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Production and characterization of monoclonal antibodies against grass carp CD4-1 and CD4-2

Aquaculture and Fisheries 2024年第2期9卷 154-163页

作者： Bangjie Wang Yuting Qin Zhaosheng Sun Xiaozhen Zhu Zhao Jia Kangyong Chen Danjie Liu Junya Wang Jun Zou Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources Ministry of EducationShanghai Ocean UniversityShanghai201306China International Research Center for Marine Biosciences at Shanghai Ocean University Ministry of Science and TechnologyShanghai201306China National Demonstration Center for Experimental Fisheries Science Education Shanghai Ocean UniversityShanghai201306China Laboratory for Marine Biology and Biotechnology Qingdao National Laboratory for Marine Science and TechnologyQingdao266071China

CD4 T helper cells are an important group of cells in the immune system of vertebrates and express CD4 receptor on the cell surface.In mammals,the CD4 receptor is encoded by a single copy gene,whilst in fish,two copies of cd4 genes,namely cd4-1 and cd4-2,are found.In this study,the ectodomains of grass carp(Ctenopharyngodon idella,Ci)CD4-1 and CD4-2 were expressed in the E.coli cells and used to generate monoclonal antibodies in mice.Western blotting,confocal microscopy and flow cytometry were performed to characterize the monoclonal antibodies.It has been shown that the CiCD4-1 and CiCD4-2 monoclonal antibodies had good specificity to react with the recombinant ectodomains of CiCD4-1 and CiCD4-2 expressed in the CHO-S cells and the native CD4 molecules of grass carp.The CiCD4-1 monoclonal antibody did not recognize CiCD4-2 and verse versa.In addition,the CD4-1 and CD4-2 monoclonal antibodies specifically recognized the CD4-1 and CD4-2 receptors expressed in the HEK293 cells and native molecules of fish cells.Further,the percentages of lymphocytes in immune tissues of healthy fish were analyzed by flow cytometry.It was found that 17.6%of lymphocytes were CD4-1^(+)cells and 22.5%were CD4-2^(+)in the head kidney.In the spleen,13.1%of lymphocytes were CD4-1^(+)and 18.6%were CD4-2^(+)while 7.3%of blood lymphocytes were CD4-1^(+)cells and 8.8%were CD4-2^(+)cells.The availability of the CD4-1 and CD4-2 monoclonal antibodies provides antibody-based tools for further elucidation of the functions of CD4 T cells in grass carp.

关键词： Grass carp CD4 monoclonal antibodies Flow cytometry Confocal microscopy

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Hepatitis B virus reactivation in patients treated with monoclonal antibodies

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World Journal of Virology 2024年第1期13卷 33-37页

作者： Silvia De Pauli Martina Grando Giovanni Miotti Marco Zeppieri Department of Internal Medicine Azienda Sanitaria Friuli OccidentaleSan Vito al TagliamentoPordenone 33170Italy Department of Plastic Surgery University Hospital of UdineUdine 33100Italy Department of Ophthalmology University Hospital of UdineUdine 33100Italy

Hepatitis B virus(HBV)reactivation poses a significant clinical challenge,espe-cially in patients undergoing immunosuppressive therapies,including mono-clonal antibody treatments.This manuscript briefly explores the complex rela-tionship between monoclonal antibody therapy and HBV reactivation,drawing upon current literature and clinical case studies.It delves into the mechanisms underlying this phenomenon,highlighting the importance of risk assessment,monitoring,and prophylactic measures for patients at risk.The manuscript aims to enhance the understanding of HBV reactivation in the context of monoclonal antibody therapy,ultimately facilitating informed clinical decision-making and improved patient care.This paper will also briefly review the definition of HBV activation,assess the risks of reactivation,especially in patients treated with monoclonal antibodies,and consider management for patients with regard to screening,prophylaxis,and treatment.A better understanding of patients at risk can help clinicians provide optimum management to ensure successful patient outcomes and prevent morbidity.

