{"title":"马 IL-1β 的单克隆抗体可定量检测马体内成熟的 IL-1β。","authors":"Susanna Babasyan, Alicia Rollins, Bettina Wagner","doi":"10.1016/j.vetimm.2024.110805","DOIUrl":null,"url":null,"abstract":"<div><p>Interleukin-1β (IL-1β) is one of the key mediators of inflammation during innate immune responses. Mature bioactive IL-1β mediates essential host defense mechanisms but also has a mechanistic role in several autoinflammatory and degenerative diseases. In horses, specific and sensitive assays for IL-1β are crucial for immunological research on inflammatory processes and diseases. In this article, we describe the development of four monoclonal antibodies (mAbs) against equine IL-1β. The specificity of the new IL-1β mAbs was confirmed using a panel of equine recombinant cytokines and chemokines. The mAbs were validated for detection of native mature IL-1β in a fluorescent bead-based assay and for staining of IL-1β-producing immune cells by flow cytometry. The bead-based assay for equine IL-1β had a linear quantification range between 60<!--> <!-->pg/ml to 960<!--> <!-->ng/ml. Horse peripheral blood mononuclear cells (PBMC) secreted IL-1β after lipopolysaccharide (LPS) stimulation in time and dose dependent manner as quantified by the new equine IL-1β bead-based assay. A comparison of two commercial equine IL-1β ELISA kits with the new IL-1β fluorescent bead-based assay revealed that the bead-based assay improved the quantification of native equine IL-1β in LPS stimulated PBMC supernatants by detecting it with high intensity and a broad linear quantification range, while both ELISAs resulted in low signals and poor native IL-1β recognition. Intracellular staining and flow cytometric analysis confirmed that the main cellular source of IL-1β in equine PBMC after LPS stimulation were CD14<sup>+</sup> monocytes. IL-1β secretion from PBMC was inhibited by a caspase inhibitor but protein translation within the cells was not, supporting the accumulation of pro-IL-1β within the cells even when proteolytic cleavage for IL-1β activation is missing. This confirmed the importance of specific mAbs for analyzing the biologically active, mature IL-1β in horses.</p></div>","PeriodicalId":23511,"journal":{"name":"Veterinary immunology and immunopathology","volume":"274 ","pages":"Article 110805"},"PeriodicalIF":1.4000,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0165242724000916/pdfft?md5=f91443a7738b5cf6e23890889a1aae98&pid=1-s2.0-S0165242724000916-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Monoclonal antibodies for equine IL-1β enable the quantification of mature IL-1β in horses\",\"authors\":\"Susanna Babasyan, Alicia Rollins, Bettina Wagner\",\"doi\":\"10.1016/j.vetimm.2024.110805\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Interleukin-1β (IL-1β) is one of the key mediators of inflammation during innate immune responses. Mature bioactive IL-1β mediates essential host defense mechanisms but also has a mechanistic role in several autoinflammatory and degenerative diseases. In horses, specific and sensitive assays for IL-1β are crucial for immunological research on inflammatory processes and diseases. In this article, we describe the development of four monoclonal antibodies (mAbs) against equine IL-1β. The specificity of the new IL-1β mAbs was confirmed using a panel of equine recombinant cytokines and chemokines. The mAbs were validated for detection of native mature IL-1β in a fluorescent bead-based assay and for staining of IL-1β-producing immune cells by flow cytometry. The bead-based assay for equine IL-1β had a linear quantification range between 60<!--> <!-->pg/ml to 960<!--> <!-->ng/ml. Horse peripheral blood mononuclear cells (PBMC) secreted IL-1β after lipopolysaccharide (LPS) stimulation in time and dose dependent manner as quantified by the new equine IL-1β bead-based assay. A comparison of two commercial equine IL-1β ELISA kits with the new IL-1β fluorescent bead-based assay revealed that the bead-based assay improved the quantification of native equine IL-1β in LPS stimulated PBMC supernatants by detecting it with high intensity and a broad linear quantification range, while both ELISAs resulted in low signals and poor native IL-1β recognition. Intracellular staining and flow cytometric analysis confirmed that the main cellular source of IL-1β in equine PBMC after LPS stimulation were CD14<sup>+</sup> monocytes. IL-1β secretion from PBMC was inhibited by a caspase inhibitor but protein translation within the cells was not, supporting the accumulation of pro-IL-1β within the cells even when proteolytic cleavage for IL-1β activation is missing. This confirmed the importance of specific mAbs for analyzing the biologically active, mature IL-1β in horses.