RESUMO

Immunological methods to detect SARS-CoV-2 seroconversion in humans are important to track COVID-19 cases and the humoral response to SARS-CoV-2 infections and immunization to future vaccines. The aim of this work was to develop a simple chromogenic magnetic bead-based immunoassay which allows rapid, inexpensive, and quantitative detection of human antibodies against SARS-CoV-2 in serum, plasma, or blood. Recombinant 6xHis-tagged SARS-CoV-2 Nucleocapsid protein was mobilized on the surface of Ni2+ magnetic beads and challenged with serum or blood samples obtained from controls or COVID-19 cases. The beads were washed, incubated with anti-human IgG-HPR conjugate, and immersed into a solution containing a chromogenic HPR substrate. Bead transfer and homogenization between solutions was aided by a simple low-cost device. The method was validated by two independent laboratories, and the performance to detect SARS-CoV-2 seroconversion in humans was in the same range as obtained using the gold standard immunoassays ELISA and Luminex, though requiring only a fraction of consumables, instrumentation, time to deliver results, and volume of sample. Furthermore, the results obtained with the method described can be visually interpreted without compromising accuracy as demonstrated by validation at a point-of-care unit. The magnetic bead immunoassay throughput can be customized on demand and is readily adapted to be used with any other 6xHis tagged protein or peptide as antigen to track other diseases.

Assuntos

RESUMO

Gram-negative bacterial endophytes have attracted research interest caused by their advantageous over epiphytic bacteria in plant nutrition and protection. However, research on these typically Gram-negative endophytes has deficiencies concerning the role of cultivation and pre-formulation strategies on further plant colonisation capabilities. Besides, the influence of cultivation conditions and osmotic stress within bacterial endophytes on their phosphate solubilising ability has not yet been addressed. By pre-conditioning cells with an osmoadaptation and a hydroxyectoine accumulation approach, this research aimed at enhancing the capability of the plant growth promoting bacterium Kosakonia radicincitans strain DSM 16656T to both solubilise phosphate and colonise plant seedlings. The results showed that halotolerant bacterial phenotypes increased the root-colonising capability by approximately 3-fold and presented growth-promoting effects in radish plants. Interestingly, findings also demonstrated that salt stress in the culture media along with the accumulation of hydroxyectoine led to an increase in the in vitro phosphate-solubilising ability by affecting the production of acid phosphatases, from 1.24 to 3.34 U mg-1 for non-salt stressed cells and hydroxyectoine-added cells respectively. Thus, this approach provides a useful knowledge upon which the salt stress and compatible solutes in bacteria endophytes can confer phenotypic adaptations to support the eco-physiological performance concerning phosphate-solubilising abilities and endosphere establishment.