This study was conducted aiming to compare the conventional microbiological method to detect Salmonella in broiler parts with the Immunomagnetic Separation method (IMS) followed by plate isolation and also the IMS associated with Rappaport-Vassiliadis broth (RV). The IMS was performed following a pre- enrichment step in buffered peptone water. Sixty-one samples (raw broiler parts) were tested and the results showed that the use of the IMS method alone allowed the isolation of Salmonella in 9 of the tested samples, while the association IMS/RV detected the agent in 30 samples. The conventional microbiological method was able to isolate the agent in 25 opportunities. These results allowed to conclude that the IMS/RV association presented an increased sensitivity and permitted a better isolation of Salmonella. The conclusion was that other means of isolation, in particular those which do not interfere with the growth of bead bounded Salmonella, should be searched.

Abstract The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900 -PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 101 CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina.

O desenvolvimento de nanocompósitos porosos e magnéticos tem recebido grande atenção nos últimos anos. Atualmente, o campo da biotecnologia tem buscado empregar compósitos magnéticos em processos de separação e purificação celular, além de outras aplicações. Já nas pesquisas com energia limpa, tem-se buscado empregar materiais carbonosos como adsorventes e sensores de gases, especialmente no encapsulamento de hidrogênio. Neste trabalho, foram sintetizados compósitos magnéticos porosos contendo nanopartículas metálicas embebidas numa matriz de carbono, através do método dos precursores poliméricos. Os compósitos sintetizados foram caracterizados através de difração de raios X (DRX), fluorescência de raios X por energia dispersiva (EDX), fisissorção de N2 (BET e BJH), técnicas de microscopia eletrônica (MET e MEV) e espectroscopia na região do infravermelho (FT-IR). Os compósitos apresentaram nanopartículas dos metais, responsáveis pelo comportamento ferromagnético e pelos sítios de quimissorção, embebidas na matriz de carbono amorfo, responsável pela porosidade e pelos sítios de fisissorção. Ensaios de redução termoprogramada e microscopia de imunofluorescência comprovaram a potencial aplicação dos compósitos nos processos de separação imunomagnética e encapsulamento de hidrogênio.

This paper addresses a sensitive method for the detection of mycobacteria in hemodialysis water samples based on a magneto-actuated immunoassay with optical readout. In this approach, micro (2.8 μm) sized magnetic particles were modified with an antibody against the lipoarabinomannan (LAM) located in the mycobacterial cell wall. The system relies on the immunocapturing of the mycobacteria with the tailored antiLAM magnetic particles to pre-concentrate the bacteria from the hemodialysis samples throughout an immunological reaction. The performance of the immunomagnetic separation on the magnetic carrier was evaluated using confocal microscopy to study the binding pattern, as well as a magneto-actuated immunoassay with optical readout for the rapid detection of the bacteria in spiked hemodialysis samples. In this approach, the antiLAM polyclonal antibody was labeled with fluorescein isothiocyanate. The optical readout was achieved by the incubation with a secondary anti-fluorescein antibody labeled with peroxidase as optical reporter. The magneto-actuated immunoassay was able to detect mycobacteria contamination in hemodialysis water at a limit of detection of 13 CFU mL-1 in a total assay time of 3 h without any previous culturing pre-enrichment step.

The conventional methods used to detect the Listeria monocytogenes in foods are laborious and expensive, requiring several days for final identification. Monoclonal antibody (MAb) based immunoassays are highly specific and rapid to perform, especially when MAbs are raised to conserved virulence factors in the pathogen. Among diverse virulence factors of L. monocytogenes, the surface protein internalin A (InlA) is one of the most well-known and characterized protein, being an excellent target as it is highly exposed on the surface and exclusive of pathogenic species. In this work we report the production, characterization and use of a panel of MAbs against InlA (2D12, 3B7, 4E4), and a MAb (3F8) which specifically recognizes all bacteria belonging the genus Listeria. MAbs were produced by the immunization of BALB/c mice with a recombinant InlA together with heat killed L. monocytogenes. The MAbs produced showed excellent reativities by indirect ELISA, Western blot and immunofluorescence. A Cy5 conjugated anti-InlA MAb-2D12 was used as detection antibody for L. monocytogenes in a sandwich-like fiber optic immunoassay. Using MAb-2D12 as capture antibody on the waveguides, the limit of detection was ~3 x 102 CFU.mL-1, and when MAb-3F8 was used for capture the limit of detection was ~1 x 105 CFU.mL-1. Furthermore, MAbs 2D12 and 3F8 were used to coat paramagnetic beads and tested in the immunomagnetic separation (IMS) of L. monocytogenes from pure cultures, and artificially contaminated cheeses and hotdogs. After IMS capture, bacteria were released from the beads, used in the fiber optic assay or plated on agar for counting. In parallel, the capture of L. monocytogenes was confirmed by real-time qPCR and light-scattering technology (BARDOT). Using IMS to concentrate and separate L. monocytogenes, followed by a fiber optic platform, it was possible to detect in less than 22 h, approximately 40 CFU/g of L. monocytogenesi, even in the presence of L. innocua in cheese and hot dogs artificially contaminated. In addition, using mass spectrometry (MALDI-TOF-MS) the protein to which MAb-3F8 binds, was identified as fructose 1,6-bisphosphate aldolase (FBA). The results presented in this work indicate that using both systems together, the IMS and fiber optic immunosensor, were more reliable and faster, and could be applied in the routinely for detection of L. monocytogenes in food. Moreover, both MAbs have the potential to useful in others biosensor platforms, as well as in other detection and functionality immunoassays for InlA and FBA in Listeria.

