化学发光ELISA检测可溶型CD100(sCD100)方法的建立及其应用

Establishment of a chemiluminescent immunoassay (CLIA) and its application

目的建立可溶型CD100(sCD100)的微孔板式化学发光检测试剂盒,并初步用于临床脑脊液标本的检测。方法利用空军军医大学免疫学教研室制备的2株抗CD100单克隆抗体(mAb),在夹心ELISA的基础上,优化实验条件,建立sCD100化学发光免疫检测方法;对该方法的灵敏度、精密度进行评价;并检测了18例脑脊液标本的sCD100水平。结果化学发光法检测sCD100在0.098 ng/mL^12.5 ng/mL范围内线性关系良好,检测敏感性能达到0.12 ng/mL。批内变异系数为3.8%~6.6%,批间变异率为6.2%~14.1%。用该方法检测病毒性脑炎患者脑脊液,平均sCD100水平显著升高,2例患者标本分别高出正常均值9.4及13.8倍。结论成功建立了快速、敏感和稳定的sCD100化学发光检测法,可用于脑脊液标本中微量sCD100的定量分析。

Objective To establish a chemiluminescent immunoassay（ CLIA） for the detection of soluble CD100（ sCD100） and evaluate its preliminary clinical application for the detection of sCD100 in clinical cerebrospinal fluid samples.Methods Ascites were prepared using two hybridomas secreting monoclonal antibody（ m Ab） to CD100,and the antibodies were purified. Based on sandwich ELISA,the experiment conditions were optimized and the CLIA for detecting sCD100 was established. The sensitivity and stability of CLIA were evaluated. The level of sCD100 in cerebrospinal fluid samples of patients（ n = 18） was detected. Results CLIA exhibited high performance within a dynamic range 0. 098-12. 5 ng/m L,and the limit of detection（ LOD） was 0. 12 ng/m L. The intra-assay coefficient variations（ CV） were between 3. 8%-6. 6% and inter-assay CV were 6. 2%-14. 1%. Using CLIA,we examined the level of sCD100 in cerebrospinal fluid of viral encephalitis patients. The results showed the level of sCD100 in the patients were higher than that in normal controls. Two cases of them were 9. 4 and 13. 8 times higher than the normal mean value of control group. Conclusion A rapid,sensitive and stable CLIA for detecting sCD100 has been successfully established,which can be used for the quantitative detection of trace sCD100 in cerebrospinal fluid samples.