an optimized protocol. Several positive fragments were isolated and identified as being VL fragments. These were then fully characterized. A reproducible competitive ELISA was developed using the magnetic beads — ciprofloxacin as support and the phages displaying the VL fragment as recognition entity. This assay showed limits of detection and quantification of 9.3 nM and 33 nM, respectively. Also, competitive ELISAs with ciprofloxacin homologues and other molecules showed cross-reactivities lower than 12%.info:eu-repo/semantics/publishedVersio

Doenças de feitiço: as Minas setecentistas e o imaginário das doenças Diseases from witchcraft: eighteenth century Minas Gerais captaincy/Brazil and the imaginary of the diseases

O presente artigo objetiva discutir a crença e o consenso em torno da ideia partilhada, entre aqueles que viveram nas Minas no curso do século XVIII, de que determinados indivíduos poderiam, por meio de feitiços, provocar uma série de males. Assim, as doenças de feitiço, conforme aparecem na documentação compulsada, pareciam bastante assíduas. Procurei igualmente analisar como eram descritos tais achaques provocados pelos feitiços: tolhimentos, dores, ligamentos, dentre outros. Estes se faziam presentes tanto nas denúncias levadas ao conhecimento de membros do clero no curso das devassas eclesiásticas (documentação sob a guarda do Arquivo Eclesiástico da Arquidiocese de Mariana) como em tratados médicos publicados, sobretudo, nas primeiras décadas do setecentos.<br>This article aims to discuss the belief and the consensus around the idea shared among those who lived in Minas in the course of the eighteenth century that certain individuals could, by spells, cause a variety of ailments. Thus, the spell of illness, as it appears in the documentation compelling seemed very assiduous. I also tried to analyze how they were described such ailments caused by spells: stunting, pain, ligaments, among others. These were present both in the complaints brought to the attention of members of the clergy during the wanton ecclesiastical (documentation in the custody of the ecclesiastical archives of the Archdiocese of Mariana) as published in medical treatises, especially in the first decades of the 18th century

Núcleos de Ensino da Unesp: artigos 2013: volume 2: metodologias de ensino e a apropriação de conhecimento pelos alunos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Núcleos de Ensino da Unesp: artigos 2010: volume 4: as disciplinas escolares, os temas transversais e o processo de educação

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved