细胞角蛋白19片段抗原21-1含量化学发光微粒子定量免疫检测方法的建立
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摘要
目的建立细胞角蛋白19片段抗原21-1(cytokeratin 19 fragment 21-1,CYFRA21-1,简称CY21-1)含量的化学发光微粒子免疫检测方法(chemiluminescent microparticle immunoassay,CMIA)。方法原核表达制备重组CY21-1(r CY21-1),用其免疫BALB/c小鼠后,收集小鼠脾脏细胞,常规杂交瘤技术与Sp2/0进行融合,制备CY21-1单抗。间接ELISA法筛选可稳定分泌CY21-1抗体的杂交瘤细胞,分别标记微性磁珠(magnetic beads,MB)和吖啶酯(acridinium ester,AE),筛选可特异检测CY21-1的单抗配对,建立CY21-1 CMIA,同时验证方法的线性及灵敏度,并与同类试剂盒进行比较。结果经筛选获得了一组能特异检测CY21-1的单抗配对MB*26B5-3E4*AE,建立的CY21-1 CMIA对检测CY21-1校准品的线性范围为0.1~1 000 ng/m L,R~2=0.992 3,检测灵敏度为0.076 ng/m L。该方法与美国雅培公司生产的Abbott CY21-1 CMIA诊断试剂盒平行检测250份临床血清标本的结果具有良好的相关性(R~2=0.961 6)。结论成功建立了CY21-1 CMIA,且具有良好的线性及灵敏度,为我国肺癌的临床筛查和早期诊断奠定了基础。
Objective To develop a chemiluminescent microparticle immunoassay(CMIA) for cytokeratin 19 fragment21-1(CYFRA21-1,CY21-1 for short) content. Methods Recombinant CY21-1 antigen was prepared by prokaryotic expression and used for immunization of BALB/c mice. The splenocytes of mice were collected and fused with Sp2/0 cells by routine hybridoma technique. The hybridoma cells stably secreting the monoclonal antibody(m Ab)against CY21-1 were screened by indirect ELISA and labeled with magnetic beads(MB) and acridinium ester(AE) respectively. The m Ab pair for specific detection of CY21-1 were screened,based on which CY21-1 CMIA was developed,verified for linearity and sensitivity,and compared with the kits of same kind. Results An antibody pair MB*26 B5-3 E4*AE was obtained,with which CY21-1 CMIA was developed. The linear range of the developed method for determination of CY21-1 calibrator was 0. 1 ~ 1 000 ng/m L,with a R~2 value of 0. 992 3,while the sensitivity was 0. 076 ng/m L. The determination results of 250 human serum samples by the developed method showed good correlation with that by commercial Abbott CY21-1 CMIA kit(R~2 = 0. 961 6). Conclusion CY21-1 CMIA was developed successfully,which showed high linearity and sensitivity. It laid a foundation of clinical screening and early diagnosis of lung cancer in China.
Objective To develop a chemiluminescent microparticle immunoassay(CMIA) for cytokeratin 19 fragment21-1(CYFRA21-1,CY21-1 for short) content. Methods Recombinant CY21-1 antigen was prepared by prokaryotic expression and used for immunization of BALB/c mice. The splenocytes of mice were collected and fused with Sp2/0 cells by routine hybridoma technique. The hybridoma cells stably secreting the monoclonal antibody(m Ab)against CY21-1 were screened by indirect ELISA and labeled with magnetic beads(MB) and acridinium ester(AE) respectively. The m Ab pair for specific detection of CY21-1 were screened,based on which CY21-1 CMIA was developed,verified for linearity and sensitivity,and compared with the kits of same kind. Results An antibody pair MB*26 B5-3 E4*AE was obtained,with which CY21-1 CMIA was developed. The linear range of the developed method for determination of CY21-1 calibrator was 0. 1 ~ 1 000 ng/m L,with a R~2 value of 0. 992 3,while the sensitivity was 0. 076 ng/m L. The determination results of 250 human serum samples by the developed method showed good correlation with that by commercial Abbott CY21-1 CMIA kit(R~2 = 0. 961 6). Conclusion CY21-1 CMIA was developed successfully,which showed high linearity and sensitivity. It laid a foundation of clinical screening and early diagnosis of lung cancer in China.
引文
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[13]DUAN M T,LAN L Z.Comparison of the effects of chemical luminescence method and enzyme-linked immunosorbent assay on the detection of HIV in blood[J].Mod Instr Med Treat,2016,22(5):78-80.(in Chinese)段美婷,兰良子.化学发光法与酶联免疫吸附法进行血HIV检测效果评价[J].现代仪器与医疗,2016,22(5):78-80.
