中性粒细胞明胶酶相关脂质运载蛋白的单克隆抗体制备及化学发光免疫定量检测试剂研究  

Preparation of monoclonal antibodies against neutrophil gelatinase-associated lipocalin(NGAL) and development of an antibody-based chemiluminescence immune quantification assay

作　　者：齐家龙[1] 邵佳[1] 彭宽[1] 黄明聪 邓丽雯 李少伟[1,3] 张军[1,3] 夏宁邵[1,3] 顾颖[1,3] 

机构地区：[1]厦门大学分子疫苗学与分子诊断学国家重点实验室公共卫生学院,福建厦门361102 [2]厦门万泰凯瑞生物技术有限公司,福建厦门361000 [3]厦门大学国家传染病诊断试剂与疫苗工程技术研究中心生命科学学院,福建厦门361102

出　　处：《中国生化药物杂志》2015年第4期5-9,共5页Chinese Journal of Biochemical Pharmaceutics

基　　金：国家高技术研究发展计划(863计划;2011AA02A101)

摘　　要：目的获得中性粒细胞明胶酶相关脂质运载蛋白（NGAL）的单克隆抗体,并对其应用于化学发光定量试剂进行研究。方法利用原核表达系统表达的NGAL重组抗原,经过Ni2＋-金属螯合层析纯化后用于免疫6~8周龄的雌性BALB/c小鼠,完成免疫程序后将其脾脏细胞与杂交瘤细胞进行融合,通过间接法ELISA筛选抗NGAL的单克隆抗体,并结合Western blot鉴定抗体与天然NGAL的反应性。结果获得了由原核表达系统表达的NGAL重组抗原,具有较高的纯度,可形成二硫键交联的二聚体形式;重组抗原具有较好的免疫原性,免疫小鼠多抗血清效价达10^6以上;筛选获得30株单克隆抗体,其中单抗23C12、38D10的EC50分别为0.034、0.022 g/mL;经NGAL标准品及临床尿液样本初步筛选,由38D10/23C12-SAE组成的抗体配对能够很好的适用于基于化学法发光的免疫反应定量检测试剂的开发;尿液阳性样本与阴性样本检测结果显示具有显著的统计学差异（P〈0.001）;对病患组定量结果与Abbott公司的尿NGAL测定试剂盒（化学发光微粒子免疫检测法）的相关系数大于0.97,线性范围为10~1500 ng/mL,检测灵敏度为0.63 ng/mL。结论本研究成功制备了NGAL重组抗原,获得了NGAL特异单克隆抗体,并开发了NGAL化学发光免疫定量试剂,该试剂检测能力与国际主流试剂相当。Objective To obtain monoclonal antibodies （ mAbs ） against neutrophil gelatinase-associated lipocalin （ NGAL ） and a chemiluminescense immune quantification assay based one paired mAbs.Methods Six-to-eight weeks old female BALB/c mice were immunized with the purified recombinant human NGAL antigen（ rhNGAL） that was produced by the Escherichia coil expression system.The spleen was fused with hybridoma for screening anti-NGAL monoclonal antibodies by indirect ELISA.Western blot was implemented to identify the reactivity with native NGAL. Results The rhNGAL antigen was found to form disulfide cross-linked dimers and present excellent immunogenicity.The reaction titer of the immune serum of NGAL immunized mice was about 10^6.Thirty mAbs were screened by indirect ELISA, hereinto;the EC50 values of mAb23C12 and 38D10 were 0.034 g/mL, 0.022 g/mL respectively.The antibodies pair, 38D10/23C12-SAE labeled with AcridiniumEster（AE）, were shown to work well in chemiluminescense immune response quantitative detection which was screened by NGAL standardand clinical urine samples.This detection can resolve positive and negative samples with a statistically significant difference （P〈0.0001）.And the correlation coefficient R2between NGAL quantitative results＆amp;nbsp;and that of the Abbott＆#39;s NGAL chemiluminescence immune assay kit was greater than 0.97.The detection linear range was 10-1500 ng/mL, analytical sensitivity of the method was 0.63 ng/mL.Conclusion Highly purified rhNGAL antigen and specific anti-NGAL monoclonal antibodies are generated in this study.The detection capability of method is comparable with that of the international commercial kit.

关 键 词：中性粒细胞明胶酶相关脂质运载蛋白 单克隆抗体 化学发光 定量试剂 

分 类 号：S853.7[农业科学—临床兽医学]