The Establishment And Application Of ELISPOT Method For Detection NDV Induced Specific IFN-γ, IL-4

In the course of the virus immune, not only need humoral immunity , but also need to cell-mediated immunity. Such as newcastle disease immunization, cell-mediated immunity and humoral immune coordination to play a role . Ghumman (1976) and others found ,after immune the activated and inactivated vaccine of Newcastle disease 2-3 days, the lymphocytes can be detected in DNA synthesis. Agrawal (1991), Used eukocyte migration inhibitory test to evaluate Newcastle disease vaccine inoculation, found that there is cellular immune response after inoculation. But, the body cellular immune and humoral immunity changes in how it does? Why immunization of Newcastle disease usually is a failure? These problems have without a clear answer.ELISPOT is a very advanced immunological detection technology. Combined with the merits of the cell culture technology and ELISA technology .It can analysis the cytokines (such as IFN-γ, TNF-α) . This method is sensitive, specific and easy to repeat. ELISPOT technology was applied to the cellular immune research of Newcastle disease in this paper, The detection of the NDV induced specific IFN-γ,Il-4 ELISPOT method was established by selecting the most appropriate spleen lymphocyte concentration, incubation time, and the stimulate antigen volume .The results showed that the specification of the ELISPOT experiment was closely related to incubation time and stimulate antigens volume. In a certain range, the longer the incubation time, the more stimulating antigen, the lower the specificity of the experiment .When the amount of antigen 5ug/well, the incubation time for 7 h, the specificity is strongest. Spots increase with the cell concentration increased, when the mouse spleen lymphocytes concentration of 106/well, the most conducive to spots counting and the lowest experimental error. With the ELISPOT method, has been established, to detect IFN-γ, Il-4 of NDV immune mice, found that the Production of IFN-γ, Il-4 and serum antibody, there is a certain time difference between serum antibody positive in the early stage , IFN-γsecretion dropped to the lowest level, Il-4 rose to the highest level.