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LSPR-based immunoassay for the specific detection of HspX protein biomarker related to Tuberculosis disease

AutorPeláez-Gutierrez, E. Cristina; Estévez, M. Carmen ; Lechuga, Laura M.
Fecha de publicación2016
Citación3rd Institut Pasteur International Network Symposium (2016)
ResumenTuberculosis is a disease that has caused the higher rate of morbidity and mortality worldwide. The World Health Organization estimates that there are nine million new cases and three million deaths attributed to the disease each year, mainly occurring in developing countries. Advances in screening and diagnostic techniques and the introduction of novel therapies have substantially increased patient survival rates during the last years. However, there are limitations related to the inefficiency of real-time detection: techniques and laboratory methods have low specificity which leads to misdiagnosis, instruments are still expensive and highly trained staff is still needed. To contribute to the detection of tuberculosis the HspX protein has been used as a biomarker. It is a 16 kDa protein known as α-crystallin homologous protein (encoded by the gene hspx 435 bp). It was originally described as a dominant antigen of M. tuberculosis observed in the serum of patients with asymptomatic infection. For this reason, it is considered a strongly associated element with latent tuberculosis. Some studies suggest that HspX protein plays an important role in the pathogenesis of tuberculosis, as it is required for the growth of M. tuberculosis in human macrophages cultures and reduces the inactivated gene expression. Due to its high immunogenicity, the hspX protein has been used in the development of new diagnostic tests or in the construction of new vaccines. The use of this biomarker for biosensing strategies could offer a valuable alternative for diagnosis of tuberculosis for early detection and rapid quantification. In the present study, we have obtained HspX recombinant protein using molecular biology techniques: clones were transformed into E. coli TOP10, on solid Luria-Bertani (LB) with ampicillin whose concentration is 100 mg/mL, incubated for 16 hours at 37 °C. A cell lysate for protein extraction, evaluated by electrophoresis in polyacrylamide gel and whose detection is performed by western blot with antibodies mouse IgG was performed. The protein was purified by affinity chromatography with a Ni-NTA resin 50% and was obtained with PBS buffer 1X, pH 7.5 and 250 mM Imidazole. Subsequently we have developed a label free nanoplasmonic biosensor based immunoassay methodology for determination of this protein in serum. The strategy has been optimized and evaluated, allowing a direct and rapid quantification of HspX protein using highly specific monoclonal antibodies. Several experimental conditions have been studied in order to assess the viability of performing the measurements in serum and/or plasma. Preliminary results show a Limit of Detection (LOD) of 9.96 nM (159 ng/mL) approximately and the possibility to directly perform diluted serum evaluations.
DescripciónResumen del trabajo presentado al 3rd Institut Pasteur International Network Symposium, celebrado en Paris (Francia) del 29 de noviembre al 2 de diciembre de 2016.
URIhttp://hdl.handle.net/10261/160996
Aparece en las colecciones: (CIN2) Comunicaciones congresos
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