Preparation of anti-largemouth bass ranavirus egg yolk antibody and establishment of indirect ELISA method

Largemouth bass ranavirus (LMBV) is a major pathogenic agent in largemouth bass culture in China, which mainly causes viral canker disease and restricts the healthy development of largemouth bass culture. In order to explore the potential role of egg yolk antibody in the prevention and control of LMBV, we immunized the LMBV inactivated vaccine to laying hens and prepared the egg yolk immunoglobulin against LMBV (Anti-LMBV IgY). Besides, we established an indirect enzyme linked immuno sorbent assay (ELISA) method for detecting the titer of anti-LMBV IgY by screening trapping concentration, encapsulation condition and sealing condition. Then we evaluated the titer of anti-LMBV IgY at different time points post LMBV inactivated vaccine immunization by using the established method. The results show that 1‰ β-propanolactone could inactivate LMBV at 4 ℃ for 72 h completely. For indirect ELISA, being coated with 10⁵ TCID₅₀ inactivated virus, incubated at 37 ℃ for 2 h, and blocked with 5% bovine serum albumin at 37 ℃ for 2 h could reduce the background values of negative control effectively. In addition, no cross reaction had been detected between inactivate LMBV with other egg yolk extract or anti-LMBV IgY with cells, indicating that the indirect ELISA method had high specificity. Finally, we detected specific anti-LMBV IgY titer at 48 d post LMBV inactivated vaccine immunization; the titer increased to a peak of 1:12800 after 58 d post immunization, and last up to 128 d. To sum up, the indirect ELISA method in this study can real-time monitor the titer level of anti-LMBV IgY, determine the efficient immunization program and the duration of high-immunity egg collection, facilitate the development and application of LMBV egg yolk antibody products, and provide a potential solution for the prevention and treatment of LMBV.