Development Of Quantitative Sandwich ELISA Detection And Research On Immunity Enhancement Of Goat IL-18

Interleukin-18 is a member of the IL-1 cytokine superfamily and distributed widely and possesses variety of bioactive functions. The immunity regulation and tumor-resistant system to the organisms were payed close attention by the researchers. The human medical researches show that IL-18 plays an important role in the resistance to a variety of diseases, especially in tumor-resistant immunity. The Mabs have more superiority than the polyclonal antibodies and were applied to the detection, diagnosis and treatment of variety of diseases. The Mabs can provide powerful tools for the analysis of variety of micrograms'epidemiology and greatly enhance the diagnosis and treatment of clinical diseases.In this study, the Mabs were prepared and studied. The gene of goat mature IL-18 proteins was amplified and cloned into prokaryotic expression vector pET-28a(+). By the identification and sequencing, the recombinant plasmid pET-28a-gIL-18 was constructed. The recombinant plasmid pET-28a-gIL-18 was transformed into Rosetta (DE3) competent cells and induced and obtain the expression of the recombinant proteins. Using the feature of the His tags in the vector, the recombinant gIL-18 proteins were purified by Ni-NTA Resin affinity chromatography. The purified proteins were used for the immunity of the mice, screening and identification of the Mabs.The mice were immunized using purified gIL-18 proteins. And, the indirect ELISA detection was constructed for detection of mice'antibodies and identification of Mabs. The titers of mice were up to 1:107 before the fusion at the satisfaction of the conditions of the fusion. The splenocytes were fused with SP2/0 myeloma cells under the effect of PEG. The fusion cells were detected by indirect ELISA. The positive hybridoma cells were sub-cloned twice by limited dilution methods to obtain the hybridoma cells secreting antibodies stably. Finally, the two Mabs (2E8 and 4C4) were obtained. The ascites fluids were harvested and purified by ammonium sulfate precipitation followed by Protein G affinity chromatography. The purified IgG was obtained and analyzed by SDS-PAGE.The identification of isotype of Mabs showed that the two Mabs were IgG1 isotype. The titers of 2E8 and 4C4 were 1:10~5 and 1:10~4, respectively. ELISA additity test showed that the binding of two Mabs, 2E8 and 4C4, was additive and both bound to the distinct epitopes. Western blot showed that two Mabs, 2E8 and 4C4, could react with recombinant gIL-18 proteins specifically and anti-His Mabs could also react with recombinant gIL-18 proteins specifically and could not react with hIFN-γproteins. Moreover, compared with the polyclonal antibody, the Mabs had high specificial and clear bands. The Mabs could be used to detect the expression of recombinant eukaryotic plasmid pcDNA3.1-gIL-18 transfected into the 293FT cells specifically by IFA. IFA displayed intense green fluorescence. Also, the expression of recombinant Bacmid-gIL-18 transfected into sf9 was detected specifically by IFA.The goat IL-18 quantitative sandwich ELISA detection was constructed using two Mabs. The Mab 2E8 was marked with HRP and the titer of HRP-2E8-IgG was identified as 1:6000.The other Mab 4C4 was used as the capture antibody. By the phalanx trial to optimize the concentration of coated antibody (4C4), dilution of HRP-2E8-IgG and condition of ELISA, the goat IL-18 quantitative sandwich ELISA detection was constructed. The standard curve of goat IL-18 was determined by the quantitative sandwich ELISA and the minimum detection was 16pg/mL. This ELISA to detect hIFN-γand hIL-18 showed that there were no cross reaction and the minimum detection were below 16pg/mL. Thus, the quantitative sandwich ELISA detection for goat IL-18 was specific.The quantitative sandwich ELISA was applied to detect the IL-18 levels of the goat PBMC at the stimulation of LPS and the sera and whey of the healthy cows and cows infected with mastitis. The results showed that the detection values of goat PBMC were 85~267 pg/mL. Compared with negative control cells, goat IL-18 levels were improved. For detection of sera, the detection values of healthy cows were 44~135 pg/mL.And, the detection values of cows infected with mastitis were 111~534 pg/mL and were improved compared with the forth. For detection of whey, the detection values of healthy cows were 83~167 pg/mL. And, the detection values of cows infected with mastitis were 171~658 pg/mL and the values of IL-18 were improved. The quantitative sandwich ELISA could detect IL-18 levels quantitatively at the inflammation in vitro and in vivo and set the basis for the diagnosis and treatment of variety of diseases.And, to study the immunity enhancement of IL-18, rBgIL-18 proteins were used as adjuvants to co-administrate goats with the vaccine (FMD bivalent inactive vaccine of O type and Asian 1 type). The goats were distributed as four groups:Ⅰgroup: rBgIL-18 + vaccine; Ⅱgroup: pre-rBgIL-18 + vaccine;Ⅲgroup: vaccine;Ⅳgroup: control. The results were analyzed at the level of humoral and cellular immunity to interpret the immunity enhancement of IL-18.The titers at variety of times post-immunization in sera were detected by FMD O type and Asian 1 type ELISA kit. The results showed that compared with pre-immunization,Ⅰ,ⅡandⅢgroup had higher titers of antibodies and maintained high titer levels for longer times andⅠgroup could arrive at higher titer levels faster. Compared with the control, the immunity levels ofⅠgroup were better and had higher titers and maintained high titer levels for longer times. The results ofⅡgroup were next to that ofⅠgroup. Moreover, the cellular immunity was detected by MTT to analyze the proliferation of lymphocytes. The results showed that in contrast with the control, PI values were greatly improved and the lymphocytes could proliferate at the stimulation of vaccine.And, the analysis of the blood cells showed that compared with pre-immunization, the lymphocytes of goats were significantly proliferated and the proportion of the lymphocytes were also improved. Thus, as immunity adjuvant, IL-18 plays an important role in enhancing immunity and immunity effects.