| Interleukin18(Interleukin-18, IL-18) is mainly secreted by monocytes-macrophagesystem, belonging to the IL-1family in structure, similar to the function of IL-12and hassynergistic effects with IL-12. IL-18is a very important cytokine which regulates innateimmunity and acquired immunity. Dynamic changes of IL-18in the body have an importantsignificance to understand the occurrence, development, prognosis of infectious diseases andthe responsive dynamics of the body protective immune, our research group had also reportedthere is a correlation between the chicken IL-18transcription level and avian tumor virusinfection. In view of this, it is necessary to establish the detection method of IL-18proteinlevel in the body. So far, there is no home-made double-antibody ELISA kits of maturechicken interleukin-18(mChIL-18) though the relative products of lots of animals have beendeveloped. The purpose of this study is to establish ChIL-18double sandwich ELISA byusing anti-ChIL-18monoclonal or polyclonal antibody, which can facilitate the studies onimmune mechanism and immune function of chicken.The recombinant ChIL-18protein was expressed in E.coli and purified. One hybridomacell lines(1B10) stably secreting monoclonal antibody (mAb) specific to mChIL-18wereestablished and characterized by immunizing BALB/c mice with purified recombinantmChIL-18as an antigen. The mAb that is subtype IgM had an ascites titers of1:3.2×104.Western-blot analysis confirmed that the mAb specifically recognized mChIL-18only. IFAassay revealed that the mAb could react with293T cells transfected by pcDNA3.1-mChIL18.ELISA showed that1B10and1G9preserved in lab aim at the same epitope.Double sandwich ELISA for mature chicken interleukin18(mChIL-18) was developedusing mAb2E6as a capture antibody and HRP-labeled mAb1G9as a detection antibody andthen the expression levels of mChIL-18in serum samples of SPF chickens infected withreticuloendotheliosis virus(REV)were investigated by using this method. The results were asfollows: The optimal concentration of capture antibody was8μg/mL, the workingconcentration of detection antibody was1:800, the best dilution of the samples was1:400, andthe detection limit of the sandwich ELISA for mChIL-18was31.5pg/mL, there was no crossreaction with other antigen protein including chicken cytokines; mChIL-18expression levelswere higher in the serum samples of SPF chickens from treatment group than those in thecontrol group at day7,14,21,28,35,42and49, but only the samples of14d were significantly changed(p<0.05).In addition, double sandwich ELISA for mature chicken interleukin18(mChIL-18) wasdeveloped using anti-ChIL-18polyclonal antibody as a capture antibody and HRP-labeledmAb1G9as a detection antibody and then the expression levels of mChIL-18in serumsamples of SPF chickens infected with REV were investigated by using this method. Theresults were as follows: The optimal concentration of capture antibody was20μg/mL, theworking concentration of detection antibody was1:800, the best dilution of the samples was1:400, and there was no cross reaction with other antigen protein including chicken cytokines;mChIL-18expression levels were higher in the serum samples of SPF chickens fromtreatment group than those in the control group at day7,14,21,28,35,42and49, but only thesamples of42d were significantly changed(p<0.05).The results above show that two kinds of double sandwich ELISA of mChIL-18have beensuccessfully developed, which can be used to help understand the role of this cytokine invarious chicken diseases and develop specific cell-mediated immunityassay. |
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