肿瘤蛋白p185的纯化及乳腺癌患者血清中p185蛋白BA-ELISA检测方法的建立
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摘要
目的从转染Her-2/neu基因的T6-17细胞上清中纯化p185蛋白作为阳性抗原对照,利用生物素-亲和素酶联免疫法(Biotin-Avidin ELISA,BA-ELISA法)检测乳腺癌患者血清中p185蛋白,探讨血清p185蛋白在乳腺癌诊断中的意义。方法采用溴化氰活化的Sepharose 4B为基质,通过交联A18 mAb,用亲和层析的技术纯化p185蛋白。将收集的T6-17细胞上清循环过亲和层析柱,经醋酸溶液解离与抗体结合的p185蛋白,收集到蛋白洗脱峰,浓缩后用ELISA检测p185的免疫反应性,并经SDS-PAGE和Western blot进一步鉴定。利用自备的生物素化Her-2/neu单克隆抗体,并利用亲和层析法从T6-17细胞培养上清中纯化的p185蛋白作为阳性对照,采用BA-ELISA方法对乳腺癌和正常对照血清进行p185蛋白检测,同时对乳腺癌患者组织进行p185蛋白免疫组化染色,并对实验结果进行统计学分析。结果从转基因细胞上清中成功获取p185肿瘤抗原;用BA-ELISA方法检测了122例乳腺癌患者血清,血清p185阳性数为39例,检测了110例正常人血清,血清p185阳性数为0例;122例乳腺癌患者中免疫组化p185阳性数为43例,其中血清p185升高38例。结论初步建立了乳腺癌患者血清中p185蛋白的BA-ELISA检测方法,结果显示患者血清中p185蛋白对乳腺癌具有重要的诊断意义,血清p185蛋白和组织p185蛋白呈高度相关。
Objective Use p185 by affinity chromatography from the culture supernatant of T6-17 which transfected the Her-2/neu gene as the positive control, to detected p185 in breast cancer patients' serum by BA-ELSIA, and discuss the significance of serum P185 in breast cancer diagnosis. Methods The p185 oncoprotein was purified by using an affinity chromatography column. In this experiment, the anti-p185 oncoprotein monoclonal antibody was obtained from A18 hybridoma ascite with octoic acid-saturated ammonium sulfate precipitation. The antibody activity was coupled with CNBr-activated Sepharose 4B as solid phase medium to pack the affinity chromatography column. The p185 oncoprotein antigen was binded with the anti-p185 antibody when the Culture supernatant run through the column. The gradient salt solution(0.2M HAC PH2.5)as elute buffer to dissociate the p185 antigen from the solid phase medium and elution fraction was collected. After be concentrated, the p185 antigen protein was identified by using ELISA、SDS-PAGE and Western blot. Use the biotinylation Her-2/neu mcAb made by ourselves, and P185 by affinity chromatography from the culture supernatant of T6-17 as the positive control, to detect the p185 in breast cancer patients’serum and normal control serum by BA-ELSIA, meanwhile perform IHC in these patients tissues to detect P185,then analyze these experimental results by statistics software. Results The oncoprotein p185 was purified successfully from the Culture supernatant of transgene T6-17 cells; detect 122 cases of breast cancer patients' serum by BA-ELISA method, 39 cases are serum p185 positive; detect 110 cases of normal human serum by BA-ELISA method, 0 case is serum p185 positive; detect 122 cases of breast cancer patients' tissues by IHC, 43 cases are p185 positive, in which 38 cases serum p185 increased. Conclusion The BA-ELISA method to detect p185 in breast cancer patients’serum has taken shape initially, the serum p185 is significant to breast cancer in diagnosis, and the serum p185 level is highly relevant to tissue expression level of p185.
