Chinese-German J Clin Oncol

October 2013, Vol. 12, No. 10, P473–P476

DOI 10.1007/s10330-013-1231-x

Preparation of monoclonal antibody to P53 and its clinical application* Wenqing Wei1, Junhua Wu2, Jing Liu1, Yuxia Wang2 1 2

General Hospital of Beijing Military Area Command, Beijing 100700, China Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing 100850, China Received: 30 July 2013 / Revised: 12 August 2013 / Accepted: 25 September 2013 © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2013 Abstract Objective: The aim of this study was to prepare monoclonal antibody against P53, a kind of tumor suppressor protein,and use the antibody initially in clinical immunoassay. Methods: Monoclonal antibody was prepared and identified via the classic protocol of monoclonal antibody preparation. Identified monoclonal antibodies were purified by affinity chromatography. Antibody titer was determined by enzyme linked immunosorbent assay (ELISA). The specific binding activity of antibody was detected by Western blotting and immunohistochemistry. Results: Three strains of monoclonal antibodies named 1P15, 2P37 and 3P40 were obtained and purified by affinity chromatography. The purity of antibodies was higher than 90%. The titers of antibodies were more than 1: 6000. Western blot and immunohistochemistry assay showed that the specific antibody can combine with endogenous P53 protein in the tumor cell lines and determine the expression of P53 in tumor tissue. Conclusion: Three strains of monoclonal antibodies with high affinity to P53 were successfully established, which can be used for detecting the expression of P53 in tumor cells or tissue. Key words

P53 protein; monoclonal antibody; tumor

Tumor suppressor protein P53 is involved in many pathophysiological processes including growth, development, proliferation, ageing and apoptosis [1, 2]. P53 protein plays important function in maintaining genome stability, repairing cell damage and inducing cell apoptosis. Researches show that the function of P53 was closely related to tumorigenesis, more than half of the solid tumor occurrences gene mutation or loss of function of P53 [3–5] . Therefore the function reconstruction of P53 protein as the research target for tumor therapy has attracted wide attention both at home and abroad [6–8]. Preparation of monoclonal antibody against P53 might have important significance in workover the P53 protein in the development of tumor genesis. Recombinant human wild-type P53 expressed in E.coli. BL21 was used as antigen to immunize Balb/c mice and the classical hybridoma cell fusion protocol was applied to screen the monoclonal antibody against P53 protein. The binding activity and the detection sensitivity of the obtained monoclonal antibody were analyzed by enzyme-linked immunosorbent assay (ELISA) and WestCorrespondence to: Wenqing Wei. Email: [email protected] * Supported by grants from the National Natural Science Foundation of China (No. 30973562) and National Basic Research Program of China (No. 2010CB933904).

ern blot. Western blot and immunohistochemistry assay whether the specific antibody can combine with P53 protein in the tumor cell lines or in tumor tissue, which might provide technical means to immunoassay the biological agent based on P53 protein.

Materials and methods Animal and main reagents BALB/c mice, provided by experimental animal center of Military Medical Science Academy, animal license No.: SCXK (Army) 2007-004; rProtein A-Sepharose TM 4 Fast Flow and Protein G-Sepharose TM 4 Fast Flow (Amersham Company, USA); horseradish peroxidase labeled Goat-anti-mouse IgG, N-four methyl benzidine (TMB) (Beijing Zhongshan Jinqiao Biotech Corporation, China); cellulose nitrate membrane (Millipore Company, USA); 96 cell culture plate and ELISA plate (Costar Company, USA). Methods Preparation and purification of monoclonal antibodies to P53 Using classical hybridoma techniques, 3 hybridoma cell strains stably secreting monoclonal antibody against P53 were obtained, named 1P15, 2P37 and 3P40. The

