Preparation and application of monoclonal antibodies against Herpes simplex virus-1

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1 doi /j. issn X Ⅰ R392 A X HSV- HSV- ELISA HSV- HSV- BALB /c F6 HRP 2B HSV- ELISA HSV- ELISA ~ 2 μg /ml R 2 = μg /ml CV < 0% 85. 6% ~ 07. % HSV- HSV- ELISA HSV- HSV- Preparation and application of monoclonal antibodies against Herpes simplex virus- YIN Bing-Qian JIA Ji-Zong ZHAO Feng-Qiang HAN Jin-Le HUANG Cheng-Hao YE Xiang-Zhong ZHANG Jing- Hai. College of Life Science and Biological Pharmacy Shenyang Pharmaceutical University Shenyang 006 China Abstract Objective To prepare and screen monoclonal antibodies against Herpes simplex virus- HSV- and develop a double antibody sandwich quantitative enzyme-linked immunosorbent assay Q-ELISA for detection of HSV- particle. This method was used to control the quality of viral particle in the developing and manufacturing process of HSV-. Methods BALB /c mice was immunized with HSV- to prepare monoclonal antibodies. A double antibody sandwich Q-ELISA was developed to determine concentration of HSV- particle which was based on the neutralizing monoclonal antibody F6 as capture antibody and 2B as HRP-conjugated antibody. The performance of the reagent was evaluated including specificity sensitivity precision accuracy and linear. And the relation between the amount of virus detected by this method and the virus titer was analyzed by regression analysis method. Results The Q- ELISA for HSV- particle was developed. The quantitation scope was μg /ml the coefficient correlation was the limit of detection was μg /ml the recovery was between 85. 6% and 07. % the variation coefficient was lower than 0% and the reagent does not react with other samples except HSV- antigen. This method has a good correlation with virus titer. Conclusion The Q- ELISA for HSV- particle was successfully developed which provide a new approach for rapid and quantitative detection of HSV- antigen. Key words HSV- Oncolytic Particle integrity Virus titer - HSV- HSV- HSV- a jinghaizhang@ foxmail. com ybq050@ sina. com yxzh_hb@ 63. com HSV- 80 nm DNA HSV- 52 kb HSV- ICP34. 5 HSV- HSV-

2 Ⅰ RR ICP SDS-PAGE 2% HSV- 90 V 30 min 20 V 45 min ICP4 ICP27 2B F6 SDS HSV Western blot 293 IL-2 GM-CSF B7-3 HSV- gb ~ gk SDS- PAGE 90 V 00 ma HSV h PBST 5 HSV- min / 3 HRP IgG 5 37 h PBST 5 min / 3 HSV- AEC HSV- ARPE HSV- ARPE ELISA HSV- HSV ELISPOT 05 U2OS % CO 2 24 h... HSV- F6 2B 00 μl / 37 h PBS 5 GAM-HRP HSV min VZV CMV. 4 ELISA A 6 CA6 7 EV7 HAV.. 3 ARPE ATCC ph μg /ml μl / 37 2 h PBST T.. 4 BSA 0. 05% Tween20 2% BSA 0 HRP Sigma BCA mmol /L PB 37 2 h pierce HRP 2 00 μl / what min PBST 300 μl / man. 2 HSV- min PBST 300 μl / 5 SPF BALB /c 00 μg / 2 50 μg / / 4 HSV- 00 μg / % 48 h ELISPOT 50 μl % 5 min PBS 00 μl 0. % Triton 5 min PBS 30min PBS 5 TMB F6 HRP 2B 6 F6 50 mmol /L 5 HRP 2B 00 μl / TMB 00 μl / 37 5 min 2 mol /L H 2 SO 4 50 μl / 450 /630 SP2 /0 PEG HSV-. 5. HSV- Protein A 2 μg /ml μg /ml 0. 5 μg /ml 3

3 F6 50 mmol /L ph μg /ml μg /ml μg /ml 0. 5 μg /ml 3 HSV h 5 00 μl / min 5 TMB μl / 450 /630 A HSV A μg /ml OD μl / 37 2 h 200 μl / 37 2h 00 μl / 00 μl / 37 5 min 2 mol /L H 2 SO % ~ 07. % ARPE CMV VZV < 0%. 6 HSV- HSV- 2 2 HSV- Western blot Fig. 2 Western blot of monoclonal antibody against 2. HSV- HSV- Note 2B.. HSV- virus 2. ARPE cell protein 3. HSV- gc. F6.. HSV- Virus 2. ARPE cell protein 3. HSV- ~ 3 gb-gk. Fig. HSV- SDS SDS-PAGE of monoclonal antibody against HSV- Note M. Marker. 2B 2. F6. 3 Fig. 3 HSV- ELISPOT The epitope identification of the monoclonal antibody against HSV- Note. Glycoprotein B 2. Glycoprotein C 3. Glycoprotein D 4. Glycoprotein E 5. Glycoprotein G 6. Glycoprotein H 7. Glycoprotein I 8. Glycoprotein J 9. Glycoprotein K 0. Glycoprotein L. Glycoprotein M 2. Cell protein. Tab. Characteristics of two HSV- monoclonal antibodies Name Purpose Antibody titer Antibody affinity Neutralizing titer 400 PFU /ml Western blot Epitope 2B HRP sign % 32 gc gc F6 Coating % 4 NA gb Note NA. Do not react with each kind of glycoproteins including gb-gk. ELISPOT