Objective Use p185 by affinity chromatography from the culture supernatant of T6-17 which transfected the Her-2/neu gene as the positive control, to detected p185 in breast cancer patients' serum by BA-ELSIA, and discuss the significance of serum P185 in breast cancer diagnosis. Methods The p185 oncoprotein was purified by using an affinity chromatography column. In this experiment, the anti-p185 oncoprotein monoclonal antibody was obtained from A18 hybridoma ascite with octoic acid-saturated ammonium sulfate precipitation. The antibody activity was coupled with CNBr-activated Sepharose 4B as solid phase medium to pack the affinity chromatography column. The p185 oncoprotein antigen was binded with the anti-p185 antibody when the Culture supernatant run through the column. The gradient salt solution(0.2M HAC PH2.5)as elute buffer to dissociate the p185 antigen from the solid phase medium and elution fraction was collected. After be concentrated, the p185 antigen protein was identified by using ELISA、SDS-PAGE and Western blot. Use the biotinylation Her-2/neu mcAb made by ourselves, and P185 by affinity chromatography from the culture supernatant of T6-17 as the positive control, to detect the p185 in breast cancer patients’serum and normal control serum by BA-ELSIA, meanwhile perform IHC in these patients tissues to detect P185,then analyze these experimental results by statistics software. Results The oncoprotein p185 was purified successfully from the Culture supernatant of transgene T6-17 cells; detect 122 cases of breast cancer patients' serum by BA-ELISA method, 39 cases are serum p185 positive; detect 110 cases of normal human serum by BA-ELISA method, 0 case is serum p185 positive; detect 122 cases of breast cancer patients' tissues by IHC, 43 cases are p185 positive, in which 38 cases serum p185 increased. Conclusion The BA-ELISA method to detect p185 in breast cancer patients’serum has taken shape initially, the serum p185 is significant to breast cancer in diagnosis, and the serum p185 level is highly relevant to tissue expression level of p185.
引文
1.孙硕,李官成.人类肿瘤抗原的鉴定及应用进展[J].国外医学肿瘤学分册, 2005; 32(10): 23-725.
2.王廷华,邹晓莉.蛋白质理论与技术[M].北京:科学出版社, 2005: 123.
3. Hennion MC, Pichon V. Immuno-based sample preparation for trace analysis [J]. J Chromatogr A, 2003, 1000(1-2): 29-52.
4. Schecnter A L, Stern D F, Vaidyanathan L, et al, The neu oncogene an erbB related gene encoding a 185,000 Mr tumor antigen [J]. Nature, 1984, 312: 513-516.
5. Gusterson BA, Gelber RD, Goldhirsch A, et al. Prognostic importance of c-erbB-2 expression in breast cancer [J]. J Clin Oncol, 1992, 10: 1049-1056.
6. Kurebayashi J. Biological and clinical significance of HER2 overexpression in breast cancer [J]. Breast Cancer, 2001, 8: 45-51.
7. Nandini Dendukuri, Karim Khetani, et al, Testing for HER2-positive breast cancer:a systematic review and cost-effectiveness analysis [J]. CMAJ?May 8, 2007?176(10).
8.王琛,李昀,李平,等.表面表位包埋法制备肿瘤表面抗原P185 neuc-erbB-2单克隆抗体及其性质研究[J].中国免疫学杂志, 2000, 16(10): 539-541.
9.杨吉成,宋礼华,周建华,等.医用细胞工程[M].上海交通大学出版社, 2001: 129-138.
10.朱立平,陈学清,张建中.免疫学常用实验方法[M].北京:人民军医出版社,2003: 342-357.
11. Vladimir I, Muronetz, Timo K.Isolation of antigens and antibodies by affinity chromatography [J]. Chromatography B, 2003, 790(1-2): 53-66.
12.李晓华,秦慧莲,何球藻.肿瘤抗原p185蛋白的纯化及其鉴定[J].中国免疫学杂志, 2000, 15(7): 381-383.
13. Narhi LO, Caughey DJ, Horan T, et al. Effect of three elution buffers on the recovery and structure of monoclonal antibodies [J]. Anal Biochem, 1997; 253(2): 236-245.
