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目的评价不同储存条件对结核亚单位疫苗参考品(简称参考品)免疫原性的影响。方法按以下抗原与佐剂比例配制1人份疫苗参考品:[10μg Ag85b蛋白+10μg EC蛋白+50μg Ploy IC+0.2 mg Al(OH)3]/0.2 ml。将疫苗参考品于-70及4℃分别保存1、6、9、12个月,并于每个时间点免疫BALB/c小鼠,实验分4组:PBS组、冷藏组(4℃保存参考品)、冻存组(-70℃保存参考品)及现配组,均经小鼠后肢内侧肌肉注射,0.2 ml/只,共免疫3次,每次间隔10 d,末次注射10 d后,收集小鼠脾淋巴细胞,并经眼球采血,分离血清。ELISPOT法检测抗原特异性的IL-2、IFNγ水平,CCK-8法检测细胞增殖水平,间接ELISA法检测血清Ig G抗体效价。结果经Ag85b和EC刺激,现配组各月间的斑点形成细胞数(spot forming cells,SFCs)及脾细胞增殖指数(stimulating indox,SI)差异无统计学意义(P>0.05),且均显著高于PBS组(P<0.05),表明免疫学检验模型成立且稳定,各试验组与现配组结果具有可比性。经4℃冷藏12个月,参考品的Ag85b及EC特异性IL-2、IFNγ相对水平及脾淋巴细胞相对增殖水平均显著低于冷藏1个月的参考品(P<0.01),经-70℃冻存12个月,参考品的Ag85b、EC特异性IL-2、IFNγ相对水平及脾淋巴细胞相对增殖水平与冻存1个月的参考品差异无统计学意义(P>0.05)。与现配组比较,冷藏组及冻存组Ag85b和EC特异性抗体水平相对平稳,各月间差异均无统计学意义(P>0.05),且两组间差异无统计学意义(P>0.05)。结论 -70℃冻存12个月内的参考品的细胞及体液免疫水平均未明显降低,可作为该参考品的长期保存条件。不同保存条件对疫苗免疫保护力的影响需进一步考察。
Objective To evaluate the effect of different storage conditions on the immunogenicity of TB subunit vaccine reference (referred to as reference). Methods One serving of vaccine reference was formulated according to the following ratio of antigen to adjuvant: [10 μg Ag85b protein + 10 μg EC protein + 50 μg Ploy IC + 0.2 mg Al (OH) 3] /0.2 ml. The vaccine reference samples were stored at -70 and 4 ℃ for 1, 6, 9, and 12 months respectively. BALB / c mice were immunized at each time point. The experiment was divided into 4 groups: PBS group, (Control reference substance at -70 ℃) and the mice in the control group were injected intramuscularly with 0.2 ml intramuscular injection of hindlimb every three days for 10 days. After 10 days of the last injection, Mouse splenic lymphocytes were collected and blood was collected by eyeball to separate the serum. Antigen-specific IL-2 and IFNγ levels were detected by ELISPOT assay. Cell proliferation was measured by CCK-8 assay. Serum Ig G antibody titers were measured by indirect ELISA. Results There were no significant differences in the number of spot forming cells (SFCs) and the proliferating index of spleen cells between the three groups (P> 0.05) after stimulation with Ag85b and EC. Higher than that of PBS group (P <0.05), indicating that the immunological test model was established and stable, the results of each test group and the match with the group is comparable. After being stored at 4 ℃ for 12 months, the relative levels of Ag85b and EC-specific IL-2 and IFNγ and the relative proliferation of splenic lymphocytes in reference samples were significantly lower than those of 1-month refrigerated samples (P <0.01) (P> 0.05). There was no significant difference in the relative levels of EC-specific IL-2, IFNγ and the relative proliferation of splenic lymphocytes among the reference samples for one month after cryopreservation. Compared with the control group, the levels of Ag85b and EC specific antibodies in the frozen group and the frozen group were relatively stable, with no significant difference in each month (P> 0.05), and there was no significant difference between the two groups (P> 0.05 ). Conclusion The cellular and humoral immunity levels of the reference samples frozen at -70 ℃ for 12 months have not significantly decreased, which can be used as long-term storage conditions for the reference samples. The influence of different storage conditions on the vaccine protective immunity needs further investigation.