474

cultured cells of the 3 cell strains were collected and injected into BALB/c mice intraperitoneally. Harvesting ascites, then 1P15 was purified with rProtein A-Sepharose TM 4 Fast Flow, 2P37 and 3P40 purified with Protein G-Sepharose TM 4 Fast Flow. The titer of purified antibody determined by ELISA The 96 well microtiter plate was coated with P53 protein antigen at a final concentration of 4 μg/mL, 100 μL/ well, kept at 4 ℃ overnight. Discarding the excess antigen, the microtiter plate was blocked with 5% skim milk and incubated at 37 ℃ for 40 min. The purified antibody diluted at 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800 was added 100 μL per hole and incubated at 37 ℃ for 1 h. Washing away the excess antibody, 100 μL HRP labeled Goat-anti-mouse IgG was added and incubated at 37 ℃ for 1 h. Coloration by TMB, the absorbance value of 450 nm (A450 nm) was read. Determination of binding specificity of the antibody and P53 protein by Western blotting The handled P53 protein samples was loaded onto 12% SDS-PAGE and blotted onto nitrocellulose membrane (Amersham) with transfer buffer (25 mM Tris, 190 mM glycine, 20% MeOH, 0.05% SDS, pH 8.3). The membrane was blocked with 5% skim milk in TBST (10 mM Tris, 100mM NaCl, 0.1% Tween 20, pH7.5) for 1 h and incubated with each McAb diluted 1:1000 (v/v) in TBST at 37 ℃ for 1 h. Then the membrane was washed, incubated with HRP conjugated goat anti-mouse IgG antibody diluted 1:1000 (v/v) in TBST at 37 ℃ for 45 min. After being washed with TBST for 5 min × 6, the membrane was detected by ECL. Western blot determination of binding activity of 3P40 with intracellular P53 protein The cultured MDA-MB-231 cells (breast cancer cell, belong to p53 mutant cell line) and H1299 cells (nonsmall lung cancer cell, belong to p53 deficiency cell line), were inoculated into 6 well plates with 1 × 105 cells per well, cultured at 37 ℃, 5% CO2 for 24 h. The cells were collected and the total protein was extracted. Thirty μg protein sample was detected in accordance with the above Western blotting procedure. Immunohistochemical determination of binding activity of 3P40 with P53 protein in tumor tissues Recruiting 126 cases of tumor specimens from 200901-10 to 2010-12-10 preserved in the clinical pathology department of our hospital, all data had complete record and clear diagnosis, including 19 cases of esophageal cancer, 25 cases of breast cancer, 23 cases of non-small cell lung cancer, 18 cases of gastric cancer, 16 cases of colon cancer, 12 cases of bladder cancer and 13 cases of prostate cancer. All specimens were sliced 4 μm thickness, stained by Envision method, observed by HE staining, PBS instead of the first antibody as negative control. P53 protein is a nuclear staining, positive staining is light yellow,

www.springerlink.com/content/1613-9089

brown or tan (Figs. 1, 2). At least 5 fields of each slice were chosen to calculate cells under the microscope, result assessment as follows: positive cells < 5%, negative; 5%–24%, positive (+); 25%–50% (++); > 50% (+++).

Results Purification of the monoclonal antibodies to P53 Protein G-Sepharose TM 4 Fast Flow can highly purify 2P37 and 3P40, while rProtein A-Sepharose TM 4 Fast Flow could highly purify 1P15. The purity of the antibody reached above 90% with SDS-PAGE analysis. Binding activity of monoclonal antibodies against P53 Fig. 3 showed that the 2P37, 3P40 and 1P15 were able to combine with the P53 protein. Three antibodies titers were measured by ELISA, among them 3P40 was the highest, up to 1:12000 above. Fig. 4 showed, 3 McAbs could be applied to detect P53 protein with Western blot. Binding activity of P53 monoclonal antibody with endogenous P53 protein in tumor cells Fig. 5 showed that 3P40 could specifically recognize P53 protein in MDA-MB-231 cell with a positive result. While H1299 cells belonging to the p53 defect type, so the result of Western blot was negative, illustrating the combination of 3P40 with endogenous P53 protein in the tumor cells at high specificity. P53 monoclonal antibodies were used for immunohistochemical determination of P53 protein in tumor tissue specimens Immunohistochemical assay 126 cases of tumor tissue specimens, including 19 cases of esophageal cancer, 25 cases of breast cancer, 23 cases of non-small cell lung cancer, 18 cases of gastric cancer, 16 cases of colon cancer, 12 cases of bladder cancer and 13 cases of prostate cancer, the positive rates were 63.2%, 64.0%, 60.9%, 33.3%, 68.8%, 83.3% and 69.2% respectively. Among the specimens, the positive rate of bladder cancer was the highest (83.3%), gastric cancer was the lowest (33.3%). Fig. 1 and Fig. 2 showed P53 positive results in the gastric cancer and lung cancer.

Discussion P53 is so far found one of the highest correlated factors with the human tumors. P53 is involved in DNA damage repair, cell cycle regulation, apoptosis and inhibiting angiogenesis, known as the “gene guard” [9]. Mutations in the p53 gene will cause the loss of function, leading to the

Chinese-German J Clin Oncol, October 2013, Vol. 12, No. 10

475

Fig. 4 Binding activity of the monoclonal antibodies to P53 protein by Western blotting Fig. 1 The expression of P53 in gastric cancer (SP × 400)

Fig. 5 Binding specificity of 3P40 to endogenous P53 protein from tumor cells identified by Western blotting. M: Marker; 1: MDA-MB-231 cell; 2: H1299 cell Fig. 2 The expression of P53 in lung cancer (SP × 400)

