RESUMO
An electrochemical impedimetric immunosensor was developed for the detection of the neuropeptide Corticotropin Releasing Hormone (CRH) based on the immobilization of half-antibody fragments on gold nanoparticles (AuNp). Then, the optimal conditions for the obtainment of AuNp through electroplating on a bare gold electrode were studied. The results showed that the obtainment of AuNp at a fixed potential of -0.2â¯V for 330â¯s, at 80⯰C and 2·10-3 mol·L-1 of HAuCl4 generates an adequate nanostructured surface and is a highly reproducible method. Also, the optimal conditions for immobilizing the half-antibody on AuNp were studied. The interaction of the CRH with the recognition layer of the immobilized half-antibody on the nanostructured surface was carried out by incubation at 4⯰C for 2â¯h. A dissolution of [Fe(CN)6]4-/[Fe(CN)6]3- as a redox probe was used to study the electrochemical responses of the nanostructured surface and the immobilization processes of the half-antibody and detection of CRH, using cyclic voltammetry and electrochemical impedance spectroscopy. An immunosensor was obtained for the specific detection of CRH, within a range of 10.0-80.0⯵gâ¯mL-1, with a limit of detection of 2.7⯵gâ¯mL-1 and a limit of quantification of 9.2⯵gâ¯mL-1. Additionally, the association constant between the CRH and the immobilized half-antibody was calculated at 1.96·105 M-1.
Assuntos
Técnicas Biossensoriais , Hormônio Liberador da Corticotropina/isolamento & purificação , Espectroscopia Dielétrica , Fragmentos de Imunoglobulinas/química , Anticorpos/química , Anticorpos/imunologia , Anticorpos Imobilizados/química , Hormônio Liberador da Corticotropina/química , Hormônio Liberador da Corticotropina/imunologia , Técnicas Eletroquímicas , Humanos , Imunoensaio , Fragmentos de Imunoglobulinas/imunologia , Limite de Detecção , Nanopartículas Metálicas/química , NanoestruturasRESUMO
A growing body of evidence suggests that corticotrophin releasing hormone (CRH) may exert direct modulatory effects on immune cells. In the present study we assessed the effects of its precursor molecule, proCRH, on interleukin-6 (IL-6) release by human peripheral blood mononuclear cells (MNC). Human MNC were incubated with the corresponding stimuli for 24 hr. The supernatants were collected and IL-6 measured by ELISA. Conditioned medium from CHO-K1 cells stably transfected with the recombinant plasmid pEE14/rat pre-proCRH cDNA was used as the source of proCRH. Western blot analysis of this medium, using an antibody specific for the intact precursor, showed that no proCRH degradation products were present. The proCRH had an inhibitory effect on basal and LPS-stimulated release of IL-6. These results suggest that the full length CRH precursor may possess immunomodulatory properties.