摘要
目的制备抗人糖抗原19-9(CA19-9)单克隆抗体(mAb),建立双抗体夹心检测CA19-9化学发光体系。方法 CA19-9免疫小鼠后,通过细胞融合技术,获得抗CA19-9的mAb。鉴定其抗体的纯度、效价、特异性和配对,建立双抗体夹心化学发光体系。在包被缓冲液、包被浓度及加样模式上优化化学发光体系,对该体系进行准确度、最低检测限、线性、重复性评估以及血清样本的检测。结果获得4株名称为#1-1、#2-1、#3-1和#4-1的mAb。抗体效价均大于10^-8,与CA125、CA15-3、CA72-4均无交叉反应。其中#3-1 mAb与#2-1-HRP建立的双抗体夹心体系经优化后,其检测范围为0~1 000 U/mL;最低检测限为2.0 U/mL;线性相关系数为0.9999。与罗氏试剂检测结果对比,Kappa系数大于0.75;相关系数大于0.9。结论成功制备了抗人CA19-9 mAb,建立了双抗体夹心检测CA19-9化学发光体系。
Objective To prepare the monoclonal antibodies(mAbs) against human carbohydrate antigen 19-9(CA19-9) and establish a double-antibody sandwich chemiluminescent immunoassay(CLIA) detection system for CA19-9 in the human serum. Methods BALB /c mice were immunized with human CA19-9 antigen. The mAbs were obtained by hybridoma technique. The purity,titer,specificity and pairing of the mAbs were characterized and the sandwich CLIA system was established. The system was evaluated in its accuracy,limit of detection,linearity and repetitiveness after optimization of coating buffer,coating concentration and pipetting mode,and the serum sample was tested with it. Results Four mAbs named #1-1,#2-1,#3-1 and # 4-1 were obtained against human CA19-9. The titers of the anti-CA19-9 mAbs were above10^-8. The mAbs had nearly no cross-reaction with CA125,CA15-3 and CA72-4. The double-antibody sandwich CLIA system was established by #3-1 mAb and #2-1 mAb-HRP. After optimization,its property was detection range of 0- 1 000 U /mL,limit detection of 2. 0 U /mL,linear correlation coefficient of 0. 9999. The results which were contrasted with Roche test showed that: Kappa 0. 75,r( correlation coefficient) 0. 9. Conclusion The mAbs against human CA9-9 have been prepared and a sandwich CLIA system for detecting CA19-9 in the human serum has been established successfully.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2014年第12期1282-1286,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
黑龙江省应用技术研究与开发项目(GC13C122)
黑龙江省发展高新技术产业专项资金(FW11C201)