细胞角蛋白19单克隆抗体的制备及组织芯片法评价
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摘要
目的利用组织芯片法对自制细胞角蛋白19(cytokeratin 19,CK19)单克隆抗体进行临床评价。方法通过CK19抗原免疫BALB/c小鼠,经细胞融合及筛选,免疫组化初筛获得CK19单克隆抗体细胞株。进一步制备腹水并纯化CK19抗体,对该抗体及商品化对照抗体进行组织芯片法评价。结果经过细胞融合共筛出1株较好的杂交瘤细胞株2H5,纯化后的抗体纯度达95%,SDS-PAGE分析可见相对分子质量约55 000和25 000的重链和轻链条带,该抗体与对照抗体经组织芯片法评价,两者相关系数r=0. 994 7。结论经组织芯片法评价,自制CK19单克隆抗体与对照抗体相比无明显差异,为后续开展大量临床组织研究提供了依据。
Objective To evaluate the prepared monoclonal antibody(McAb) against cytokeratin 19(CK19) by tissue microarray. Methods BALB/c mice were immunized with CK19 antigen,based on which the cell strain secreting McAb was obtained by cell fusion and screening. The murine ascites was prepared,from which the McAb was purified and evaluated by tissue microarray,using the commercial McAb as control. Results Hybridoma cell strain 2 H5 was screened by cell fusion. The secreted McAb reached a purity of 95% after purification and showed heavy and light chains with relative molecular masses of about 55 000 and about 25 000 respectively on SDS-PAGE profile. The correlation coefficient(r)of tissue microarray results of prepared and commercial McAbs was 0. 994 7. Conclusion There was no significant difference between the tissue microarray results of prepared and commercial McAbs against CK19,which provided a basis for large-scale study on clinical tissue specimens.
Objective To evaluate the prepared monoclonal antibody(McAb) against cytokeratin 19(CK19) by tissue microarray. Methods BALB/c mice were immunized with CK19 antigen,based on which the cell strain secreting McAb was obtained by cell fusion and screening. The murine ascites was prepared,from which the McAb was purified and evaluated by tissue microarray,using the commercial McAb as control. Results Hybridoma cell strain 2 H5 was screened by cell fusion. The secreted McAb reached a purity of 95% after purification and showed heavy and light chains with relative molecular masses of about 55 000 and about 25 000 respectively on SDS-PAGE profile. The correlation coefficient(r)of tissue microarray results of prepared and commercial McAbs was 0. 994 7. Conclusion There was no significant difference between the tissue microarray results of prepared and commercial McAbs against CK19,which provided a basis for large-scale study on clinical tissue specimens.
引文
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[8]张强,王爱萍,谭力铭.肺癌血清标志物CK19单克隆抗体制备及初步应用[J].西藏科技,2015,40(7):31-35.
[9] CHAVAN S S,RAVINDRA S,PRASAD M. Breast biomarkers-comparison on whole section and tissue microarray section[J]. J Clin Diag Res,2017,11(3):EC40-EC44.
[10] CARDANO M,DIAFERIA G R,FALAVIGNA M,et al. Cell and tissue microarray technologies for protein and nucleic acid expression profiling[J]. J Histochem Cytochem,2013,61(2):116-124.
[11] NOLTE S,ZLOBEC I,LUGLI A,et al. Construction and analysis of tissue microarrays in the era of digital pathology:a pilot study targeting CDX1 and CDX2 in a colon cancer cohort of612 patients[J]. J Pathol Clin Res,2017,3(1):58-70.
[2] YANG R N,YANG S H,CHANG C C,et al. Upregulation of fecal cytokeratin 19 is associated with prognosis in older colorectal cancer patients[J]. Genetic Test Mol Biomarkers,2010,14(5):703-708.
[3] HUANG D,XU W,XU X,et al. EMT influences the expression of CK19 in pleural effusion-derived lung cancer cells and their invasion and metastasis[J]. Oncol Lett,2016,12(6):5052-5058.
[4] CHEN Y F,YANG H W,MO Q G,et al. Micrometastasis detection in breast cancer of patients by biological markers CK19,hMAM and SBEM[J]. Chin J Cancer Prev Treat,2014,21(3):198-202.(in Chinese)陈月凤,杨华伟,莫钦国,等. CK19和hMAM及SBEM检测乳腺癌微转移价值分析[J].中华肿瘤防治杂志,2014,21(3):198-202.
[5] LU W,LIANG K C,WU H G,et al. Correlation of CK19,lung specific protein mRNA X expression with micrometastasis and prognosis in primary non small cell lung cancer[J]. J Clin Pulmon Med,2017,22(5):802-805.(in Chinese)陆薇,梁克诚,吴汉刚,等.Ⅰ期非小细胞肺癌淋巴结中ck19、肺特异性蛋白X m RNA表达与微转移及预后的相关性研究[J].临床肺科杂志,2017,22(5):802-805.
[6] ZHANG M L,HUANG Y N,LU L,et al. Study on the lymph node metastasis of gastric antrum carcinoma in PN0 with CK19marker[J]. Ningxia Med J,2017,39(1):36-38.(in Chinese)张明亮,黄允宁,卢林,等.以CK19为标记物研究pN0胃窦癌区域淋巴结微转移[J].宁夏医学杂志,2017,39(1):36-38.
[7] GIOVANELLA L,CERIANI L,GIARDINA G,et al. Serum cytokeratin fragment 21. 1(CYFRA 21. 1)as tumour marker for breast cancer:comparison with carbohydrate antigen 15. 3(CA15. 3)and carcinoembryonic antigen(CEA)[J]. Clin Chem Lab Med,2002,40(3):298-303.
[8]张强,王爱萍,谭力铭.肺癌血清标志物CK19单克隆抗体制备及初步应用[J].西藏科技,2015,40(7):31-35.
[9] CHAVAN S S,RAVINDRA S,PRASAD M. Breast biomarkers-comparison on whole section and tissue microarray section[J]. J Clin Diag Res,2017,11(3):EC40-EC44.
[10] CARDANO M,DIAFERIA G R,FALAVIGNA M,et al. Cell and tissue microarray technologies for protein and nucleic acid expression profiling[J]. J Histochem Cytochem,2013,61(2):116-124.
[11] NOLTE S,ZLOBEC I,LUGLI A,et al. Construction and analysis of tissue microarrays in the era of digital pathology:a pilot study targeting CDX1 and CDX2 in a colon cancer cohort of612 patients[J]. J Pathol Clin Res,2017,3(1):58-70.