Production And Identification Of The Monoclonal Hybridoma Against Goat IFN-Gamma

Gamma Interferon(IFN-γ) is a kind of important cytokine which could reflect the level of cellular immunity, and it was always measured by ELISA kits which needed the most critical reagent monoclonal antibody against IFN-γ. There was no report about monoclonal antibody against goat IFN-γ so far, and it could not be measured by using the ELISA kits for other species because of the high species specificity. For these reasons, BALB/c mice were immunized with purified prokaryotic recombinant goat IFN-γ(r IFN-γ), and then the serum was collected and used to establish and optimize an ELISA method for detecting antibodies against goat IFN-γ; and the splenocytes of immunized mice were isolated and fused with SP2/0 myeloma cells to produce hybridoma cells which used for filtering ones secreting monoclonal antibodies against goat IFN-γ. The results were as follows:1. The ELISA method was optimized when the antigen was coated at 4 for 12 h with a ℃concentration of 10 ug/ml, immune serum was incubated for 60 min, and HRP conjugated goat anti-mouse antibody was diluted following the proportion of 1:5000. After immunized three times, the titer of serum could raise up to 105;2. After treating SP2/0 myeloma cells with 8-AG twice, the fusion rate increased effectively, which reached 60%;3. A strain of hybridoma cell named 5D5B8 was obtained, and the secreted monoclonal antibodies could recognize r IFN-γ, rather than the fusion protein tags of p ET32 a vector. What’s more, the serum of sick goats and the culture supernatant of lymphocytes stimulated with Con A could react with the monoclonal antibodies;4. The selected strain of hybridoma cell was able to secrete IFN-γ after continuous passage and repeated cryopreservation, and the OD450 value of the culture supernatant was stable at 1.0;5. The titers of the selected strain of hybridoma cell culture supernatant, mice ascites, and purified monoclonal antibodies were 29, 107, and 106 respectively.The results suggested that an ELISA method for detecting antibodies against goat IFN-gamma and a protocol for production, identification of the monoclonal hybridoma were successfully established in the present study. A strain of hybridoma stably secrete monoclonal antibodies against goat IFN-gamma which could be used for detection of goat IFN-gamma at the protein level was obtained.