RESUMEN
Monoclonal antibodies (mAbs) against black rockfish Sebastes schlegeli serum immunoglobulin M (IgM) were developed, which showed a specific reaction with the heavy chain of S. schlegeli IgM in Western blotting and with surface IgM positive (sIgM+ ) lymphocytes in indirect immunofluorescence. mAb 2A6 was employed to investigate the antibody and sIgM+ lymphocyte responses of S. schlegeli injected with inactivated Edwardsiella tarda, by ELISA and flow cytometry. Compared with controls, the level of specific antibodies and the percentage of sIgM+ lymphocytes both increased in the immunized fish and simultaneously reached their peaks at day 35 after immunization.
Asunto(s)
Anticuerpos Monoclonales , Inmunoglobulina M/inmunología , Perciformes/inmunología , Animales , Especificidad de Anticuerpos , Biomarcadores , Western Blotting , Ensayo de Inmunoadsorción Enzimática/veterinariaRESUMEN
Lymphocystis disease virus (LCDV) enters and infects the gill cells of flounder (Paralichthys olivaceus) via a 27.8 kDa membrane protein receptor. In the present study, immunohistochemistry was performed to locate the tissue distribution of this molecule in healthy flounder and showed that it was widely distributed in the tissues tested. Indirect enzyme-linked immunosorbent assay (ELISA) showed that the expression of the receptor in healthy flounder was highest in the gills and stomach, then in the skin, intestine and liver, followed by the spleen, head kidney, heart, ovary and brain and finally the kidney. On LCDV infection, ELISA indicated that the expression of the receptor, as determined by ELISA, was significantly upregulated in all tissues of LCDV-infected flounder compared with controls, but this expression decreased over the 4 weeks post infection. In contrast, real-time quantitative polymerase chain reaction demonstrated that the copy number of the LCDV gene in the tissues increased with time post infection, and that viral loads were higher in the tissues with higher expressions of the receptor. These results point to a correlation between high expression of the 27.8 kDa receptor and efficient LCDV propagation. The wide tissue distribution of the receptor might be one reason why LCDV can infect various tissues leading to systemic infection.
Asunto(s)
Infecciones por Virus ADN/veterinaria , Enfermedades de los Peces/metabolismo , Proteínas de la Membrana/biosíntesis , Animales , Infecciones por Virus ADN/metabolismo , Ensayo de Inmunoadsorción Enzimática , Lenguado , Iridoviridae , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
AIMS: To develop a gold immunochromatographic test strip for on-site rapid detection of lymphocystis disease virus (LCDV). METHODS AND RESULTS: Monodispersional colloidal gold and gold-labelled anti-LCDV monoclonal antibody (McAb) 2D11 were prepared and characterized by UV-visible spectroscopy and transmission electron microscopy. Gold-labelled probe was used as the detection antibody, and goat anti-mouse IgG at the control line and anti-LCDV McAb 1A8 at the test line of the test strip served as the capture antibody. The positive results could be easily judged by the presence of a red test line with naked eye within 10 min. The test strip, in good agreement with enzyme-linked immunosorbent assay and dot-blotting in sensitivity and LCDV detection, gave a detection limit of 1 µg ml(-1) of LCDV and was stable for 6 months at room temperature and 12 months at 4°C. CONCLUSIONS: The test strip was specific, simple and convenient for rapid detection of LCDV presenting good stability and reproducibility. SIGNIFICANCE AND IMPACT OF THE STUDY: This ready-to-use test strip allows on-site rapid detection of LCDV in fish without the requirement of specialized equipments and professional personnel, which could augment the practical application for diagnosis of LCDV even in disadvantage areas.