Epidermal stem cells isolation struggle remains, mainly due to the yet essential requirement of well-defined approaches and markers. The herein proposed methodology integrates an assemblage of strategies to accomplish the enrichment of the interfollicular epidermal stem cells multipotent fraction and their subsequent separation from the remaining primary human keratinocytes culture. Those include rapid adherence of freshly isolated human keratinocytes to collagen type IV through the β 1-integrin ligand and Rho- Associated Protein Kinase Inhibitor Y- 27632 administration to the cultures, followed by an immunomagnetic separation to obtain populations based in the combined CD49f bri /CD71 dim expression. Flow cytometry is the supporting method to analyze the effect of the treatments over the expression rate of early epidermal markers keratins19/5/14 and in correlation to CD49f bri /CD71 dim subpopulations. The stepby- step methodology herein described indulges the boosting and consecutive puri fi cation and separation of interfollicular epidermal stem cells from human keratinocytes cultures.

Circulating tumor cells have an active role in the formation of metastasis. However, their scarce number in blood of cancer patients raises numerous technological challenges. Technologies for detection of these cells and the conceptions of metastatic progression are the cancer investigation allies for a new dimension of clinical research that demands modern and clinically viable trial designs. This dissertation aims for the development of functionalized polymer beads with encapsulated magnetic nanoparticles for detection and quantification of circulating tumor cells and their in vitro culturing after isolation from blood samples. The project was divided into two main tasks: depletion of CD45 white cells for culture in vitro, and development of nanocomposites for cell detection and quantification. For depletion of CD45 white cells, two kinds of beads were studied, the commercially available Dynabeads® and beads prepared at International Iberian Nanotechnology Laboratory (Braga, Portugal). For Dynabeads®, the number of depleted cells increased with the total amount of beads, whereas for INL beads this does not apply. For preparation of the polymer beads, a nanoprecipitation method was used, with Poly(L-Lactide-co-glycolide) and superparamagnetic iron oxide nanoparticles as the main materials. The polymer was chosen due to its biodegradability. Since the magnetic beads will not be removed prior to in vitro culturing of the cells, biodegradability is an important aspect to avoid negative effects on cell growth. Initially, a preliminary study was performed to determine the optimal formulation for beads production. The best conditions included as non-solvent a mixture of ethanol and acetone in a volume ratio of 20:80, a solvent and non-solvent ratio of 0.02, and an amount of magnetic nanoparticles ten times smaller than the amount of polymer. The beads were characterized by Dynamic Light Scattering, in terms of particle size, polydispersity index and zeta potential; magnetic content through Thermogravimetric Analysis; and morphological features by Scanning Electron Microscopy. They presented average size values of 232 ± 14.30 nm. Their polydispersity index and zeta potential was 0.155 ± 0.046 and -48.66 ± 3.72 mV, respectively. The beads successfully encapsulated the iron oxide nanoparticles, with values of magnetic content of 51.5 ± 23 wt%. Lastly, the efficiency of nanocomposite beads was assessed by an immunomagnetic cell separation test. Beads formulated by nanoprecipitation were able to isolate 47 ± 16 % of cells. This dissertation focuses on polymeric beads for detection and quantification of cells. It has been demonstrated that biodegradable Poly(L-Lactide-co-glycolide) beads containing superparamagnetic iron oxide nanoparticles are an appropriate cell separation vehicle for cell culturing.