</p></div>\",\"PeriodicalId\":23511,\"journal\":{\"name\":\"Veterinary immunology and immunopathology\",\"volume\":\"274 \",\"pages\":\"Article 110805\"},\"PeriodicalIF\":1.4000,\"publicationDate\":\"2024-07-03\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S0165242724000916/pdfft?md5=f91443a7738b5cf6e23890889a1aae98&pid=1-s2.0-S0165242724000916-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Veterinary immunology and immunopathology\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0165242724000916\",\"RegionNum\":3,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"IMMUNOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Veterinary immunology and immunopathology","FirstCategoryId":"97","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0165242724000916","RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"IMMUNOLOGY","Score":null,"Total":0}
Monoclonal antibodies for equine IL-1β enable the quantification of mature IL-1β in horses
Interleukin-1β (IL-1β) is one of the key mediators of inflammation during innate immune responses. Mature bioactive IL-1β mediates essential host defense mechanisms but also has a mechanistic role in several autoinflammatory and degenerative diseases. In horses, specific and sensitive assays for IL-1β are crucial for immunological research on inflammatory processes and diseases. In this article, we describe the development of four monoclonal antibodies (mAbs) against equine IL-1β. The specificity of the new IL-1β mAbs was confirmed using a panel of equine recombinant cytokines and chemokines. The mAbs were validated for detection of native mature IL-1β in a fluorescent bead-based assay and for staining of IL-1β-producing immune cells by flow cytometry. The bead-based assay for equine IL-1β had a linear quantification range between 60 pg/ml to 960 ng/ml. Horse peripheral blood mononuclear cells (PBMC) secreted IL-1β after lipopolysaccharide (LPS) stimulation in time and dose dependent manner as quantified by the new equine IL-1β bead-based assay. A comparison of two commercial equine IL-1β ELISA kits with the new IL-1β fluorescent bead-based assay revealed that the bead-based assay improved the quantification of native equine IL-1β in LPS stimulated PBMC supernatants by detecting it with high intensity and a broad linear quantification range, while both ELISAs resulted in low signals and poor native IL-1β recognition. Intracellular staining and flow cytometric analysis confirmed that the main cellular source of IL-1β in equine PBMC after LPS stimulation were CD14+ monocytes. IL-1β secretion from PBMC was inhibited by a caspase inhibitor but protein translation within the cells was not, supporting the accumulation of pro-IL-1β within the cells even when proteolytic cleavage for IL-1β activation is missing. This confirmed the importance of specific mAbs for analyzing the biologically active, mature IL-1β in horses.
期刊介绍:
The journal reports basic, comparative and clinical immunology as they pertain to the animal species designated here: livestock, poultry, and fish species that are major food animals and companion animals such as cats, dogs, horses and camels, and wildlife species that act as reservoirs for food, companion or human infectious diseases, or as models for human disease.
Rodent models of infectious diseases that are of importance in the animal species indicated above,when the disease requires a level of containment that is not readily available for larger animal experimentation (ABSL3), will be considered. Papers on rabbits, lizards, guinea pigs, badgers, armadillos, elephants, antelope, and buffalo will be reviewed if the research advances our fundamental understanding of immunology, or if they act as a reservoir of infectious disease for the primary animal species designated above, or for humans. Manuscripts employing other species will be reviewed if justified as fitting into the categories above.
The following topics are appropriate: biology of cells and mechanisms of the immune system, immunochemistry, immunodeficiencies, immunodiagnosis, immunogenetics, immunopathology, immunology of infectious disease and tumors, immunoprophylaxis including vaccine development and delivery, immunological aspects of pregnancy including passive immunity, autoimmuity, neuroimmunology, and transplanatation immunology. Manuscripts that describe new genes and development of tools such as monoclonal antibodies are also of interest when part of a larger biological study. Studies employing extracts or constituents (plant extracts, feed additives or microbiome) must be sufficiently defined to be reproduced in other laboratories and also provide evidence for possible mechanisms and not simply show an effect on the immune system.