Production of recombinant electron transfer flavoprotein beta subunit protein and its application in a lateral flow assay for early diagnosis of leptospirosis.

IF 5.5 3区 医学 Q1 IMMUNOLOGY
Uraiwan Kositanont, Charin Thawornkuno, Athisri Sitthipunya, Worawan Dachavichitlead, Chanwit Tribuddharat, Suppalak Brameld, Galayanee Doungchawee, Benchaporn Lertanantawong, Chatchawan Srisawat
{"title":"Production of recombinant electron transfer flavoprotein beta subunit protein and its application in a lateral flow assay for early diagnosis of leptospirosis.","authors":"Uraiwan Kositanont, Charin Thawornkuno, Athisri Sitthipunya, Worawan Dachavichitlead, Chanwit Tribuddharat, Suppalak Brameld, Galayanee Doungchawee, Benchaporn Lertanantawong, Chatchawan Srisawat","doi":"10.1007/s00430-025-00822-6","DOIUrl":null,"url":null,"abstract":"<p><p>Leptospirosis is a major cause of acute febrile illness, often presenting with non-specific symptoms that can lead to misdiagnosis. Early laboratory diagnosis is essential for confirmation to avoid misdiagnosis and ensure appropriate management. This study aimed to identify and produce a recombinant protein, approximately 25 kDa, with high antigenicity for diagnostic applications. The 25 kDa protein from Leptospira interrogans was identified as electron transfer flavoprotein beta subunits (Etfβ) and exhibited 98% nucleotide and 99% amino acid homology to the reference strain. Lateral flow assays (LFAs) using recombinant Etfβ (rEtfβ) as antigens were developed to detect specific antibodies, namely rEtfβ-IgM and rEtfβ-IgG, and evaluated their performance against the standard microscopic agglutination test (MAT). Testing 33 paired serum samples from confirmed leptospirosis cases and 24 controls revealed sensitivities of 69.7% for IgM and 57.6% for IgG. However, the combined assays yielded enhanced diagnostic accuracy, achieving a sensitivity of 94.0%, specificity of 95.8%, positive predictive value of 96.9%, negative predictive value of 92.0%, and percent agreement of 94.7% (kappa value of 0.89). Also, the combined LFAs demonstrated 66.7% in initial serum samples whose MAT results were negative, enhancing the capacity for early diagnosis. In conclusion, the developed rapid tests demonstrated strong diagnostic capability, particularly in early-phase leptospirosis, distinguishing between initial and recurrent infections. Importantly, rEtfβ-IgG identified a subset of patients lacking detectable IgM. Thus, integrating rEtfβ-IgM and rEtfβ-IgG is recommended to improve sensitivity and accuracy in endemic populations. The rEtfβ is a promising target for future antigen-based diagnostic strategies for leptospirosis. The rEtfβ antigen shows promise as a target for future development of antigen-based diagnostic strategies for leptospirosis.</p>","PeriodicalId":18369,"journal":{"name":"Medical Microbiology and Immunology","volume":"214 1","pages":"12"},"PeriodicalIF":5.5000,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Medical Microbiology and Immunology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1007/s00430-025-00822-6","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"IMMUNOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

Leptospirosis is a major cause of acute febrile illness, often presenting with non-specific symptoms that can lead to misdiagnosis. Early laboratory diagnosis is essential for confirmation to avoid misdiagnosis and ensure appropriate management. This study aimed to identify and produce a recombinant protein, approximately 25 kDa, with high antigenicity for diagnostic applications. The 25 kDa protein from Leptospira interrogans was identified as electron transfer flavoprotein beta subunits (Etfβ) and exhibited 98% nucleotide and 99% amino acid homology to the reference strain. Lateral flow assays (LFAs) using recombinant Etfβ (rEtfβ) as antigens were developed to detect specific antibodies, namely rEtfβ-IgM and rEtfβ-IgG, and evaluated their performance against the standard microscopic agglutination test (MAT). Testing 33 paired serum samples from confirmed leptospirosis cases and 24 controls revealed sensitivities of 69.7% for IgM and 57.6% for IgG. However, the combined assays yielded enhanced diagnostic accuracy, achieving a sensitivity of 94.0%, specificity of 95.8%, positive predictive value of 96.9%, negative predictive value of 92.0%, and percent agreement of 94.7% (kappa value of 0.89). Also, the combined LFAs demonstrated 66.7% in initial serum samples whose MAT results were negative, enhancing the capacity for early diagnosis. In conclusion, the developed rapid tests demonstrated strong diagnostic capability, particularly in early-phase leptospirosis, distinguishing between initial and recurrent infections. Importantly, rEtfβ-IgG identified a subset of patients lacking detectable IgM. Thus, integrating rEtfβ-IgM and rEtfβ-IgG is recommended to improve sensitivity and accuracy in endemic populations. The rEtfβ is a promising target for future antigen-based diagnostic strategies for leptospirosis. The rEtfβ antigen shows promise as a target for future development of antigen-based diagnostic strategies for leptospirosis.