14. Subramanian A. Immunoaffinity chromatography [J]. Mol Biotechnol, 2002; 20(1): 41-47.
15. Richard R, Burgess and Nancy E. Thompson advances in gentle immunoaffinity chromatography [J]. Cur Opin Biotechnol 2002, 13(4): 304-308.
16.柳晓燕,姚晓玲,宋玉,等.结肠癌相关抗原的制备及其临床意义[J].细胞与分子免疫学杂志, 2007, 23 (4): 335-337.
17.张宁,罗志明,邓洁明,等.妇女乳腺癌发病与相关因素的研究[J].国际医药卫生导报, 2006, 12(2): 6-8.
18.蔡明香,王骐.血清肿瘤标志物检测的临床意义[J].铁道医学, 2000, 28(1): 29-30.
19.吴孟超,沈锋.肝癌研究的现状和展望[J].国外医学肿瘤学分册, 2000, 27(1):17-19.
20.吴孟超.原发性肝癌的诊断和治疗进展[J].中华外科杂志, 1998, 36(9): 515-517.
21. Ogunbiyi JO. Hepatocellular carcinoma in the developing world [J]. Semin Oncol, 2001, 28(2): 179-187.
22.王红阳.原发性肝细胞性肝癌分子机理研究的几个热点问题[J].中华实验外科杂志, 2001, 18(2): 105-107.
23.顾平.生物标志物在乳腺癌研究中的应用[J].上海交通大学学报(医学版), 2007, 27(11): 1396-1399.
24.李琳.乳腺癌标记物研究现状和进展[J].国际肿瘤学杂志2007, 34(9): 673-676
25.董志伟,谷酰之.临床肿瘤学[M].北京:人民卫生出版社, 2002.
26. Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the Her-2 /neu oncongene [J]. Science, 1987, 235: 177-182.
27. Brandt -Rauf PW , Luo JC, Carney Wp, et al. Detection of Increased Amounts of the Extracellular Domain of the c-erbB-2 Oncoprotein in Serum during Pulmonary Carcinogenesis in Humans [J]. Int-J-Cancer, 1994: 156.
28. Eitzel K. Elavated Serum c-erbB-2 Antigen Levels and Decreased Response to Hormone Therapy of Breast Cancer [J]. J Clin Oncol, 1995, 13(5): 1129-1135.
29. Shigeru I, Hisanao O, Kokichi S, et al. Determination of Cytosol C-erbB2 Protein in Breast Cancer by Sandwich Enzyme Immunoassay [J]. Jpn J Clin, 1998, 28(2): 92-102.
30.冼志红,丛文铭,等.用DAKO HercepTest检测肝细胞癌HER-2/neu蛋白的表达[J].中华医学研究杂志, 2005, 5(10): 32-35.
31.姚阳,官伟宁,吴强,等.抗p185单克隆抗体的制备及其在乳腺癌诊断中的应用[J].中国肿瘤杂志, 2000, 22(5): 394.
32.沈关心,周汝麟.现代免疫学实验技术[M].武汉:湖北科学技术出版社, 2002: 157.
33. Pegram MD, Psuletti G, Slamon DJ. HER-2/neu as a predictive marker of response to breast cancer therapy [J].Breast Caner Res Treat, 1998, 52(1-3): 765-772.
1. Schecnter A L, Stern D F, Vaidyanathan L, et al. The neu oncogene an erbB related gene encoding a 185,000 Mr tumor antigen [J]. Nature,1984, 312: 513-516.
2. Gusterson BA, Gelber RD, Goldhirsch A, et al. Prognostic importance of c-erbB-2 expression in breast cancer [J]. J Clin Oncol, 1992, 10: 1049-1056.
3. Kurebayashi J. Biological and clinical significance of HER2 over expression in breast cancer [J]. Breast Cancer, 2001, 8:45-51.
