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Preparation of single chain variable fragment of MG_7 mAb by phage display technology

World Journal of Gastroenterology 2001年第4期7卷 510-514页

作者： Zhao-Cai Yu Jie Ding Yong-Zhan Nie Dai-Ming Fan Xue-Yong Zhang Department of Gastroenterology,Xijing Hospital,Fourth Military Medical University,Xi’an 710032,Shaanxi Province,China Department of Gastroenterology Xijing Hospital Fourth Military Medical University Xi'an 710032 Shaanxi Province China.

AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produc

关键词： Peptide Library Animals Antibodies, Monoclonal Antibody Specificity Cell Line enzyme-linked immunosorbent assay Gene Therapy Genetic Screening Humans Hybridomas Immunoglobulin Variable Region Mice Recombination, Genetic Stomach Neoplasms

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Levels of v5 and v6 CD44 splice variants in serum of patients with colorectal cancer are not correlated with pT stage,histopathological grade of malignancy and clinical features

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World Journal of Gastroenterology 2004年第4期10卷 583-585页

作者： Bogdan Zalewski 2nd Department of General and Gastroenterological Surgery Medical University of BialystokPoland

AIM:This study was designed to compare the levels of v5 and v6 splice variants of CD44 evaluated using EITSA test in the serum of patients with colorectal cancer in different stages of progression of the disease estimated in pT stage according to WHO score,histopathological grade of malignancy and some clinicopathological features. METHODS:The serum obtained from 114 persons with colorectal adenocarcinomas was examined using ELISA method,pT stage and grade of malignancy of the tumour were examined in formalin fixed and paraffin embedded materials obtained during operation. RESULTS:Only the level of CD44 v5 in the serum of patients before operation with G2 pT4 tumour was lower than that in other probes and the difference was statistically significant. We did not find any other correlations between the level of v5 and v6 CD44 variants and other evaluated parameters. CONCLUSION:The level of CD44 v5 and v6 estimated by ELISA test in the serum can not be used as a prognostic factor in colorectal cancer.

关键词： Alternative Splicing Adenocarcinoma Adult Aged Aged, 80 and over Antigens, CD44 Colorectal Neoplasms Disease Progression enzyme-linked immunosorbent assay Female Humans Male Middle Aged Predictive Value of Tests Prognosis Tumor Markers, Biological

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Evaluation of the role of H pylori infection in pathogenesis of gastric cancer by immunoblot assay

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World Journal of Gastroenterology 2006年第43期12卷 7029-7032页

作者： Kuo-Ching Yang Alexander Chu Chao-Sheng Liao Yu-Min Lin Gen-Min Wang Division of Gastroenterology Department of Internal Medicine Shin Kong Wu Ho-Su MemorialHospitalTaipei Taiwan China Taipei Medical University Taipei TaiwanChina

AIM： To elucidate the different serological reactions to H pylori using the immunoblotting technique for further understanding of its pathogenic role in gastric cancer. METHODS： A total of 54 patients were divided into two groups after upper gastrointestinal endoscopy： normal control group （25 patients） and gastric cancer group （29 patients）. Both groups were further divided into Hpylori （＋） and H pylori （-） subgroups based on the results of CLO test, Giemsa staining and culture. Sera were further analyzed with the immunoblotting technique （HelicoBIot 2.0, Genelabs Diagnostics, Singapore）. RESULTS： The positive rate of the immunoblotting test was as high as 88.9% in the H pylori （-） gastric cancer group and only 14.3% in the H pylori （-） normal control group with a statistically significant difference. CONCLUSION： The prevalence of H pylori infection is higher in gastric cancer patients than in the normal controls, suggesting that H pylori may play a role in the pathogenesis of gastric cancer.

关键词： Western blot Immunoblotting Gastric cancer Hpylori enzyme-linked immunosorbent assay

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Interaction of hepatitis C virus envelope glycoprotein E2 with the large extracellular loop of tupaia CD81

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World Journal of Gastroenterology 2009年第2期15卷 240-244页

作者： Zhan-Fei Tian Hong Shen Xi-Hua Fu Yi-Chun Chen Hubert E Blum Thomas F Baumert Xi-Ping Zhao Department of Infectious Diseases 161 Hospital of PLA Wuhan 430030 Hubei Province China Department of Infectious Diseases Tongji Hospital Tongji Medical College Huazhong Science & Technology University Wuhan 430030 Hubei Province China Department of Medicine II University of Freiburg D-79106 Freiburg Germany Inserm U748 Universit6 Louis Pasteur Service d'Hepatogastroenterologie 3 Rue Koeberle F-67000 Strasbourg France

AIM: To further analyze the interaction of tupaia CD81 with hepatitis C virus (HCV) envelope protein E2. METHODS: A tupaia CD81 large extracellular loop (CD81 LEL), which binds to HCV E2 protein, was cloned and expressed as a GST-fusion protein, and interaction of HCV E2 protein with a tupaia CD81 LEL was evaluated by enzyme-linked immunosorbent assay (EIA). RESULTS: Although tupaia and human CD81 LEL differed in 6 amino acid changes, tupaia CD81 LEL was strongly recognized by anti-CD81 antibodies against human CD81 LEL conformation-dependent epitopes. Investigating LEL CD81-E2 interactions by EIA, we demonstrated that binding of tupaia CD81 LEL GST fusion protein to recombinant HCV E2 protein was markedly reduced compared to binding of human CD81 LEL GST fusion protein to recombinant HCV E2 protein. CONCLUSION: These data suggest that the structural differences in-between the tupaia and human CD81 may alter the interaction of the large extracellular loop with HCV envelope glycoprotein E2. These findings may be important for the understanding of the mechanisms of binding and entry of HCV to PTHs.

