增强结核病诊断：提高靶向PPE17表位的scFv抗体的特异性

IF 2.5 4区生物学 Q1 ANATOMY & MORPHOLOGY

Tissue & cell Pub Date : 2025-04-29 DOI:10.1016/j.tice.2025.102933

Kamran Heidarnejad , Mehrzad Bahtouee , Seyed Nooreddin Faraji , Setareh Moazen , Farhad Abbasi , Amirhossein Sahebkar , Foroogh Nejatollahi

{"title":"增强结核病诊断：提高靶向PPE17表位的scFv抗体的特异性","authors":"Kamran Heidarnejad , Mehrzad Bahtouee , Seyed Nooreddin Faraji , Setareh Moazen , Farhad Abbasi , Amirhossein Sahebkar , Foroogh Nejatollahi","doi":"10.1016/j.tice.2025.102933","DOIUrl":null,"url":null,"abstract":"<div><h3>Background</h3><div>Serological assays have demonstrated enhanced simplicity, accuracy, and effectiveness in detecting Mycobacterium Tuberculosis (Mtb) antigens. The proline-proline-glutamic acid 17 (PPE17) antigen, specifically localized on the surface of Mtb, has been identified as unique to the Mtb species. The unique properties of single-chain antibodies make them well-suited for accurate diagnostic applications. In this study, specialized single-chain antibodies (scFvs) targeting PPE17 were employed to create a precise indirect immunofluorescent assay for diagnosing pulmonary tuberculosis (TB).</div></div><div><h3>Methods</h3><div>To select an immunodominant epitope of PPE17 in silico analysis was applied. The sequence was evaluated using the BLAST algorithm. A phage antibody display library of scFv was applied and two scFvs were isolated against the epitope by panning process. Specific clones were distinguished through PCR and DNA fingerprinting techniques. The reactivity of the chosen scFvs towards the selected epitope was assessed by ELISA. An Indirect Immunofluorescence Assay (IFA) was performed on 50 positive and 50 negative TB sputum smears, which were confirmed through both culture and genotype methods, to evaluate the performance of anti-PPE17 scFvs in accurately and rapidly detecting TB-positive smears, and TB-negative and Nocardia smears serving as negative controls for comparison.</div></div><div><h3>Results</h3><div>An immunodominant epitope of the PPE17 antigen consisting of amino acids 27–39, was identified. Two specific anti-PPE17-scFvs with frequencies of 25 % and 20 % were selected. ELISA results confirmed the reactivity of the scFvs against the epitope. Immunofluorescence assays demonstrated positive results for both antibodies when tested against positive TB sputum smears, whereas no positive results were obtained in tests against TB-negative and Nocardia smears.</div></div><div><h3>Conclusion</h3><div>A fast and accurate indirect immunofluorescence assay was developed to identify Mtb bacteria in TB sputum smears using specific anti-PPE17 scFvs. The results illustrated the capability of both scFvs in detecting Mtb in TB samples and differentiating Mtb from Nocardia smears. This suggests the potential for a novel diagnostic test that ensures precise TB detection in sputum samples, thereby preventing any potential misdiagnosis of tuberculosis.</div></div>","PeriodicalId":23201,"journal":{"name":"Tissue & cell","volume":"96 ","pages":"Article 102933"},"PeriodicalIF":2.5000,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Enhancing TB diagnosis: Improving specificity with scFv antibodies targeting the PPE17 epitope\",\"authors\":\"Kamran Heidarnejad , Mehrzad Bahtouee , Seyed Nooreddin Faraji , Setareh Moazen , Farhad Abbasi , Amirhossein Sahebkar , Foroogh Nejatollahi\",\"doi\":\"10.1016/j.tice.2025.102933\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Background</h3><div>Serological assays have demonstrated enhanced simplicity, accuracy, and effectiveness in detecting Mycobacterium Tuberculosis (Mtb) antigens. The proline-proline-glutamic acid 17 (PPE17) antigen, specifically localized on the surface of Mtb, has been identified as unique to the Mtb species. The unique properties of single-chain antibodies make them well-suited for accurate diagnostic applications. In this study, specialized single-chain antibodies (scFvs) targeting PPE17 were employed to create a precise indirect immunofluorescent assay for diagnosing pulmonary tuberculosis (TB).</div></div><div><h3>Methods</h3><div>To select an immunodominant epitope of PPE17 in silico analysis was applied. The sequence was evaluated using the BLAST algorithm. A phage antibody display library of scFv was applied and two scFvs were isolated against the epitope by panning process. Specific clones were distinguished through PCR and DNA fingerprinting techniques. The reactivity of the chosen scFvs towards the selected epitope was assessed by ELISA. An Indirect Immunofluorescence Assay (IFA) was performed on 50 positive and 50 negative TB sputum smears, which were confirmed through both culture and genotype methods, to evaluate the performance of anti-PPE17 scFvs in accurately and rapidly detecting TB-positive smears, and TB-negative and Nocardia smears serving as negative controls for comparison.</div></div><div><h3>Results</h3><div>An immunodominant epitope of the PPE17 antigen consisting of amino acids 27–39, was identified. Two specific anti-PPE17-scFvs with frequencies of 25 % and 20 % were selected. ELISA results confirmed the reactivity of the scFvs against the epitope. Immunofluorescence assays demonstrated positive results for both antibodies when tested against positive TB sputum smears, whereas no positive results were obtained in tests against TB-negative and Nocardia smears.</div></div><div><h3>Conclusion</h3><div>A fast and accurate indirect immunofluorescence assay was developed to identify Mtb bacteria in TB sputum smears using specific anti-PPE17 scFvs. The results illustrated the capability of both scFvs in detecting Mtb in TB samples and differentiating Mtb from Nocardia smears. This suggests the potential for a novel diagnostic test that ensures precise TB detection in sputum samples, thereby preventing any potential misdiagnosis of tuberculosis.</div></div>\",\"PeriodicalId\":23201,\"journal\":{\"name\":\"Tissue & cell\",\"volume\":\"96 \",\"pages\":\"Article 102933\"},\"PeriodicalIF\":2.5000,\"publicationDate\":\"2025-04-29\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Tissue & cell\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0040816625002137\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"ANATOMY & MORPHOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Tissue & cell","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0040816625002137","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ANATOMY & MORPHOLOGY","Score":null,"Total":0}

