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目的筛选能增强特异性抗原早期分泌抗原靶6蛋白(Early secretory antigenic target 6,ESAT6)-培养滤出液蛋白-10(Culture filtrate protein 10,CFP-10)融合蛋白(E1C0)诱导小鼠细胞免疫应答的佐剂,建立基于细胞免疫应答的小鼠模型,以评价基于体外干扰素γ释放分析(IFNγrelease assay,IGRA)结核诊断方法中特异性刺激抗原E1C0的活性。方法建立小鼠IFNγ双抗体夹心SABC-ELISA检测系统,并验证系统的线性、灵敏度、重复性和特异性。将BALB/c小鼠随机分为7组:E1C0+单磷酸类脂A(Monophosphoryl lipid A,MPL)+双十八烷基二甲基溴化铵(Dimethyl dioctadecylammonium bromide,DDA)组、E1C0+DDA组、E1C0+MPL组、E1C0+弗氏不完全佐剂(IFA)组、E1C0组、生理盐水组和MPL+DDA联合组,每组6只,经小鼠后肢内侧皮下免疫3次,间隔2周,免疫剂量为:E1C0 100μg/只,MPL 25μg/只,DDA 250μg/只,IFA 100μl/只。末次免疫4周后处死小鼠,无菌取脾,分离脾淋巴细胞,加入E1C0进行培养,MTT法检测特异性淋巴细胞增殖反应,ELISA法检测培养上清中IFNγ水平。采用筛选出的最佳佐剂与抗原组合免疫3批BALB/c小鼠,进行IFNγ诱生测定。结果检测系统的线性范围为:40～2 560 pg/ml(R>0.98);灵敏度为40 pg/ml;变异系数(CV)<15%,检测大鼠、豚鼠和兔血清IFNγ均为阴性;E1C0+MPL+DDA组、E1C0+IFA组和E1C0+DDA组小鼠特异性淋巴细胞增殖刺激指数均明显高于生理盐水组(P<0.01);E1C0+MPL+DDA组小鼠的脾淋巴细胞经E1C0体外刺激后,产生的IFNγ水平明显高于其他组(P<0.001);重复试验结果显示,E1C0+MPL+DDA组3批小鼠免疫后,脾淋巴细胞诱生的IFNγ水平差异无统计学意义(P>0.05)。结论 E1C0与MPL和DDA联合免疫所诱导的小鼠Th1型细胞免疫应答最强,成功建立了用于评价刺激抗原E1C0活性的小鼠模型。
Objective To screen the cellular immune responses induced by early secretory antigenic target 6 (ESAT6) - Culture filtrate protein 10 (CFP-10) fusion protein (E1C0) Responsive adjuvant to establish a mouse model based on the cellular immune response to evaluate the activity of the specific stimulation of the antigen E1C0 based on the diagnostic method of the IFN gammarelease assay (IGRA) tuberculosis. Methods Mouse IFNγ double antibody sandwich ELISA system was established and its linearity, sensitivity, reproducibility and specificity were verified. BALB / c mice were randomly divided into 7 groups: E1C0 + monophosphoryl lipid A (MPL) + dioctadecyl dimethyl ammonium bromide (DDA) group, E1C0 + DDA group , E1C0 + MPL group, E1C0 + incomplete Freund’s adjuvant (IFA) group, E1C0 group, saline group and MPL + DDA group. Each group had 6 mice in each group. Immunization dose: E1C0 100μg / only, MPL 25μg / only, DDA 250μg / only, IFA 100μl / only. Four weeks after the last immunization, the mice were sacrificed and the spleen was removed aseptically. Splenic lymphocytes were isolated and cultured with E1C0. Specific lymphocyte proliferation was detected by MTT assay and IFNγ level in culture supernatants by ELISA. Three batches of BALB / c mice were immunized with the selected optimal adjuvant and antigen for IFNγ induction. Results The linear range of the detection system was 40-2 560 pg / ml (R> 0.98), the sensitivity was 40 pg / ml and the coefficient of variation (CV) was <15% The specific lymphocyte proliferation stimulating index of E1C0 + MPL + DDA group, E1C0 + IFA group and E1C0 + DDA group were significantly higher than those of saline group (P <0.01). The spleen lymphocytes of mice in E1C0 + MPL + DDA group IFNγ levels in E1C0 + MPL + DDA group were significantly higher than those in the other groups (P <0.001). The results of repeated experiments showed that there was no statistical difference in IFNγ levels induced by splenic lymphocytes among three batches of mice immunized with E1C0 + MPL + DDA Significance (P> 0.05). Conclusion The combination of E1C0 with MPL and DDA co-immunization induced the strongest Th1-type immune response in mice, and successfully established a mouse model for evaluating the activity of stimulating antigen E1C0.