Characterization of new anti-IL-6 antibodies revealed high potency candidates for intracellular cytokine detection and specific targeting of IL-6 receptor binding sites.

Interleukin-6 (IL-6) expression and secretion, induced by inflammatory processes, stimulate the acute phase response cascade. The overexpression of IL-6 contributes to a variety of inflammatory diseases, e.g. rheumatoid arthritis, Castleman's disease, multiple myeloma, and prostate cancer. Screening for high amounts of IL-6 in the patients' blood serum can be crucial for an adequate treatment. In this study, five novel murine monoclonal antibodies (mAbs) reactive to human IL-6 were generated. The mAbs were characterized for potential diagnostic purposes and recombinant antibodies were derived thereof. Initial epitope mapping using a combination of blocking experiments and Hyper-IL-6, a fusion protein consisting of IL-6 and the soluble IL-6 receptor revealed distinct but overlapping binding sites. At least one of the mAbs was found to interact with the region of IL-6/ IL-R complex formation. Three mAbs were applied successfully in intracellular staining by flow cytometry, whereas one of the mAbs showed comparable binding as a reference reagent. Furthermore, the mAbs were tested for applications in various immunological assays such as ELISA, Western blot and surface plasmon resonance spectroscopy (SPR), using IL-6 from commercial sources as well as in-house produced protein (IL-6_IME). The limit of detection was determined by sandwich ELISA (0.5 ng/mL, SD ±0.005). Our results also demonstrated that the recombinant IL-6 produced was functional and correctly folded. These findings support the use of the generated mAb clones as promising candidates for application in various immunological assays for diagnostic and scientific purposes.

Fine Tuning Antibody Conjugation Methods using SNAP-tag Technology.

BACKGROUND: Targeted imaging and therapy (theranostics) is a promising approach for the simultaneous improvement of cancer diagnosis, prognosis and management. Therapeutic and imaging reagents are coupled to tumor-targeting molecules such as antibodies, providing a basis for truly personalized medicine. However, the development of antibody-drug conjugates with acceptable pharmaceutical properties is a complex process and several parameters must be optimized, such as the controlled conjugation method and the drug-to-antibody ratio. OBJECTIVE: The major aim of this work is to address fundamental key challenges for the development of versatile technology platform for generating homogenous immunotheranostic reagent. METHOD: We conjugated the theranostics reagent IRDye700dx to a recombinant antibody fusion protein containing a self-labeling protein (SNAP-tag) which provides a unique reaction site. RESULTS: The resulting conjugate was suitable for the imaging of cancer cells expressing the epidermal growth factor receptor and demonstrated potent phototherapeutic and imaging activities against them. CONCLUSION: Here, we describe a simple, rapid and robust site-directed labeling method that can be used to generate homogeneous immunoconjugate with defined pharmacological properties.

Qualidade microbiológica de alimentos servidos em restaurantes self-service / Microbiological quality of food from self-service restaurants

Os serviços de alimentação coletiva têm se expandido em todo o mundo, sendo os restaurantes do tipo self-service a preferência atual dos consumidores. Considerando-se a importância da qualidade higiênica dos alimentos para a saúde da população, foi investigada a microbiota presente em alimentos prontos para o consumo. No segundo semestre de 2008, foram coletadas 20 amostras de refeições, à base de carne, prontas para o consumo, em diferentes restaurantes self-service da cidade de Araçatuba/SP. As análises bacteriológicas realizadas seguiram as metodologias convencionais e os resultados foram comparados com os padrões regulamentados pela legislação brasileira vigente. Das amostras analisadas, 90% foram positivas para coliformes a 35ºC. Coliformes a 45°C foram detectados em 55% das amostras e, destes, em 63,63% foi confirmada a ocorrência de Escherichia coli. A presença de estafilococos coagulase-positiva foi verificada em 10% das amostras; Salmonella spp. e Bacillus cereus não foram detectados. Não foram pesquisados os clostrídios sulfito redutores a 42ºC. Este estudo aponta a necessidade de atenção rigorosa quanto às condições sanitárias de preparo e exposição dos alimentos prontos para consumo, uma vez que a ingestão de produtos contaminados constitui um potencial risco para a saúde pública.

Collective food services have been increasing worldwide, and the self-service restaurant has been the current preference by consumers. Considering the importance of hygienic quality of food, the microbiologicalcomposition of ready-to-eat food was assessed. In the second semester of 2008, 20 samples of meals, mainlymeat-based foods, were collected from different self-service restaurants in Araçatuba city, SP. Bacteriologicalanalyses were performed following the conventional methodologies, and the results were compared with thestandards established by the effective Brazilian legislation. Coliforms at 35ºC were detected in 90% of analyzedsamples. Coliforms at 45°C were found in 55% of the samples and, among these, in 63.63%, the occurrence of Escherichia coli was confirmed. Coagulase-positive staphylococci were detected in 10% of samples and no sample showed Salmonella spp. or Bacillus cereus contamination. Sulfite reducing clostridia at 42oC were not investigated in this study. These findings indicate the need for a rigorous approach for improving the sanitary conditions during preparation and presentation of ready-to-eat food, as the consumption of contaminated products represents a potential risk to public health.