4. Nandini Dendukuri, Karim Khetani, et al. Testing for HER2-positive breast cancer: a systematic review and cost-effectiveness analysis [J].CMAJ?May 8, 2007?176(10).
5.沈镇宇,邵志敏.现代乳腺肿瘤学进展[M].上海:上海科学技术文献出版社, 2002: 381-404.
6.朱孝峰,刘宗潮,谢冰芬,等. SUCI02选择性抑制Her-2/neu受体酪氨酸激酶磷酸化及其对Her-2/neu过表达乳腺癌细胞生长的影响[J].癌症, 2003, 22(8): 790-794.
7. Datts SR, Brunet A, Greenberg ME. Cellular survival:A play in three Akts [J]. GenesDev, 1999, 13(22): 2905-2927.
8. Dubskal, Anderal, SheardMA, et al. Her-2 singaling downregulating by tastuzunab and suppression of the PI3K/AKT pathway: An unexpected effect on TRA IL-induced apoptosis [J] .Febs Lett, 2005, 579(19): 49-58.
9. Das R, Mahabelshwar GH, Kundu GC, et al. Osteopontin stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase type plasminogen activator through phospatidylinositol 3-kinase /Akt signaling pathways in breast cancer cell [J]. Biol Chen, 2003, 278(31): 28593-28606.
10. Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the Her-2 /neu oncongene [J]. Science, 1987, 235:177-182.
11. Pegram MD, Psuletti G, Slamon DJ. HER-2/neu as a predictive marker of response to breast cancer therapy [J]. Breast Caner Res Treat, 1998, 52(1-3): 765-772.
12. Geisler S, Lonning PE, Aas T, et al. Influence of TP53 gene alterations and c-erbB-2 expression on the response to treatment with doxorubicin in locally advanced breast cancer [J]. Cancer Res, 2001, 6(6): 2505-2512.
13.冼志红,丛文铭,等.用DAKO HercepTest检测肝细胞癌HER-2/neu蛋白的表达[J]中华医学研究杂志, 2005, 5(10): 32-35.
14. Nahta R, Yu D, Hung MC, Hortobagyi GN, Esteva FJ: Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer. Nat Clin Pract Oncol 2006, 3: 269-280.
15. Gonzalez-Angulo AM, Hortobagyi GN, Esteva FJ: Adjuvant therapy with trastuzumab for HER-2/neu-positive breast cancer. Oncologist 2006, 11: 857-867.
16. Stever SH. For the herceptin multinational in vestigator study group. Overview of the trastuzumab (Herceptin) amtin Her-2 monoclonal antibody clinical program in Her-2 over expression in breast cancer [J]. Sem in Oncol, 1999, 26 (4 Supp 112): 71.
17. De Laurentiis M, Cancello G, Zinno L, et al. Targeting HER2 as a therapeutic strategy for breast cancer: a paradigmatic shift of drug development in oncology [J]. Oncol, 2005, 16 (Supp l 4): 7-13.
2.王廷华,邹晓莉.蛋白质理论与技术[M].北京:科学出版社, 2005: 123.
3. Hennion MC, Pichon V. Immuno-based sample preparation for trace analysis [J]. J Chromatogr A, 2003, 1000(1-2): 29-52.
4. Schecnter A L, Stern D F, Vaidyanathan L, et al, The neu oncogene an erbB related gene encoding a 185,000 Mr tumor antigen [J]. Nature, 1984, 312: 513-516.
5. Gusterson BA, Gelber RD, Goldhirsch A, et al. Prognostic importance of c-erbB-2 expression in breast cancer [J]. J Clin Oncol, 1992, 10: 1049-1056.
6. Kurebayashi J. Biological and clinical significance of HER2 overexpression in breast cancer [J]. Breast Cancer, 2001, 8: 45-51.
7. Nandini Dendukuri, Karim Khetani, et al, Testing for HER2-positive breast cancer:a systematic review and cost-effectiveness analysis [J]. CMAJ?May 8, 2007?176(10).
