| Interleukin-18 (IL-18) is an important cytokine, which can act on T helper 1-type T cells and strongly induce them to produce IFN-γin combination with IL-12. Pleiotropic effects of IL-18 have recently been reported. It plays an important role in the development of cell-mediated immune responses, defending the infection of microbial and resisting the tumor. Therefore, IL-18 may be used as an immune regulatory, anti-microbes and/or anti-tumor agent in clinic. So far, the products of human, mouse, monkey, porcine, bovine, goat IL-18 and the monoclonal or polyclonal antibody reagents against them have been developed, as well as the relative double-antibody ELISA kits. However, the function and relative studies of equine IL-18 (EIL-18) is still poorly understood. Even no any products of EIL-18, anti-EIL-18 monoclonal or polyclonal antibody or double-antibody ELISA kits come out yet.In this study, the DNA of EIL-l8 was amplified by RT-PCR from the total RNA extracted from ConA stimulated equine PBMCs, and subsequently cloned into the vector pMD18-T, sequenced and named pMD-EIL-18. Then the genes encoding precursor and mature EIL-l8 were amplified from pMD-EIL-18 by PCR and subcloned into pET-28a(+) and pET-26b(+) respectively and recombinant plasmids (pET-pEIL-18, pET-EIL-18 and pET-mEIL-18) were obtained after sequencing confirmation. High levels of rpEIL-18, rEIL-18 and rmEIL-18 proteins were successfully expressed in E. coli strain BL21 (DE3) transformed with pET-pEIL-18, pET-EIL-18 and pET-mEIL-18. The SDS-PAGE and Western-blot analysis indicated that the fusion proteins were 25kD, 20kD and 18kD in molecular weight and had immunological reaction with rabbit against human IL-18 antibody. High-purity of recombinant proteins were recovered after purification with a Ni2+NTA column or CNBr-activated Sepharose 4B conjugated anti-EIL-18 McAb column. To identify whether or not rpEIL-18, rEIL-18 and rmEIL-18 proteins have biological activity, the method of real-time quantitative RT-PCR was established to detect the mRNA products of equine IFN-γfrom equine PBMCs stimulated with these recombinant proteins in vitro. The results showed that rEIL-18 and rmEIL-18 had a synergistic effect to induce the expression of IFN-γgene in equine PBMCs with the presence of recombinant human IL-12. All these results also indicated that the rEIL-18 and rmEIL-18 expressed in this study had biological activity commensurate with the native molecule. However, the rpEIL-18 had no such activity.Rabbits were immunized subcutaneously with purified rEIL-18 proteins and high titers of PcAbs against EIL-18 were obtained subsequently. Meanwhile, BALB/c mice were immunized intraperitoneally with the same antigens for EIL-18 McAb preparation. After murine myeloma cells were fused with the splenocytes from the immunized mice, an indirect ELISA using the rAcEIL-18 protein derived from baculovirus expression system as antigen was carried to screen antibody-producing hybridomas. Consequently, nine McAbs against EIL-18 were got and Western-blot and IFA analysis showed all of them had positive reaction with rEIL-18 and rAcEIL-18 respectively. These 9 McAbs were used to react with mEIL-18 1-78aa and mEIL-18 79-157aa respectively in specific ELISA tests. As a result, five of them showed positive reaction to mEIL-18 1-78aa, the others had positive reaction to mEIL-18 79-157aa. McAb subtype identification kit was used to make sure which subtype these 9 McAb belonged to. The results showed three of them were IgM isotype and the rest ones belonged to IgG1 isotype, and the light chains of all McAbs wereκchain. Additional study displayed that the S1D7 McAb could effectively neutralize the bioactivity of rEIL-18 and rmEIL-18.The McAbs and PcAb against EIL-18 were purified with extraction and immunoaffinity chromatography methods. And S6A3, B1G1 McAbs and PcAb were chosen to be biotinylated. After all of McAbs and PcAb were screened for the matched-pairs of double-antibody ELISA, a sandwich ELISA was developed to detect the EIL-18 by using purified S6A3 McAb as a capture antibody and the biotinylated B1G1 McAb as a detection antibody. And the limit of detection for the concentration of EIL-18 was 15pg/mL by this method.Sera samples from health and EIAV-infected horse, pig, bovine and sheep were employed to measure the levels of IL-18 using the method established in this study. The results showed no cross-reaction occurred to pig, bovine and sheep sera samples in this testing method. What's more, the IL-18 level from healthy horse serum was 40-80 pg/mL, while that was 70-150 pg/mL for EIAV-infected horse. Because of cross-reaction, porcine and bovine IL-18 quantitative sandwich ELISA was preliminarily established according to the EIL-18 quantitative sandwich ELISA. A sandwich ELISA for the detection of porcine IL-18 was developed using purified S1C6 McAb as a capture antibody and the biotinylated PcAb against EIL-18 as a detection antibody. The limit of detection for porcine IL-18 concentration was 30pg/mL in this sandwich ELISA. Additionally, the sandwich ELISA for the detection of bovine IL-18 was also preliminary established by using purified S5F1 McAb as a capture antibody and the biotinylated PcAb against EIL-18 as a detection antibody.
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