微量溶血分光光度法测定气管炎疫苗的免疫活性

Determination of immune activity of tracheitis vaccine by using micro-quantitative hemolysis spectrophotometry

目的采用微量溶血分光光度法测定气管炎疫苗的免疫活性。方法将BALB/c小鼠随机分为5组:空白对照组、阴性对照组及试验1、2、3组,阴性对照组免疫生理盐水,试验1、2、3组分别免疫3批气管炎疫苗,免疫途径为小鼠颈背部皮下,免疫剂量为0.5 ml/只,免疫时间为第1、7、14天,除空白对照组外,所有小鼠第22天经腹腔注射20%的绵羊红细胞(sheep red blood cell,SRBC)悬液,0.2 ml/只。4 d后处死小鼠,取脾脏,制备脾细胞悬液,与SRBC结合,于37℃,在补体的参与下,使抗体形成细胞周围的SRBC溶解,通过酶标仪测定抗体形成细胞介导的溶血反应中,SRBC溶解后释放血红蛋白的量。另设7组小鼠:空白对照组、阴性对照组及试验4、5、6、7、8组,阴性对照组免疫生理盐水,试验4、5、6、7、8组分别免疫不同浓度(6×108、3×108、1.5×108、0.75×108、0.375×108/ml)的气管炎疫苗,按上述方法检测不同浓度气管炎疫苗的免疫活性。结果在抗体形成细胞介导的溶血反应中,3个试验组SRBC溶解后释放的血红蛋白的量均明显高于阴性对照组(P<0.05)。随着气管炎疫苗浓度的降低,SRBC溶解后释放的血红蛋白的量也呈下降趋势,气管炎疫苗浓度在1.5×108/ml以上时,其免疫活性明显高于阴性对照组(P<0.05);低于0.75×108/ml时,与阴性对照组比较,差异无统计学意义(P>0.05)。结论微量溶血分光光度法可用于检测气管炎疫苗的免疫活性。气管炎疫苗浓度在1.5×108/ml以上时,即可增强小鼠免疫功能。

Objective To determine the immune activity of tracheitis vaccine by using micro-quantitative hemolysis spectrophotometry.Methods BALB / c mice were divided into one blank control,one negative control and three test groups.The mice in blank control group were unimmunized,while those in negative control group were immunized s.c.with physiological saline,and those in test groups 1,2 and 3 with three batches of tracheitis vaccine,0.5 ml for each,on days 1,7 and 14.The mice,except those in blank control group,were injected i.p.with 20% sheep red blood cell（SRBC）suspension on day 22 0.2 ml for each,and killed 4 d later,of which the spleens were prepared into splenocyte suspension.The splenotye suspension was incubated with SRBCs at 37 ℃ to dissolve the SRBCs around the antibodyforming cells with the participation of complement.The hemoglobin released after dissolution of SRBCs in the hemolytic reaction immediate by antibody-forming cells was measured by microplate reader.Another seventy mice were divided into seven groups,10 for each.The mice in blank control group were unimmunized,while those in negative control group were immunized with physiological saline,and those in test groups 4,5,6,7 and 8 tracheitis vaccine at concentrations of 6 × 108,3 × 108,1.5 × 108,0.75 × 108 and 0.375 × 108 bacteria / ml respectively.The immune activities of vaccine at various concentrations were determined by above-mentioned method.Results The hemoglobin levels released after dissolution of SRBCs in the hemolytic reaction immediate by antibody-forming cells were significantly higher in three test groups than in negative control group（P 〈 0.05）.However,the level showed a decreasing tendency with the decreased concentration of tracheitis vaccine.The immune activities of tracheitis vaccine at concentrations of more than 1.5 ×108bacteria / ml were significantly higher than（P 〈 0.05）,while those at concentrations of less than 0.75 × 108 bacteria / ml showed no significant difference with,that in negative control group（P 〉 0.05）.Conclusion Micro-quantitative hemolysis spectrophotometry may be used for the determination of immune activity of tracheitis vaccine.The vaccine at concentrations of more than 1.5 × 108 bacteria / ml enhanced the immune function of mice.