798 205 3 doi 0. 3969 /j. issn. 000-484X. 205. 06. 07 Ⅰ 2 3 006 R392 A 000-484X 205 06-0798-05 HSV- HSV- ELISA HSV- HSV- BALB /c F6 HRP 2B HSV- ELISA HSV- ELISA 0. 25 ~ 2 μg /ml R 2 = 0. 995 5 0. 25 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 0. 25-2 μg /ml the coefficient correlation was 0. 995 5 the limit of detection was 0. 25 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 02206 2 36005 3 E-mail jinghaizhang@ foxmail. com 989 - E-mail ybq050@ sina. com 967 - E-mail yxzh_hb@ 63. com HSV- 80 nm DNA HSV- 52 kb HSV- ICP34. 5 HSV- HSV-
Ⅰ 6 799 RR ICP6 2. 3. 3. SDS-PAGE 2% HSV- 90 V 30 min 20 V 45 min ICP4 ICP27 2B F6 SDS HSV-. 3. 2 Western blot 293 IL-2 GM-CSF B7-3 HSV- gb ~ gk SDS- PAGE 90 V 00 ma HSV- 4 37 h PBST 5 HSV- min / 3 HRP IgG 5 37 h PBST 5 min / 3 HSV- AEC HSV- ARPE HSV- ARPE ELISA HSV- HSV-. 3. 3 ELISPOT 05 U2OS 96 37 5% CO 2 24 h... HSV- F6 2B 00 μl / 37 h PBS 5 GAM-HRP 37.. 2 HSV - 37 5 min VZV CMV. 4 ELISA A 6 CA6 7 EV7 HAV.. 3 ARPE ATCC ph9. 6 3 μg /ml 96 00 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- 37 60 min PBST 300 μl / man. 2 HSV- min PBST 300 μl / 5 SPF BALB /c 00 μg / 2 50 μg / / 4 HSV- 00 μg / 3. 5 4 5% 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 / 37 45 TMB 00 μl / 37 5 min 2 mol /L H 2 SO 4 50 μl / 450 /630 SP2 /0 PEG 200 7 HSV-. 5. HSV- Protein A 2 μg /ml μg /ml 0. 5 μg /ml 3
800 205 3 3 2. 2 F6 50 mmol /L ph9. 6 3. 0 μg /ml. 5. 2 2 μg /ml μg /ml 0. 5 μg /ml 3 HSV- 3 3 37 h 5 00 μl / 37 45 min 5 TMB. 5. 3 50 μl / 450 /630 A HSV- 2 0. 5 0. 25 0. 25 0 A μg /ml OD 2. 3 96 00 μl / 37 2 h 200 μl / 37 2h 00 μl / 00 μl / 37 5 min 2 mol /L H 2 SO 4 2. 3.. 5. 4 85. 6% ~ 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 2560 96. 0% 32 gc gc F6 Coating 60 73. 0% 4 NA gb Note NA. Do not react with each kind of glycoproteins including gb-gk. ELISPOT
Ⅰ 6 80 2 Tab. 2 Accuracy and precision of developed ELISA method HSV- antigen μg /ml Recovery ratio % CV % 2 95. 2-03. 3 3. 0 9. 2-02. 2 3. 8 0. 5 85. 6-07. 5. 7 HSV- 4 HSV- Fig. 4 Standard curve of internal reference of HSV- antigen 5 Fig. 5 Correlation analysis of two detection methods 2 2. 3. 2 0. 25 ~ 2 μg /ml R 2 = 0. 995 5 0. 25 μg /ml 4 POT F6 gb 2B 2. 3. 3 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- 8 9 0. 05 2. 3. 4 r = 0. 93 5 HSV- HSV- Anthony Orvedahl Diane Alexander Amgen T-Vec Zsolt Tallóczy et al HSV- ICP34. 5 Confers Neurovirulence by Targeting the Beclin Auto-
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