Ciencias Exactas y Ciencias de la Salud

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Design of methods to monitor and evaluate Clostridium chauvoei under industrial settings for generating a fermentation map
(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2022-06-01) Cepeda Pérez, Daniela; Licona Cassani, Cuauhtémoc; puemcuervo, emipsanchez; Mendoza, Uri; Mayolo Deloisa, Karla; Orellana Montecino, Camila; Torres Acosta, Mario Antonio; School of Engineering and Sciences; Campus Monterrey
Blackleg is a disease that affects cattle caused by the anaerobic and spore-forming bacteria Clostridium chauvoei. It possesses several virulence factors that act in synergy to cause the typical lesions. Flagellin (FliC) is a cell surface antigen that provides protective immunity to vaccinated animals against C. chauvoei infection. It is related to virulence and pathogenicity, thus, demonstrating a relationship between flagellar antigen quantity and final vaccine efficacy for blackleg disease, as demonstrated in several immunoassays. Clostridium chauvoei toxin A (CctA) is a pore-forming toxin secreted to the supernatant. It causes lysis by perforating the cell membrane, and it is known to be the main virulence factor of the pathogen. Other proteins such as sialidase and hyaluronidase are considered the main virulence factors of Clostridium chauvoei. Because blackleg is of economic importance, and as a disease that advances rapidly, vaccination has been available as a prevention method. However, since its development, the vaccine remains as a whole-cell formalin-inactivated culture due to the lack of knowledge of specific conditions to generate enough antigens to induce protection. Additionally, there is a variability of batch-to-batch immunogenic properties and final efficacy, which may be explained by the antigen’s quantity variation throughout the process. Although final batch testing is currently applied in the industry, there are no methods to monitor the fermentation dynamics and consistency of the overall production. Consistency can be achieved by setting parameters that constitute a product profile that satisfies final product requirements. The expression and purification of a recombinant FliC allowed the development of an indirect ELISA to measure the flagellin antigen quantity of Clostridium chauvoei fermentation samples. While the assay is not validated to predict the potency and final efficacy of the vaccine, it can be used as part of a battery of methods to provide a more characterized product profile to monitor trends and dynamics in the production. In addition, a rapid micro-plate bioactivity assay was developed to measure the hemolysis percentage caused by the supernatant of samples of C. chauvoei. The assays represent valuable methods to generate data and monitor the fermentation dynamics of C. chauvoei cultures under industrial conditions to achieve production consistency.
STREM-1 based sepsis detection with centrifugal microfluidics incorporating active valves and image analysis
(2017-11-17) Medrano Danés, Jonathan; Martinez Chapa, Sergio Omar; Mehdi Aeinehvand, Mohammad; Dieck Assad, Graciano; Martins Fernandes, Rute Fabiana
Sepsis is the main cause of neonate death in hospitals. According to WHO, around one million neonatal deaths are caused each year because of sepsis. Rapid and early sepsis detection, are necessary for effective treatment of the patients to reduce the mortality rate, however this is still a big challenge, especially in low income countries with a poor healthcare system. Recently, soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) biomarker has showed to be an accurate indicator for sepsis detection in neonates. When compared to conventional tests that take several days up to a week, sTREM-1 immunoassay is a faster approach for sepsis detection as it can be performed in five hours. However, the sTREM-1 immunoassay must be performed in a clinic laboratory by specialized staff and require several expensive and bulky equipment. One way to enable the sTREM-1 immunoassay outside specialized labs or at point-of-care (POC) is the integration of the complex assay into a centrifugal microfluidic platform. The disc-shaped microfluidic platform also known as lab-on-disc (LoD), employs centrifugal force from spinning of the disc to manipulate samples and reagents. The centrifugal force that exist everywhere on the disc prevents the need for several expensive syringe pumps. However, it makes simultaneous control over the retention and flow of several reagents/samples on a LoD challenging. To overcome this problem and automate sTREM-1 immunoassay for POC settings, this document presents the development of a new sacrificial polyethylene and a novel reversible valving mechanism on LoD. Based on the two valving mechanisms, a sTREM-1 immunoassay LoD is developed for sepsis detection. Integrating a series of sacrificial valves in the disc allowed for storage of reagents in the disc and their controlled release to a reaction chamber. The reversible valve, was used for the retention of each reagent in the detection chamber during incubation periods, and then transferring it to a waste chamber. A portable spinning system was developed to run the assay by microfluidic disc at POC. The successful automation of sTREM1 immunoassay on the microfluidic disc can provide a significant contribution in the reduction of complexity of the resources needed for sepsis detection at POC settings, particularly in remote areas.

Ciencias Exactas y Ciencias de la Salud

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