4 Ⅰ Tab. 2 Accuracy and precision of developed ELISA method HSV- antigen μg /ml Recovery ratio % CV % HSV- 4 HSV- Fig. 4 Standard curve of internal reference of HSV- antigen 5 Fig. 5 Correlation analysis of two detection methods ~ 2 μg /ml R 2 = μg /ml 4 POT F6 gb 2B HSV gc gc gb A450 /630 Cutoff 3 Ⅲ 8 Yervoy I 9 Medigene NV020 I /II 47. 2% 0 Feng 2 HSV- KTR27 HSV- ICP0 ICP27 HSV- HSV- HSV- 6 PCR 7 ELISA Western blot ELIS r = HSV- HSV- Anthony Orvedahl Diane Alexander Amgen T-Vec Zsolt Tallóczy et al HSV- ICP34. 5 Confers Neurovirulence by Targeting the Beclin Auto-

5 phagy Protein J. Cell Host & Microbe L Miao C Fraefel K C Sia et al The potential application of a transcriptionally regulated oncolytic herpes simplex virus for human cancer therapy J. Br J Cancer Jacqueline N. Parkera Luz-Andrea Pfistera Debra Quenellea et al Genetically engineered herpes simplex viruses that express IL-2 or GM-CSF as vaccine candidates J. Vaccine William F. Goins Shaohua Huang Justus B. Cohen et al Engineering HSV- Vectors for Gene Therapy J. Methods Mol Biol Manservigi R Argnani R Marconi P. HSV recombinant vectors for gene therapy J. Open Virol J A 6 ELISA J M Robert Hans Ingemar Andtbacka Frances A. Collichio Thomas Amatruda et al. OPTiM a multicenter randomized phase 3 trial of talimogene laherparepvec versus subcutaneous GM-CSF for the treatment of unresected stage IIIB /C and IV melanoma FC-022 J. J Deut Derma Gesellschaft Igor Puzanov Mohammed Milhem Robert Andtbacka et al. Phase results of a phase b /2 multicenter open-label trial to evaluate safety and efficacy of talimogene laherparepvec T-VEC and ipilimumab ipi vs ipi alone in previously untreated unresected stage IIIB-IV melanoma J. J Immunother Sze DY Chari R Geller D et al Abstract No. 62 Phase I /II study of the oncolytic herpes virus NV020 to treat liver-dominant metastatic colorectal cancer J. J Vasc Interve Rad Geevarghese SK Geller DA Haan HA et al. Phase I /II study of oncolytic herpes simplex virus NV020 in patients with extensively pretreated refractor colorectal cancer metastatic to the liver J. Human Gene Therapy Feng Yao Nao Murakami Oliver Bleiziffer et al. Development of a regulatable oncolytic herpes simplex virus type recombinant virus for tumor therapy J. J Virology Bataille D Epstein AL. Herpes simplex virus type replication and recombination J. Biochimie Mateu MG. Assembly stability and dynamics of virus capsids J. Arch Biochem Biophys Seyffert M de Oliveira AP Fraefel C et al Multifluorescence live analysis of herpes simplex virus type- replication J. Methods Mol Biol Michael Costello MT Sabatinl L Yungbluth P. Herpes simplex virus infections and current methods for laboratory detection J. Clinical Microbiology Newsletter Slomka MJ Emery L Munday PE et al. A comparison of PCR with virus isolation and direct antigen detection for diagnosis and typing of genital herpes J. J Med Virol Gallagher John R Atanasiu Doina Saw Wan Ting et al. Functional fluorescent protein insertions in herpes simplex virus gb report on gb conformation before and after execution of membrane fusion J. PLoS Pathogens Frink RJ Eisenberg R Cohen G et al Detailed analysis of the portion of the herpes simplex virus type genome encoding glycoprotein C J. J Virol Maylin S Laouénan C Martinot-Peignoux M et al. Role of hepatic HCV-RNA level on the severity of chronic hepatitis C and response to antiviral therapy J. J Clin Virol HCV ELISA J IgM. J J Wang TY Kuo HT Chen LC et al. Use of polymerase chain reation for early detection and management of hepations C virus infection after needlestick injury J. Ann Clin Lab Sci HBc-IgG J HCV CLIA S /CO J J M

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