关键词： Hepatitis C virus E2 protein Tupaia CD81,Bind enzyme-linked immunosorbent assay

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A comparative study on serologic profiles of virus hepatitis B

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World Journal of Gastroenterology 2001年第1期7卷 107-110页

作者： BoYOUl Cho Moran Ki Hung Bae Park Department of Preventive Medicine Hanyang University College of Medicine Seoul 133791 Korea Department of Preventive Medicine Eulji University School of Medicine Taejon 301110 Korea

INTRODUCTIONHepatitis B viral infection, one of the most-prevalent liver disorders in China and Korea, is aserious infectious disease as it has the potential ofprogressing into liver cirrhosis and primary hepaticcarcinoma. China and Korea both belong to high-risk endemic regions of viral hepatitis[1]. TheHBsAg positive rates in China ranged from 6.9% -17.9% by age, race and test methods[2-5].

关键词： Adult Age Distribution Antibodies, Viral China Comparative Study enzyme-linked immunosorbent assay Ethnic Groups Female Hepatitis B Hepatitis B Surface Antigens Hepatitis B virus Humans Korea Male Middle Aged Research Support, Non-U.S. Gov't Seroepidemiologic Studies Sex Distribution

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FK506 effects on proliferation and secretion of Schwann cells

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Neural Regeneration Research 2011年第1期6卷 29-33页

作者： Hua jun Jiang Wei Qu Feng Han Wei guo Zhang De cheng Lu Department of Orthopaedics First Affiliated Hospital Dalian Medical University Dalian 116011 Liaoning Province China

The immunosuppressive agent FK506 contributes to neural regeneration via growth cone and Schwann cell processes. However, it is difficult to rapidly harvest a large number of Schwann cells, and Schwann cell senescence is obvious following culture, proliferation, and amplification. The present study investigated the effects of various concentrations of FK506 on proliferation and secretion of cultured Schwann cells. In addition, the suitability of FK506 application as tissue engineered artificial nerves was considered. Bilateral sciatic nerves of 3-5-day old, Sprague Dawley rats were obtained, and primary cultured Schwann cells were identified by S-100 protein staining. Following subculture, Schwann cells purity was 〉 90%. After 3-5 days in culture, Schwann cell proliferation was visible in each group. Results verified that FK506 promoted proliferation of cultured Schwann cells and secretion of nerve growth factor. This effect was significant at a low concentration （1 × 10.8 mol/L）.

关键词： Schwann cells nerve growth factor MTT enzyme-linked immunosorbent assay FK506

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Von Willebrand Factor Antigen and ADAMTS13 Activity assay in Pregnant Women and Severe Preeclamptic Patients

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2010年第6期30卷 777-780页

作者：张丹丹肖娟黄浩梁陈娟娟刘涛尹宗智高单萍刘琼艾继辉陈素华 Department of Obstetrics and Gynecology Tongji HospitalTongji Medical CollegeHuazhong University of Science and Technology Department of Obstetrics and Gynecology the First Hospital of Wuhan

The present study examined von Willebrand factor (vWF) levels and ADAMTS13 activity in pregnant and severe preeclamptic women in order to shed light on the prothrombotic state in severe *** healthy women of childbearing age,22 second trimester pregnant women,30 third trimester pregnant women and 10 severe preeclamptic patients were recruited in this ***13 activity was determined by the FRETS-vWF73 assay and vWF antigen (vWF:Ag) levels by an enzyme-linked immunosorbent *** results showed that there were statistically significant differences in plasma vWF antigen levels between the severe preeclamptic and third trimester pregnant women,between third and second trimester pregnant women (P0.05).In conclusion,plasma ADAMTS13 activity is normal in severe preeclampsia despite the increased vWF:Ag *** state is involved in the pathogenesis of severe preeclampsia,as a result of endothelial injury.