8.王琛,李昀,李平,等.表面表位包埋法制备肿瘤表面抗原P185 neuc-erbB-2单克隆抗体及其性质研究[J].中国免疫学杂志, 2000, 16(10): 539-541.
9.杨吉成,宋礼华,周建华,等.医用细胞工程[M].上海交通大学出版社, 2001: 129-138.
10.朱立平,陈学清,张建中.免疫学常用实验方法[M].北京:人民军医出版社,2003: 342-357.
11. Vladimir I, Muronetz, Timo K.Isolation of antigens and antibodies by affinity chromatography [J]. Chromatography B, 2003, 790(1-2): 53-66.
12.李晓华,秦慧莲,何球藻.肿瘤抗原p185蛋白的纯化及其鉴定[J].中国免疫学杂志, 2000, 15(7): 381-383.
13. Narhi LO, Caughey DJ, Horan T, et al. Effect of three elution buffers on the recovery and structure of monoclonal antibodies [J]. Anal Biochem, 1997; 253(2): 236-245.
14. Subramanian A. Immunoaffinity chromatography [J]. Mol Biotechnol, 2002; 20(1): 41-47.
15. Richard R, Burgess and Nancy E. Thompson advances in gentle immunoaffinity chromatography [J]. Cur Opin Biotechnol 2002, 13(4): 304-308.
16.柳晓燕,姚晓玲,宋玉,等.结肠癌相关抗原的制备及其临床意义[J].细胞与分子免疫学杂志, 2007, 23 (4): 335-337.
17.张宁,罗志明,邓洁明,等.妇女乳腺癌发病与相关因素的研究[J].国际医药卫生导报, 2006, 12(2): 6-8.
18.蔡明香,王骐.血清肿瘤标志物检测的临床意义[J].铁道医学, 2000, 28(1): 29-30.
19.吴孟超,沈锋.肝癌研究的现状和展望[J].国外医学肿瘤学分册, 2000, 27(1):17-19.
20.吴孟超.原发性肝癌的诊断和治疗进展[J].中华外科杂志, 1998, 36(9): 515-517.
21. Ogunbiyi JO. Hepatocellular carcinoma in the developing world [J]. Semin Oncol, 2001, 28(2): 179-187.
22.王红阳.原发性肝细胞性肝癌分子机理研究的几个热点问题[J].中华实验外科杂志, 2001, 18(2): 105-107.
23.顾平.生物标志物在乳腺癌研究中的应用[J].上海交通大学学报(医学版), 2007, 27(11): 1396-1399.
24.李琳.乳腺癌标记物研究现状和进展[J].国际肿瘤学杂志2007, 34(9): 673-676
25.董志伟,谷酰之.临床肿瘤学[M].北京:人民卫生出版社, 2002.
26. Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the Her-2 /neu oncongene [J]. Science, 1987, 235: 177-182.
27. Brandt -Rauf PW , Luo JC, Carney Wp, et al. Detection of Increased Amounts of the Extracellular Domain of the c-erbB-2 Oncoprotein in Serum during Pulmonary Carcinogenesis in Humans [J]. Int-J-Cancer, 1994: 156.
28. Eitzel K. Elavated Serum c-erbB-2 Antigen Levels and Decreased Response to Hormone Therapy of Breast Cancer [J]. J Clin Oncol, 1995, 13(5): 1129-1135.
29. Shigeru I, Hisanao O, Kokichi S, et al. Determination of Cytosol C-erbB2 Protein in Breast Cancer by Sandwich Enzyme Immunoassay [J]. Jpn J Clin, 1998, 28(2): 92-102.
30.冼志红,丛文铭,等.用DAKO HercepTest检测肝细胞癌HER-2/neu蛋白的表达[J].中华医学研究杂志, 2005, 5(10): 32-35.
31.姚阳,官伟宁,吴强,等.抗p185单克隆抗体的制备及其在乳腺癌诊断中的应用[J].中国肿瘤杂志, 2000, 22(5): 394.