引用次数: 0

摘要

背景：血清学检测在检测结核分枝杆菌（Mtb）抗原方面已经证明了更高的简便性、准确性和有效性。脯氨酸-脯氨酸-谷氨酸17 （PPE17）抗原，特异定位于Mtb表面，已被确定为Mtb所特有。单链抗体的独特性质使它们非常适合于准确的诊断应用。本研究利用靶向PPE17的特异性单链抗体（scFvs）建立了一种诊断肺结核（TB）的精确间接免疫荧光检测方法。方法选择PPE17的免疫优势表位，应用芯片分析。利用BLAST算法对序列进行评价。应用噬菌体抗体展示文库，通过筛选法分离到2个抗原表位。通过PCR和DNA指纹图谱技术鉴定特异性克隆。所选scFvs对所选表位的反应性通过ELISA进行评估。采用间接免疫荧光法（IFA）对经培养法和基因型法确认的50例阳性和50例阴性结核痰涂片进行检测，以评估抗ppe17 scFvs准确、快速检测结核阳性涂片的性能，并将结核阴性涂片和诺卡菌涂片作为阴性对照进行比较。结果鉴定出PPE17抗原的免疫优势表位，由27 ~ 39个氨基酸组成。选择了两种特异性抗ppe17 - scfvs，频率分别为25 %和20 %。ELISA结果证实了scFvs对抗原表位的反应性。免疫荧光分析显示，在对结核痰涂片阳性进行检测时，两种抗体均呈阳性结果，而对结核阴性和诺卡菌涂片进行检测时，未获得阳性结果。结论建立了一种快速、准确的间接免疫荧光法，可用于结核痰涂片中结核分枝杆菌的特异性抗ppe17 scFvs检测。结果表明，这两种scFvs在检测结核样品中的结核分枝杆菌和从诺卡菌涂片中区分结核分枝杆菌方面具有能力。这表明有可能出现一种新的诊断测试，确保在痰样本中精确检测结核病，从而防止任何潜在的结核病误诊。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

查看原文本刊更多论文

Enhancing TB diagnosis: Improving specificity with scFv antibodies targeting the PPE17 epitope

Background

Serological assays have demonstrated enhanced simplicity, accuracy, and effectiveness in detecting Mycobacterium Tuberculosis (Mtb) antigens. The proline-proline-glutamic acid 17 (PPE17) antigen, specifically localized on the surface of Mtb, has been identified as unique to the Mtb species. The unique properties of single-chain antibodies make them well-suited for accurate diagnostic applications. In this study, specialized single-chain antibodies (scFvs) targeting PPE17 were employed to create a precise indirect immunofluorescent assay for diagnosing pulmonary tuberculosis (TB).

Methods

To select an immunodominant epitope of PPE17 in silico analysis was applied. The sequence was evaluated using the BLAST algorithm. A phage antibody display library of scFv was applied and two scFvs were isolated against the epitope by panning process. Specific clones were distinguished through PCR and DNA fingerprinting techniques. The reactivity of the chosen scFvs towards the selected epitope was assessed by ELISA. An Indirect Immunofluorescence Assay (IFA) was performed on 50 positive and 50 negative TB sputum smears, which were confirmed through both culture and genotype methods, to evaluate the performance of anti-PPE17 scFvs in accurately and rapidly detecting TB-positive smears, and TB-negative and Nocardia smears serving as negative controls for comparison.

Results

An immunodominant epitope of the PPE17 antigen consisting of amino acids 27–39, was identified. Two specific anti-PPE17-scFvs with frequencies of 25 % and 20 % were selected. ELISA results confirmed the reactivity of the scFvs against the epitope. Immunofluorescence assays demonstrated positive results for both antibodies when tested against positive TB sputum smears, whereas no positive results were obtained in tests against TB-negative and Nocardia smears.

Conclusion

A fast and accurate indirect immunofluorescence assay was developed to identify Mtb bacteria in TB sputum smears using specific anti-PPE17 scFvs. The results illustrated the capability of both scFvs in detecting Mtb in TB samples and differentiating Mtb from Nocardia smears. This suggests the potential for a novel diagnostic test that ensures precise TB detection in sputum samples, thereby preventing any potential misdiagnosis of tuberculosis.

求助全文

通过发布文献求助，成功后即可免费获取论文全文。去求助

来源期刊

Tissue & cell 医学-解剖学与形态学

CiteScore

3.90

自引率

0.00%

发文量

234

期刊介绍： Tissue and Cell is devoted to original research on the organization of cells, subcellular and extracellular components at all levels, including the grouping and interrelations of cells in tissues and organs. The journal encourages submission of ultrastructural studies that provide novel insights into structure, function and physiology of cells and tissues, in health and disease. Bioengineering and stem cells studies focused on the description of morphological and/or histological data are also welcomed. Studies investigating the effect of compounds and/or substances on structure of cells and tissues are generally outside the scope of this journal. For consideration, studies should contain a clear rationale on the use of (a) given substance(s), have a compelling morphological and structural focus and present novel incremental findings from previous literature.