关键词： Hepatitis B virus Reactivation Acute infection Chronic infection monoclonal antibodies

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monoclonal antibodies-a proven and rapidly expanding therapeutic modality for human diseases

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Protein & Cell 2010年第4期1卷 319-330页

作者： Zhiqiang An Brown Foundation Institute of Molecular Medicine University of Texas Health Science Center at HoustonHoustonTX 77030USA

The study of antibodies has been a focal point in modern biology and medicine since the early 1900s.However,progress in therapeutic antibody development was slow and intermittent until recently.The first antibody therapy,murine-derived murononab OKT3 for acute organ rejection,was approved by the US Food and Drug Administration(FDA)in 1986,more than a decade after César Milstein and Georges Köhler developed methods for the isolation of mouse monoclonal antibodies from hybridoma cells in 1975.As a result of the scientific,technological,and clinical breakthroughs in the 1980s and 1990s,the pace of therapeutic antibody discovery and development accelerated.antibodies are becoming a major drug modality with more than two dozen therapeutic antibodies in the clinic and hundreds more in development.Despite the progress,need for improvement exists at every level.Antibody therapeutics provides fertile ground for protein scientists to fulfill the dream of personalized medicine through basic scientific discovery and technological innovation.

关键词： monoclonal antibodies personalized medicine therapeutic antibodies

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monoclonal antibodies to the exon 18 encoded moiety of NCAM

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Journal of Cancer Metastasis and Treatment 2019年第7期4卷 50-55页

作者： Ann Vander Borght Mieke Duysinx Monique Ummelen Bernard AMvan der Zeijst Department of Genetics and Cell Biology and GROW School for Oncology and Developmental Biology Maastricht UniversityMaastrichtPO Box 6166200 MDThe Netherlands MUbio Products B.V. Maastricht6200 EVThe Netherlands Catherinn B.V. Manuel de Fallapad 1Rotterdam3069 MRThe Netherlands

Aim: Exon 18 expression of NCAM has been recognized as a biomarker for small cell lung cancer (SCLC). To use this finding for an improved diagnosis of SCLC and personalized treatment of patients, techniques to identify and quantitate E18, the exon 18 encoded protein moiety of NCAM, are needed. We developed three monoclonal antibodies for this purpose. Methods: The his-tagged E18 antigen was expressed in E. coli and, after purification, used to immunize mice. Hybridoma's were isolated by standard procedures and tested for their reaction with E18. Results: Three monoclonal antibodies, MUM-1, MUM-4 and MUM-6 were obtained. They reacted with E18 in western blots, with SCLC cell line NCI-H82, but not with unrelated his-tagged proteins. Only permeabilized NCI-H82 cells stained with the antibodies, confirming the intracellular position of E18. Next an enzyme-linked immunosorbent assay was developed using the earlier isolated monoclonal antibody MUMi-21B2, coated on the surface of microtiter wells as capture antibody and biotinylated MUM-6 as second antibody. Using streptavidin conjugated to horse radish peroxidase a linear dose response curve to his-tagged E18 antigen was obtained between 0 and 5 μg/mL with a sensitivity of at least 0.5 μg/mL or 50 ng/well. Conclusion: Four monoclonal antibodies are available to be used in assays for the identification and quantification of SCLC biomarker E18. This will enable the development of liquid biopsies to follow the tumor load in patients.

关键词： Small cell lung cancer tumor biomarker monoclonal antibodies NCAM exon 18

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Preparation of monoclonal antibodies Against Prion Proteins With Full-length Hamster PrP

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Biomedical and Environmental Sciences 2005年第4期18卷 273-280页

作者： XIN-LI XIAO HUI-YING JIANG JIN ZHANG JUN HAN KAI NIE XIAO-BO ZHOU YIN-XIA HUANG LAN CHEN WEI ZHOU BAO-YUN ZHANG YONG LIU XIAO-PING DONG School of Medicine Xi＇an Jiao-Tong University Zhu-Que Street 205 Xi＇an 710061 Shaanxi China National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Hngxin Rd. 100 Beijing 100052 China National Laboratory of Medical Molecular Biology Institute of Basic Medical Science Chinese Academy of Medical Sciences and Peking Union Medical College Dong Dan San Tiao 5 Beijing 100005 China

To prepare the PrP specific monoclonal antibodies （mAbs） that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases. Methods Several BALB/c mice were immunized with recombinant hamster prion protein （HaPrP）. Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry. Results The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrP^Sc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPc from several other mammalian species, including humans and cattles, lmmunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPso. Conclusion The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.