Fig. 3 The titer of the antibody assayed by ELISA

formation of tumor. There are more than 60% of the tumor caused by p53 gene mutation, p53 mutation occurred in gastric cancer, liver cancer, colorectal cancer, bladder cancer, breast cancer, prostate cancer, lymphatic hematopoietic system tumor, glioma, soft tissue sarcoma, and malignant tumor [10–17] so on. Targeting P53 protein function reconstruction for clinical anticancer drug research has important significance. Wild type human P53 protein expression in prokaryotic used as immune antigen, via classical hybridoma techniques, 3 hybridoma cell strains stably secreting monoclonal antibody against P53 were obtained in our study. ELISA and Western blotting identified that the

3 mAbs could specifically bind P53 proteins, among them the binding activity and detection sensitivity of 3P40 was the highest. 3P40 could specifically recognize the endogenous P53 protein in the tumor cells. The results of different immune staining showed that positive result mainly occurs in the nucleus, truthfully reflect the distribution of endogenous P53 protein, and consistent with the protein as a transcription factor function. The preliminary clinical application of 3P40 showed that it can combine specifically with a variety of P53 protein in tumor. Followed the further research of the function of P53 protein and P53 protein as anti-tumor drugs, specific monoclonal antibody against P53 protein will provide effective method for immune analysis and drug detection.

References 1. Lane D, Levine A. p53 Research: the past thirty years and the next thirty years. Cold Spring Harb Perspect Biol, 2010, 2: a000893. 2. Reinhardt HC, Schumacher B. The p53 network: cellular and systemic DNA damage responses in aging and cancer. Trends Genet, 2012, 28: 128–136. 3. Muller PA, Vousden KH. p53 mutations in cancer. Nat Cell Biol, 2013, 15: 2–8. 4. Strano S, Dell’Orso S, Di Agostino S, et al. Mutant p53: an oncogenic transcription factor. Oncogene, 2007, 26: 2212–2219. 5. Olivier M, Hollstein M, Hainaut P. TP53 mutations in human cancers: origins, consequences, and clinical use. Cold Spring Harb Perspect Biol, 2010, 2: a001008. 6. Wang Y, Li DD, Shao RG, et al. Construction and application of cellular screening model for identification of novel anti-cancer compounds

476 targeting p53. Chin Med Biotechnol (Chinese), 2012, 7: 333–339. 7. Zhou XJ, Xie P, Si XM, et al. Inhibited proliferation effects of recombinant adenovirus p53 injection combined with matrine on lung cancer A549 cells and its mechanism. Chin J Clinicians (Chinese), 2012, 6: 6296–6300. 8. Qi JS, Yang RM, Zhao P, et al. Interventional targeting administration of Ad-p53 combined with ultrasound irradiation in rabbit models of hepatic VX2 tumors. Chin J Med Imaging Technol (Chinese), 2013, 29: 335–338. 9. Hoffman WH, Biade S, Zilfou JT, et al. Transcriptional repression of the anti-apoptotic survivin gene by wild type p53. J Biol Chem, 2002, 277: 3247–3257. 10. Wang RJ, Zhao HX, Shi QL, et al. Influence of arterial chemoembolization on expression of p53 in bladder cancer tissue and its clinical significance. J Pract Oncol (Chinese), 2012, 27: 486–489. 11. Wang H, Wang S. Expression and its clinical significance of P53 in patients with esophageal cancer. Chin Mod Doctor (Chinese), 2012, 50: 136–137, 139. 12. Yu ZH, Ma JF, Yang CY, et al. Expression of CREB-bingding protein

www.springerlink.com/content/1613-9089

13.

14. 15. 16. 17.

and P53 in ovarian cancer and their clinical significance. Chongqing Med (Chinese), 2013, 42: 1207–1209, 1212. He CG, Huang QY, Wu HG, et al. Relationships of expression of survivin with clinicopathological factors, p53 protein, apoptosis and prognosis in gastrointestinal stromal tumors. Guangxi Med J (Chinese), 2013, 35: 16–18. Zhou SW, Huang CP, Huang DY, et al. The expression of KLF4, p21, p53, APC and CyclinD-1 in breast invasive ductal carcinoma and its significance. J Guiyang Med Col (Chinese), 2013, 38: 50–54. Su G, Tang ZM, Mo QR, et al. Expressions and the clinical significances of p53, p57(Kip2) and CD68 in esophageal squamous cell carcinoma. Chinese-German J Clin Oncol, 2013, 12: 167–170. Wang GL, Li JW. Expression and clinical significance of Clusterin, Livin and P53 in gastric cancer. Chin Med Herald (Chinese), 2012, 9: 20–22. Liu JG, Yue F, Yang JY, et al. The expression of ras-p21 and p53 protein in inguinal lymph nodes and clinical significance with penile cancer. Chinese-German J Clin Oncol, 2011,10: 337–339.