重组电子传递黄蛋白β亚基蛋白的生产及其在横向流动试验中的应用,用于钩端螺旋体病的早期诊断。
钩端螺旋体病是急性发热性疾病的主要原因,通常表现为可导致误诊的非特异性症状。早期实验室诊断是确诊的必要条件,以避免误诊和确保适当的管理。本研究旨在鉴定和生产一种重组蛋白,约25 kDa,具有高抗原性,用于诊断应用。从钩端螺旋体分离得到的25 kDa蛋白被鉴定为电子转移黄蛋白β亚基(Etfβ),与参比菌株有98%的核苷酸和99%的氨基酸同源性。建立了以重组Etfβ (rEtfβ)为抗原的侧流法(LFAs)检测rEtfβ- igm和rEtfβ- igg特异性抗体,并通过标准显微凝集试验(MAT)评价其性能。对来自确诊钩端螺旋体病病例和24名对照者的33对血清样本进行检测,结果显示IgM敏感性为69.7%,IgG敏感性为57.6%。然而,联合检测提高了诊断准确性,灵敏度为94.0%,特异性为95.8%,阳性预测值为96.9%,阴性预测值为92.0%,一致性为94.7% (kappa值为0.89)。此外,联合LFAs在MAT结果为阴性的初始血清样本中占66.7%,增强了早期诊断能力。总之,开发的快速检测显示出强大的诊断能力,特别是在早期钩端螺旋体病中,可以区分初次感染和复发感染。重要的是,rEtfβ-IgG鉴定了缺乏可检测IgM的患者亚群。因此,建议整合rEtfβ-IgM和rEtfβ-IgG,以提高流行人群的敏感性和准确性。rEtfβ是未来基于抗原的钩端螺旋体病诊断策略的一个有希望的靶标。rEtfβ抗原有望成为未来发展基于抗原的钩端螺旋体病诊断策略的靶标。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
CiteScore
10.60
自引率
0.00%
发文量
29
审稿时长
1 months
期刊介绍: Medical Microbiology and Immunology (MMIM) publishes key findings on all aspects of the interrelationship between infectious agents and the immune system of their hosts. The journal´s main focus is original research work on intrinsic, innate or adaptive immune responses to viral, bacterial, fungal and parasitic (protozoan and helminthic) infections and on the virulence of the respective infectious pathogens. MMIM covers basic, translational as well as clinical research in infectious diseases and infectious disease immunology. Basic research using cell cultures, organoid, and animal models are welcome, provided that the models have a clinical correlate and address a relevant medical question. The journal also considers manuscripts on the epidemiology of infectious diseases, including the emergence and epidemic spreading of pathogens and the development of resistance to anti-infective therapies, and on novel vaccines and other innovative measurements of prevention. The following categories of manuscripts will not be considered for publication in MMIM: submissions of preliminary work, of merely descriptive data sets without investigation of mechanisms or of limited global interest, manuscripts on existing or novel anti-infective compounds, which focus on pharmaceutical or pharmacological aspects of the drugs, manuscripts on existing or modified vaccines, unless they report on experimental or clinical efficacy studies or provide new immunological information on their mode of action, manuscripts on the diagnostics of infectious diseases, unless they offer a novel concept to solve a pending diagnostic problem, case reports or case series, unless they are embedded in a study that focuses on the anti-infectious immune response and/or on the virulence of a pathogen.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信