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Development of a test associated with magnetic nanoparticles for the diagnosis of tuberculosis.

Abstract : Mycobacterium tuberculosis causes one of the diseases with the highest mortality and morbidity rate in the Americas and around the world. In developing countries, the diagnosis of tuberculosis (TB) is based on smear microscopy and bacteriological cultures. The first method has low sensitivity, and the second take several weeks to reach a confirmatory diagnosis. The lack of a rapid diagnosis compromises the efforts to control TB, favoring its transmission to the susceptible population. Currently, magnetic nanoparticles (MNPs) functionalized with biomolecules have been used in biomedicine, due the magnetic, electrical and optical properties. In this way, applying external magnetic fields, bio-functionalized MNPs is used to detect and concentrate cells and biomolecules from biological samples.In this work we present the synthesis, characterization and bio-functionalization of magnetic nanoparticles, to develop a sandwich ELISA assay associated to MNPs to detect antigens from M. tuberculosis. For this purpose, magnetic nanoparticles were synthesized by co-precipitation method. The MNP surface was amine-silanized (MNP@Si@NH2) and characterized by physical-chemical methods.The MTB antigens evaluated in this study were: Hsp16.3, CFP10, ESAT6, MTC28, MPT64, 38 kDa protein, Ag85B and MoeX. Cloning ad expression of recombinant proteins were made in E. coli BL21 (DE3) pLysS system. Polyclonal antibodies were produced in New Zealand rabbits and BALB/C mice, previously immunized with purified recombinant antigens. Specific antibodies (ab) were immobilized in the amine-silanized MNP surfaces. The MNP@Si@ab were associated in a colorimetric sandwich ELISA assay to capture and detect native MTB antigens from sputum samples.The XRD, Mössbauer spectroscopy, zeta potential, TEM and FTIR demonstrated the successful preparation of the MNPs showing a diffraction crystal diameter of ~12.5 nm (10.48 ± 2.56 nm), superficial net charge of ᶎ: +23.57 ± 2.87 mV, characteristic patterns of magnetite and a spherical structure. Additionally, a magnetization saturation of 37.06 emu.g-1 was observed. For the functionalization of nanoparticle surfaces with antibodies, active ester method (coupling agent EDC/NHS) were used for peptide bond formation. Parameters such as time of incubation, concentration of coupling agents and surface saturation level of amine-silanized MNPs (MNP@Si@NH2), were previously standardized.Finally, antibody functionalized on MNPs were used to capture and detect recombinant and native M. tuberculosis antigens in an ELISA-MNP@Si@ab sandwich test (in a reaction time <4 h). The ESAT6 and CFP10 antigens were better discriminated in sputum pooles from patients with TB (fold value ~ 1.8). The use of MNP@Si@ab improved the detection of MTB antigens in biological samples. Our results are encouraging, but the essay requires additional evaluations such as determining cross-reactions with sputum samples from patients with other infections, performing the test with fresh sputum of TB patients, and determining the sensitivity and specificity of the method.
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Nancy León Janampa. Development of a test associated with magnetic nanoparticles for the diagnosis of tuberculosis.. Organic chemistry. Université de Bordeaux; Universidad Peruana Cayetano Heredia, 2019. English. ⟨NNT : 2019BORD0272⟩. ⟨tel-02865409⟩

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