关键词： Prions Hamster prion protein monoclonal antibodies

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Comparing different domains of analysis for the characterisation of N-glycans on monoclonal antibodies

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Journal of Pharmaceutical Analysis 2020年第1期10卷 23-34页

作者： Sara Carillo Raquel Peerez-Robles Craig Jakes Meire Ribeiro da Silva Silvia Millan Martín Amy Farrell Natalia Navas Jonathan Bones Characterisation and Comparability Laboratory NIBRT e the National Institute for Bioprocessing Research and TrainingFoster AvenueMount MerrionBlackrockCo.DublinA94 X099Ireland Department of Analytical Chemistry Biohealth Research Institute(ibs.GRANADA)University of GranadaGranadaSpain School of Chemical and Bioprocess Engineering University College DublinBelfieldDublin 4D04 V1W8Ireland

With the size of the biopharmaceutical market exponentially increasing,there is an aligned growth in the importance of data-rich analyses,not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships.In monoclonal antibodies,many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known.The importance of their function focuses analytical research efforts on the development of robust,accurate and fast methods to support drug development and quality control.Released N-glycan analysis is considered as the gold standard for glycosylation characterisation;however,it is not the only method for quantitative analysis of glycoform heterogeneity.In this study,ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies.While observing good comparability between the quantitative results generated,it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow according to application and the desired depth of data generated.

关键词： N-Glycans Biopharmaceuticals monoclonal antibodies Intact mass analysis Mass spectrometry Native mass spectrometry Glycan analysis Peptide mapping Glycopeptide analysis

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Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies

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Asian Journal of Andrology 2016年第1期18卷 108-113页

作者： Jana Capkova Alena Kubatova Lukas Ded Olina Tepla Jana Peknicova Laboratory of Reproductive Biology Institute of Biotechnology Academy of Sciences of the Czech Republic v. v. i. Prague Czech Republic Clinic Center IVF ISCARE Prague Czech Republic

Recent studies have shown that infertility affects estimated 15% of all couples. Male infertility is the primary or contributory cause in 60% of these cases. Consequently, the application of assisted reproduction is increasing. These methods could benefit from an extended evaluation of sperm quality. For this reason, we analyzed sperm proteins from 30 men with normal spermiograms and 30 men with asthenozoospermia. Ejaculates of both groups were tested by flow cytometry （FCM） and fluorescence with a set of well-characterized anti-human sperm Hs-monoclonal antibodies （MoAbs）, which were generated in our laboratory. No statistically significant differences were found between normospermics and asthenospermics in the expression of the sperm surface protein clusterin, evaluated with Hs-3 MoAb, and semenogelin, evaluated with Hs-9 MoAb. However, FCM revealed quantitative differences in the acrosomal proteins between normozoospermic and asthenozoospermic men, namely, in glyceraldehyde-3-phosphate dehydrogenase, evaluated with Hs-8 MoAb, valosin-containing protein, evaluated with Hs-14 MoAb, and ATP synthase （cAMP-dependent protein kinase II, PRKAR2A）, evaluated with MoAb Hs-36. Asthenozoospermic men displayed a highly reduced expression of intra-acrosomal proteins, with a likely decrease in sperm quality, and thus a negative impact on successful reproduction. Asthenozoospermia seems to be a complex disorder involving intra-acrosomal proteins.

关键词： asthenozoospermia flow cytometry fluorescence microscopy monoclonal antibodies sperm proteins

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Novel P22-monoclonal antibody based blocking ELISA for the detection of African swine fever virus antibodies in serum

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Biosafety and Health 2022年第4期4卷 234-243页

作者： Ghebremedhin Tsegay Weldu Tesfagaber Yuanmao Zhu Xijun He Wan Wang Zhenjiang Zhang Encheng Sun Jinya Zhang Yuntao Guan Fang Li Renqiang Liu Zhigao Bu Dongming Zhao State Key Laboratory of Veterinary Biotechnology National High Containment Facilities for Animal Diseases Control and PreventionNational African Swine Fever Para-reference LaboratoryHarbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069China Department of Veterinary Science Hamelmalo Agricultural CollegeKerenEritrea

