Development Of A Colloidal Gold Immuochromagoraphic Test Strip For Detecting Lymphocystis Disease Virusin Fish

Lymphocystis disease is a chronic, benign viral disease characterized by the appearance of pearl-like nodules consisting of enormously hypertrophied dermal cells in skin, gills, fins and even the internal organs. Lymphocystis disease virus (LCDV), the causative agent of lymphocystis disease, has a worldwide geographical distribution and has infected more than 140 species of marine and freshwater fish belonging to 42 families. Various laboratory methods are available for the diagnosis of LCDV, such as histopathological observation, cell culture inoculation, serological techniques and molecular techniques. Generally, those diagnostic approaches have respective merits in terms of accuracy, sensitivity, safety and cost. However, their performances are restricted to laboratory mainly due to the requirement of skilled technicians and sophisticated equipments. Gold immnochromatographic assy (GICA) was simple and convenient, while, monoclonal antibody had good specificity and sensitivity. Combined with both of them, this experiment developed a gold immnochromatographic test strip for detecting lymphocystis disease virus in fish to overcome the above limitations.Three strains of hybridomas (1B2, 1D7, 2D11) secreting monoclonal antibodies (McAbs) against the surface of LCDV were cultured and immuned to BALB/c mice. And then ascitic fluids were obtained and purified by caprylic acid ammonium sulphate method. The purified McAbs were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It indicated that the McAb protein was pure and contained little other protein. Analyzed by western blotting, three kinds of McAbs all belonged to IgG class. Three kinds of McAbs only reacted specifically with the LCDV showed by IFAT and dot blotting. The titers of McAb 1B2, McAb 1D7, McAb 2D11 were determined to be 1:25 600, 1:12 800, 1:25 600 by indirect ELISA. The result of additive ELISA test revealed that three kinds of McAbs recognized distinct antigen epitopes. Finally, McAb 1B2 and McAb 2D11 of high titer and good bioactivity were selected as the capture antibody and detector antibody labeled with colloidal gold for the GICA based on the principle of double antibody sandwich.Monodispersed colloidal gold with a mean diameter of approximately 20 nm was synthesized by electric heating of microwave and sodium citrate reduction. It was proved that colloidal gold was homogeneous, without fragments and agglutination characterized by visual observation, ultraviolet-visible absorption spectroscopy and transmission electron microscopy. The optimized pH value and protein amount of conjugation between colloidal gold and McAb 2D11 was confirmed as 8.2 and 18μg/ml, respectively. According to the optimized condition of conjugation, colloidal gold labeled McAb 2D11 with good immunologic reactivity was successfully prepared. Then the colloidal gold-antibody conjugation was dispensed on a porous glass fiber to form the gold conjugated released pad. Purified McAb 1B2 and goat anti-mouse IgG were coated on the nitrocellulose membrane as the control line and the test line, respectively. The test strip was assembled by the different accessory in regular sequence to detect LCDV in the samples. The various factors and conditions of the gold immunochromatographic test strip were explored, and then appropriate NC membrane and fiberglass membrane were chosen. Optimal dilution of colloidal gold-antibody conjugation diluted at the ratio of 1:2 was also ascertained. Optimum coating concentrations of McAb 1B2 and goat anti-mouse IgG were determined as 1 mg/ml and 0.5 mg/ml, respectively.In the process of diagnosis, the LCDV antigen binds the colloidal gold-antibody conjugation on the test strip and moves along the membrane. Then it binds the capture antibody at the test line on nitrocellulose membrane displaying a visible red line. If LCDV antigen exists in the sample both the control line and the test line will show a red line. If LCDV antigen does not exist in the sample a red line will only emerge at the control line. The results could be easily judged by the presence or absence of a red line on the test line with naked eye within no more than 10 min without incubation. Analysis of LCDV supernatant with a titer of 1.65×2⁹ TCID₅₀/ml, the test strip detection limit was 1.69 TCID₅₀/ml. Compared with reference methods, the sensitivity of the test strip was in a good agreement with those of ELISA and dot-blotting. This gold immunochromatographic test strip is proved to be simple, accurate and specific for rapid detection of LCDV with no requirement of specialized equipments, reagent or professional skills. Therefore, the kit is a practical tool for rapid detection of LCDV.