[14]ZHANG L J.Comparison of chemiluminescent microparticle immunoassay and enzyme-linked immunosorbent assay for determination of anti-neutrophil cytoplasm antibodies[J].Chin J Immunol,2016,32(8):1175-1178.(in Chinese)张立娟.磁微粒化学发光法与酶联免疫吸附法测定抗中性粒细胞胞浆抗体的结果比较[J].中国免疫学杂志,2016,32(8):1175-1178.
[2]银新,贺永梅,宁守斌.细胞角蛋白的临床研究进展[J].实用医学杂志,2011,27(16):3064-3066.
[3]VAN DER GAAST A,SCHOENMAKERS C H,KOK T C,et al.Evaluation of a new tumour marker in patients with nonsmall-cell lung cancer:Cyfra 21.1[J].Br J Cancer,1994,69(3):525-528.
[4]MOLINA R,AUGE J M,FILELLA X,et al.Pro-gastrin-releasing peptide(pro GRP)in patients with benign and malignant diseases:comparison with CEA,SCC,CYFRA 21-1 and NSE in patients with lung cancer[J].Anticancer Res,2005,25(3A):1773-1778.
[5]HOLDENRIEDER S,WEHNL B,HETTWER K,et al.Carcinoembryonic antigen and cytokeratin-19 fragments for assessment of therapy response in non-small cell lung cancer:a systematic review and meta-analysis[J].Br J Cancer,2017,116(8):1037-1045.
[6]WIESKOPF B,DEMANGEAT C,PUROHIT A,et al.Cyfra21-1 as a biologic marker of non-small cell lung cancer.Evalua-tion of sensitivity,specificity,and prognostic role[J].Chest,1995,108(1):163-169.
[7]STIEBER P,HASHOLZNER U,BODENM譈LLER H,et al.CYFRA 21-1.A new marker in lung cancer[J].Cancer,1993,72(3):707-713.
[8]CHEN Z Y.Application of joint detection of tumor markers such as SCC Ag,NSE,CYFRA21-1,and CEA in the diagnosis of lung cancer[J].J Clin Pulmonary Med,2013,18(1):10-11.(in Chinese)陈邹阳.肿瘤标志物SCC-Ag、NSE、CYFRA21-1、CEA联合检测在肺癌诊断中的应用价值[J].临床肺科杂志,2013,18(1):10-11.
[9]ZHANG H R,ZHAI J Y,WANG X Q,et al.Preparation and preliminary application of double antibody sandwich ELISA for CYFRA21-1[J].Chin J Immunol,2014,30(5):644-647.(in Chinese)张慧茹,翟晋豫,王雪琴,等.CYFRA21-1双抗体夹心酶联免疫检测方法的研制[J].中国免疫学杂志,2014,30(5):644-647.
[10]LU L F,GE S X,ZHANG J.Review on the development of chemiluminescence immunoassay[J].J Mol Diag Ther,2015,7(5):289-295,284.(in Chinese)陆龙飞,葛胜祥,张军.化学发光免疫分析法研究进展[J].分子诊断与治疗杂志,2015,7(5):289-295,284.
[11]CHEN W Q,ZHENG R S,ZHANG S W,et al.Report of cancer incidence and mortality in China,2012[J].China Cancer,2016,25(1):1-8.(in Chinese)陈万青,郑荣寿,张思维,等.2012年中国恶性肿瘤发病和死亡分析[J].中国肿瘤,2016,25(1):1-8.
[12]ABERLE D R,BERG C D,BLACK W C,et al.The national lung screening trial:overview and study design[J].Radiology,2011,258(1):243-253.
[13]DUAN M T,LAN L Z.Comparison of the effects of chemical luminescence method and enzyme-linked immunosorbent assay on the detection of HIV in blood[J].Mod Instr Med Treat,2016,22(5):78-80.(in Chinese)段美婷,兰良子.化学发光法与酶联免疫吸附法进行血HIV检测效果评价[J].现代仪器与医疗,2016,22(5):78-80.
[14]ZHANG L J.Comparison of chemiluminescent microparticle immunoassay and enzyme-linked immunosorbent assay for determination of anti-neutrophil cytoplasm antibodies[J].Chin J Immunol,2016,32(8):1175-1178.(in Chinese)张立娟.磁微粒化学发光法与酶联免疫吸附法测定抗中性粒细胞胞浆抗体的结果比较[J].中国免疫学杂志,2016,32(8):1175-1178.
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