{"title":"白喉链杆菌外毒素广泛中和性单克隆抗体的抗原表位鉴定","authors":"Ya.S. Kulyk","doi":"10.15407/biotech15.04.037","DOIUrl":null,"url":null,"abstract":"Background. Better and high-potency vaccines against diphtheria are urgently needed to provide broader protection against diverse strains and subtypes. Identification of novel broadly neutralizing epitopes targeted by protective antibodies could aid in such efforts. Aim. In this study we focused on the search of binding sites identification of anti diphtheria toxin monoclonal antibodies and their neutralizing activity to block binding of recombinant exotoxin derivates with host receptors. Methods. Vero cells were cultured in the complete RPMI-1640 medium under standard conditions and used for flow cytometry assay. Recombinant antigens and products of tryptic hydrolysis of CRM197 and SbB were characterized by Ni2+-NTA affinity chromatography and SDS-PAGE under reducing conditions with following ECL Western-Blot using several hybridomas clones of anti-diphtheria toxin monoclonal antibodies. Results. ECL western blot film results for clone 9.1-E11 showed the specific binding both to whole CRM197 molecule, and to almost all fragments of CRM197 formed as a result of limited proteolysis. In particular, a band corresponding to SbB in molecular weight can be identified. Thus, epitope region of the CRM197 molecule specific to 9.1-E1 mAbs is located within the structure of SbB. At the same time 16.4-E9 clone antibodies had high specificity to R-domain of SbB. In addition, both hybridoma clones antibodies have neutralizing activity against the DT binding subunit, which is a key factor in blocking between cell receptor and it ligand, C.diphtheriae exotoxin. Conclusions. The results obtained indicate that obtained antibodies are prospective for improving new diagnostic tools and therapeutic agents, which are used for treatment and understanding of the molecular mechanisms of diphtheria pathogenesis.","PeriodicalId":9267,"journal":{"name":"Biotechnologia Acta","volume":" ","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2022-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"EPITOPES IDENTIFICATION OF BROADLY NEUTRALIZING MONOCLONAL ANTIBODIES AGAINST Corynebacterium diphtheriae EXOTOXIN\",\"authors\":\"Ya.S. Kulyk\",\"doi\":\"10.15407/biotech15.04.037\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Background. Better and high-potency vaccines against diphtheria are urgently needed to provide broader protection against diverse strains and subtypes. Identification of novel broadly neutralizing epitopes targeted by protective antibodies could aid in such efforts. Aim. In this study we focused on the search of binding sites identification of anti diphtheria toxin monoclonal antibodies and their neutralizing activity to block binding of recombinant exotoxin derivates with host receptors. Methods. Vero cells were cultured in the complete RPMI-1640 medium under standard conditions and used for flow cytometry assay. Recombinant antigens and products of tryptic hydrolysis of CRM197 and SbB were characterized by Ni2+-NTA affinity chromatography and SDS-PAGE under reducing conditions with following ECL Western-Blot using several hybridomas clones of anti-diphtheria toxin monoclonal antibodies. Results. ECL western blot film results for clone 9.1-E11 showed the specific binding both to whole CRM197 molecule, and to almost all fragments of CRM197 formed as a result of limited proteolysis. In particular, a band corresponding to SbB in molecular weight can be identified. Thus, epitope region of the CRM197 molecule specific to 9.1-E1 mAbs is located within the structure of SbB. At the same time 16.4-E9 clone antibodies had high specificity to R-domain of SbB. In addition, both hybridoma clones antibodies have neutralizing activity against the DT binding subunit, which is a key factor in blocking between cell receptor and it ligand, C.diphtheriae exotoxin. Conclusions. The results obtained indicate that obtained antibodies are prospective for improving new diagnostic tools and therapeutic agents, which are used for treatment and understanding of the molecular mechanisms of diphtheria pathogenesis.\",\"PeriodicalId\":9267,\"journal\":{\"name\":\"Biotechnologia Acta\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2022-08-31\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biotechnologia Acta\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.15407/biotech15.04.037\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnologia Acta","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.15407/biotech15.04.037","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
摘要
背景。迫切需要更好和高效的白喉疫苗,以提供针对不同毒株和亚型的更广泛保护。鉴定保护性抗体靶向的新的广泛中和表位可以帮助实现这一目标。的目标。在本研究中,我们主要研究了抗白喉毒素单克隆抗体的结合位点鉴定及其阻断重组外毒素衍生物与宿主受体结合的中和活性。方法。Vero细胞在标准条件下于完整的RPMI-1640培养基中培养,并用于流式细胞术检测。利用几种抗白喉毒素单克隆抗体杂交瘤克隆,采用Ni2+-NTA亲和层析和SDS-PAGE在还原条件下对CRM197和SbB的重组抗原和产物进行了表征。结果。克隆9.1-E11的ECL western blot结果显示,该蛋白既能与CRM197的整个分子结合,又能与由于有限蛋白水解而形成的几乎所有CRM197片段结合。特别是,可以识别出分子量与SbB相对应的条带。因此,CRM197分子对9.1-E1单克隆抗体特异性的表位区域位于SbB结构内。同时,16.4-E9克隆抗体对SbB的r结构域具有较高的特异性。此外,这两种杂交瘤克隆抗体对DT结合亚基具有中和活性,DT结合亚基是阻断细胞受体与其配体白喉白喉外毒素的关键因素。结论。结果表明,所获得的抗体有望开发新的诊断工具和治疗剂,用于治疗和了解白喉发病机制的分子机制。
EPITOPES IDENTIFICATION OF BROADLY NEUTRALIZING MONOCLONAL ANTIBODIES AGAINST Corynebacterium diphtheriae EXOTOXIN
Background. Better and high-potency vaccines against diphtheria are urgently needed to provide broader protection against diverse strains and subtypes. Identification of novel broadly neutralizing epitopes targeted by protective antibodies could aid in such efforts. Aim. In this study we focused on the search of binding sites identification of anti diphtheria toxin monoclonal antibodies and their neutralizing activity to block binding of recombinant exotoxin derivates with host receptors. Methods. Vero cells were cultured in the complete RPMI-1640 medium under standard conditions and used for flow cytometry assay. Recombinant antigens and products of tryptic hydrolysis of CRM197 and SbB were characterized by Ni2+-NTA affinity chromatography and SDS-PAGE under reducing conditions with following ECL Western-Blot using several hybridomas clones of anti-diphtheria toxin monoclonal antibodies. Results. ECL western blot film results for clone 9.1-E11 showed the specific binding both to whole CRM197 molecule, and to almost all fragments of CRM197 formed as a result of limited proteolysis. In particular, a band corresponding to SbB in molecular weight can be identified. Thus, epitope region of the CRM197 molecule specific to 9.1-E1 mAbs is located within the structure of SbB. At the same time 16.4-E9 clone antibodies had high specificity to R-domain of SbB. In addition, both hybridoma clones antibodies have neutralizing activity against the DT binding subunit, which is a key factor in blocking between cell receptor and it ligand, C.diphtheriae exotoxin. Conclusions. The results obtained indicate that obtained antibodies are prospective for improving new diagnostic tools and therapeutic agents, which are used for treatment and understanding of the molecular mechanisms of diphtheria pathogenesis.