32.沈关心,周汝麟.现代免疫学实验技术[M].武汉:湖北科学技术出版社, 2002: 157.
33. Pegram MD, Psuletti G, Slamon DJ. HER-2/neu as a predictive marker of response to breast cancer therapy [J].Breast Caner Res Treat, 1998, 52(1-3): 765-772.
1. Schecnter A L, Stern D F, Vaidyanathan L, et al. The neu oncogene an erbB related gene encoding a 185,000 Mr tumor antigen [J]. Nature,1984, 312: 513-516.
2. Gusterson BA, Gelber RD, Goldhirsch A, et al. Prognostic importance of c-erbB-2 expression in breast cancer [J]. J Clin Oncol, 1992, 10: 1049-1056.
3. Kurebayashi J. Biological and clinical significance of HER2 over expression in breast cancer [J]. Breast Cancer, 2001, 8:45-51.
4. Nandini Dendukuri, Karim Khetani, et al. Testing for HER2-positive breast cancer: a systematic review and cost-effectiveness analysis [J].CMAJ?May 8, 2007?176(10).
5.沈镇宇,邵志敏.现代乳腺肿瘤学进展[M].上海:上海科学技术文献出版社, 2002: 381-404.
6.朱孝峰,刘宗潮,谢冰芬,等. SUCI02选择性抑制Her-2/neu受体酪氨酸激酶磷酸化及其对Her-2/neu过表达乳腺癌细胞生长的影响[J].癌症, 2003, 22(8): 790-794.
7. Datts SR, Brunet A, Greenberg ME. Cellular survival:A play in three Akts [J]. GenesDev, 1999, 13(22): 2905-2927.
8. Dubskal, Anderal, SheardMA, et al. Her-2 singaling downregulating by tastuzunab and suppression of the PI3K/AKT pathway: An unexpected effect on TRA IL-induced apoptosis [J] .Febs Lett, 2005, 579(19): 49-58.
9. Das R, Mahabelshwar GH, Kundu GC, et al. Osteopontin stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase type plasminogen activator through phospatidylinositol 3-kinase /Akt signaling pathways in breast cancer cell [J]. Biol Chen, 2003, 278(31): 28593-28606.
10. Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the Her-2 /neu oncongene [J]. Science, 1987, 235:177-182.
11. Pegram MD, Psuletti G, Slamon DJ. HER-2/neu as a predictive marker of response to breast cancer therapy [J]. Breast Caner Res Treat, 1998, 52(1-3): 765-772.
12. Geisler S, Lonning PE, Aas T, et al. Influence of TP53 gene alterations and c-erbB-2 expression on the response to treatment with doxorubicin in locally advanced breast cancer [J]. Cancer Res, 2001, 6(6): 2505-2512.
13.冼志红,丛文铭,等.用DAKO HercepTest检测肝细胞癌HER-2/neu蛋白的表达[J]中华医学研究杂志, 2005, 5(10): 32-35.
14. Nahta R, Yu D, Hung MC, Hortobagyi GN, Esteva FJ: Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer. Nat Clin Pract Oncol 2006, 3: 269-280.
15. Gonzalez-Angulo AM, Hortobagyi GN, Esteva FJ: Adjuvant therapy with trastuzumab for HER-2/neu-positive breast cancer. Oncologist 2006, 11: 857-867.
16. Stever SH. For the herceptin multinational in vestigator study group. Overview of the trastuzumab (Herceptin) amtin Her-2 monoclonal antibody clinical program in Her-2 over expression in breast cancer [J]. Sem in Oncol, 1999, 26 (4 Supp 112): 71.
17. De Laurentiis M, Cancello G, Zinno L, et al. Targeting HER2 as a therapeutic strategy for breast cancer: a paradigmatic shift of drug development in oncology [J]. Oncol, 2005, 16 (Supp l 4): 7-13.