关键词： von Willebrand factor ADAMTS13 enzyme-linked immunosorbent assay fluorescence resonance energy transfer

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Measurement of Serum IgG4 Levels by an Established ELISA System and Its Clinical Applications in Autoimmune Diseases

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2013年第4期33卷 611-614页

作者：孙玮高荣芬陈雨苏玉莹董凌莉 Department of Stomatology Union HospitalHuazhong University of Science and Technology Department of Rheumatology Tongji HospitalHuazhong University of Science and Technology

Summary： IgG4-related disease （IgG4-RD） is a novel and rare autoimmune disease entity. Elevated serum IgG4 level is strongly suggestive of IgG4-RD. But it is still unknown whether serum IgG4 eleva tion commonly occurs in other autoimmune diseases. In this study, the serum IgG4 levels were detected by an established enzyme-linked immunosorbent assay （ELISA） in a variety of autoimmune diseases including systemic lupus erythematosus （SLE）, Sjogren＇s syndrome （SS）, polymyositis or dermatomy- ositis （PM/DM） and IgG4-RD. To evaluate the reliability of this ELISA system, some of our samples were sent to a lab in Kanazawa Medical University, Japan, and detected by using the nephelometric as-say. The results showed that our findings were consistent with theirs. Moreover, it was found that the serum IgG4 levels were 0.23±0.16 g/L in 53 healthy controls, 0.16±0.15 g/L in 103 SLE patients, 0.22±0.18 g/L in 41 SS patients and 0.40±0.32 g/L in 21 PM/DM patients. No significant difference in the serum IgG4 level was observed among these groups （P〉0.05）. The serum IgG4 levels of two cases of IgG4-RD were 1.63 and 4.65 g/L respectively, and both decreased markedly after treatment with glucocorticoids. These data indicated that this established ELISA system can be used for detecting serum IgG4 levels. Elevated serum IgG4 levels help diagnose IgG4-RD and evaluate the curative effect of this condition rather than other autoimmune diseases.

关键词： IgG4-related disease IgG4 enzyme-linked immunosorbent assay autoimmune disease

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Preparation and Application of Monoclonal Antibodies Specific for Salicylic Acid

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Acta Botanica Sinica 2001年第11期43卷 1207-1210页

作者：王树才李国婧夏凯徐朗莱陈溥言周燮南京农业大学农学院南京210095 南京农业大学理学院南京210095 南京农业大学动物医学院南京210095

Salicylic acid (SA) is widely distributed in many monocots and dicots and has many physiological effects. It can induce heat production in the thermogenic inflorescences of Arum lily([1]), block the biosynthesis of ethylene, and more attractively, it seems to be an important natural signal molecule in the induction of systemic acquired resistance (SAR) in tobacco, cucumber and other plants([2,3]). Studies in recent years showed that SA was also intimately related to the resistance of plants to aboitic stress, for example, SA increased chilling resistance of maize seedlings. Hence, SA has been accepted as a kind of new plant hormones. Up to date, the quantification of SA usually has been performed by HPLC[4,5], which often needs a large quantity of sample and a verbose pretreatment. Compared to HPLC, immunoassays, including radio-immunoassays (RIA) and enzyme-immunoassays (EIA), are easy to perform and have been widely used in the quantification of other plant hormones, such as IAA([6]), ABA([7,8]), GAs([9]), cytokinins et al([10]), and jasmonic acid (JA)([11]), and other low-molecular-weight, none-immunogenic compounds in plants([12]). Till now, only an indirect enzyme-linked immunosorbent assay (ELISA) for SA based on polyclonal antibodies (PAbs) has been developed by our group([13]), although Bennett et al([14]) had prepared SA PAbs using 4-aminosalicylic acid linked to KLH as immunogen in goat. However, the sensitivity of the ELISA we established formerly was relatively low, and also relatively larger quantity of sample is needed than other ELISAs for plant hormones. In this paper, an ELISA for SA based on monoclonal antibody raised against SA-NH-CH2-NH-KLH was introduced, and the fluctuation of SA content in cucumber leaves after inoculated with Pseudomonas syringae pv. syringae was determined.

关键词： salicylic acid monoclonal antibody enzyme-linked immunosorbent assay Cucumis sativus

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Production and Characterization of Monoclonal Antibodies to Bluetongue Virus

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Virologica Sinica 2011年第1期26卷 8-18页

作者： Veerakyathappa Bhanuprakash Madhusudhan Hosamani Vinayagamurthy Balamurugan Pradeep Narayan Gandhale Gnanavel Venkatesan Raj Kumar Singh Division of Virology Indian Veterinary Research Institute Indian Veterinary Research Institute Project Directorate on Animal Disease Monitoring And Surveillance National Research Centre on Equines and Veterinary Type Culture Centre

In the present study,a total of 24 MAbs were produced against bluetongue virus (BTV) by polyethyleneglycol (PEG) mediated fusion method using sensitized lymphocytes and myeloma cells. All these clones were characterized for their reactivity to whole virus and recombinant BTV-VP7 protein,titres,isotypes and their reactivity with 24 BTV-serotype specific sera in cELISA. Out of 24 clones,a majority of them (n = 18) belong to various IgG subclasses and the remaining (n = 6) to the IgM class. A panel of eight clones reactive to both whole BTV and purified rVP7 protein were identified based on their reactivity in iELISA. For competitive ELISA,the clone designated as 4A10 showed better inhibition to hyperimmune serum of BTV serotype 23. However,this clone showed a variable percent of inhibition ranging from16.6% with BTV 12 serotype to 78.9% with BTV16 serotype using 24 serotype specific sera of BTV originating from guinea pig at their lowest dilutions. From the available panel of clones,only 4A10 was found to have a possible diagnostic application.

关键词： Bluetongue virus Competitive ELISA enzyme-linked immunosorbent assay Monoclonal antibody India.

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