| Interleukin-17(IL-17)is a cytokine produced mainly by activated IL-17+ cells,which not only paly a certain inflammatory effect in allergic reactions,autoimmune diseases,tumor,but also play a key role in the inflammatory response mediator in resistance to bacteria,fungi,viruses,parasitic infections.At present,the role of IL-17 in the anti-infective immunity and inflammatory response is still unclear,mainly related to the lack of caprine IL-17 monoclonal antibodies.In this study,the BABL/c mice were immunized with purified caprine rIL-17.Then the mice serum was collected and the indirect ELISA method of caprine IL-17 antibody was established.The spleen of immunized mouse was fused with SP2/0 myeloma cell,and screened which can stable secretion specificity of IL-17 monoclonal antibody hybridoma cell.The results were as follows:1.The ELISA detected method was optimized when the concentration of IL-17 was 8 ?g /m L,the mice immune serum was incubated 60 min,the enzyme labeled second antibody was diluted by 1:5000 and the reaction time was 60 min incubating at 37?;2.Two strains of hybridoma cell secreting specific rIL-17 monoclonal antibody were obtained,named B6 and H8;3.The chromosome number of B6 and H8 cell lines were 104,84;4.The antibody subtype of B6 and H8 was IgG1,and the antibody was a kappa;5.The supernatant titers of B6,H8 were 1: 28,1: 29,the ascites titers of B6,H8 were 1: 105,1: 106,the purified antibody titers were 1: 105;6.The B6 cell strains were able to identify the r IL-17 and cell culture supernatant by IL-17+293T cells and not react with pET32 a tagged proteins.The H8 cell line can only identify the rIL-17 specifically.The western blot results show that B6 has a specific immune combination with r IL-17 and cell culture supernatant by IL-17+293T cells.In this study,the indirect ELISA method for the detection of caprine IL-17 antibody was established,and a hybridoma cell-B6 which could secrete specificity IL-17 monoclonal antibody was screened. |
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