African swine fever(ASF)is a highly infectious,transboundary viral disease of domestic and wild pigs,and is currently the most serious threat to world swine production,resulting in significant economic loss.In the absence of vaccines and treatments,the control of the disease entirely depends on accurate and early diagnosis accompanied by the culling of infected pigs.Thus,a highly specific and sensitive diagnostic assay is required during an outbreak and surveillance of the disease.In this study,a highly sensitive,specific,rapid and repeatable P22-monoclonal antibody-based blocking enzyme-linked immunosorbent assay(bELISA)assay was developed for the detection of antibodies against genotype I and II African swine fever viruses(ASFVs).A total of 806 pig serum samples were tested to evaluate the performance of the diagnostic assay.To determine the PI(percent Inhibition)cut-off value,receiver-operating characteristic(ROC)analysis was applied.According to the ROC analysis of the data,98.10%specificity and 100%sensitivity were recorded when the threshold cut-off value of PI was established at 47%.In addition,the assay was able to detect ASFV antibodies as early as 9 days post-infection when serum samples from experimentally infected pigs were used.Taking all together,the results of the present study indicated that the P22-mAb based bELISA assay can be used for rapid and accurate detection of antibodies against ASFV,which could play a valuable role in the containment and prevention of ASFV as an alternative to other serological diagnostic methods.Also,this study will assist researchers to further investigate the immunogenic importance of P22 protein in ASFV infection.

关键词： monoclonal antibodies African swine fever Blocking ELISA Diagnosis P22

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Pharmacogenetics of anticancer monoclonal antibodies

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Cancer Drug Resistance 2019年第1期2卷 69-81页

作者： Dmitrii Shek Scott ARead Golo Ahlenstiel Irina Piatkov Blacktown Clinical School Western Sydney UniversityBlacktownNSW 2148Australia Storr Liver Centre The Westmead Institute for Medical ResearchThe University of SydneyWestmeadNSW 2145Australia Blacktown Hospital BlacktownNSW 2148Australia

Pharmacogenetics is the study of therapeutic and adverse responses to drugs based on an individual’s genetic background.monoclonal antibodies(mAbs)are a rapidly evolving field in cancer therapy,however a number of newly developed and highly effective mAbs(e.g.,anti-CTLA-4 and anti-PD-1)possess pharmacogenomic profiles that remain largely undefined.Since the first chemotherapeutic mAb Rituximab was approved in 1997 by the US Food and Drug Administration for cancer treatment,a broad number of other mAbs have been successfully developed and implemented into oncological practice.Nowadays,mAbs are considered as one of the most promising new approaches for cancer treatment.The efficacy of mAb treatment can however be significantly affected by genetic background,where genes responsible for antibody presentation and metabolism,for example,can seriously affect patient outcome.This review will focus on current anticancer mAb treatments,patient genetics that shape their efficacy,and the molecular pathways that bridge the two.

关键词： Pharmacogenetics pharmacogenomics immune-checkpoint proteins monoclonal antibodies cancer immunotherapy personalized medicine

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In vivo neutralization assays by monoclonal antibodies against white spot syndrome virus in crayfish (Cambarus proclarkii)

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Acta Oceanologica Sinica 2008年第2期27卷 126-132页

作者： WANG Yinan ZHAN Wenbin XING Jing JIANG Yousheng Laboratory of Pathology and Immunology of Aquatic Animals Laboratory of Maricuhure of Ministry of Education Ocean University of China Qingdao 266003 China

The neutralizing activities of eight monoclonal antibodies （MAbs） against white spot syndrome virus （WSSV）（2D2, 2B2,1D2, 1D5, 1C2, 4A1, 6A4 and 6B4） were analyzed by in vivo experiments. Gills from WSSV-infected shrimp were homogenized and ten-fold serially diluted by PBS, and then incubated with MAbs （hybridoma culture supernatant）, respectively. The mixture of WSSV and MAbs were injected into crayfish （Procambarus clarkii）. After challenge, the death rates of crayfish were counted to determine the neutralizing activities of MAbs. At the same time, the mixture of myeloma culture supernatant and WSSV or PBS was served as positive or negative control, respectively. The results showed that, at each virus dilution, the mean time to death of the crayfish injected with MAb-treated virus was significantly longer than that in the positive control, though they all showed 100% mortality within 25 d, and meanwhile, few crayfish died in the negative control. Among the eight MAbs, 2D2, 2B2, 1D2 and 1D5, especially the former two, delayed the mortality significantly, and 1 C2, 4A1 and 6A4 delayed the mortality as well but not so efficiently, while MAb 6IM was efficient only when the virus concentration increased. The results indicated that the anti-WSSV MAbs can neutralize WSSV in different virus dilutions.

关键词： crayfish WSSV monoclonal antibodies in vivo neutralization

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