Background: Cirsium japonicum (CJ) and Cirsium setosum (CS) are two widely recognized medicinal crops in Chinese herbal medicine (CHM) that are listed as authentic herbal plants in the herbal pharmacopeia. Given their strikingly similar morphological characteristics, CJ and CS are particularly susceptible to adulteration in the herbal marketplace, raising significant concerns about product integrity and consumer safety.
Purpose: To ensure the safety and efficacy of CHM, proper authentication of these herbs is a critical step in maintaining high-quality pharmaceutical production.
Methods: In this study, we developed a species-specific loop-mediated isothermal amplification (LAMP) method for the authentication of CJ and CS.
Results: We designed specific LAMP primer sets for both species using the selected DNA barcode, the internal transcribed spacer (ITS) sequence, through in silico analysis. After validating the specificity of the primers, we successfully authenticated CS and CJ using the LAMP primer sets and visualized the detection results through gel electrophoresis. The sensitivity of the LAMP method for CJ and CS authentication was established, with a limit of detection (LOD) of 100 pg for CJ and 50 pg for CS, using direct SYBR Green staining. When we applied the LAMP method to commercial Cirsium products, we found that only 25% (5 out of 20) of the samples contained CJ, while none contained CS.
Conclusion: In conclusion, we successfully established a species-specific LAMP assay for authenticating CJ and CS. This method can be used for species verification and is applicable to commercial Cirsium products for authenticity testing, contributing to quality control in pharmaceutical manufacturing.
{"title":"Species-specific isothermal nucleic acid amplification assay targeting Internal Transcribed Spacer (ITS) for rapid authentication of the medicinal crop Cirsium japonicum and Cirsium setosum in herbal markets.","authors":"Jia-An Ling, Jin-Xuan He, Chia-Hsin Lin, Shyang-Chwen Sheu, Jai-Hong Cheng, Meng-Shiou Lee","doi":"10.1016/j.ab.2026.116063","DOIUrl":"10.1016/j.ab.2026.116063","url":null,"abstract":"<p><strong>Background: </strong>Cirsium japonicum (CJ) and Cirsium setosum (CS) are two widely recognized medicinal crops in Chinese herbal medicine (CHM) that are listed as authentic herbal plants in the herbal pharmacopeia. Given their strikingly similar morphological characteristics, CJ and CS are particularly susceptible to adulteration in the herbal marketplace, raising significant concerns about product integrity and consumer safety.</p><p><strong>Purpose: </strong>To ensure the safety and efficacy of CHM, proper authentication of these herbs is a critical step in maintaining high-quality pharmaceutical production.</p><p><strong>Methods: </strong>In this study, we developed a species-specific loop-mediated isothermal amplification (LAMP) method for the authentication of CJ and CS.</p><p><strong>Results: </strong>We designed specific LAMP primer sets for both species using the selected DNA barcode, the internal transcribed spacer (ITS) sequence, through in silico analysis. After validating the specificity of the primers, we successfully authenticated CS and CJ using the LAMP primer sets and visualized the detection results through gel electrophoresis. The sensitivity of the LAMP method for CJ and CS authentication was established, with a limit of detection (LOD) of 100 pg for CJ and 50 pg for CS, using direct SYBR Green staining. When we applied the LAMP method to commercial Cirsium products, we found that only 25% (5 out of 20) of the samples contained CJ, while none contained CS.</p><p><strong>Conclusion: </strong>In conclusion, we successfully established a species-specific LAMP assay for authenticating CJ and CS. This method can be used for species verification and is applicable to commercial Cirsium products for authenticity testing, contributing to quality control in pharmaceutical manufacturing.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116063"},"PeriodicalIF":2.5,"publicationDate":"2026-01-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146099514","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-27DOI: 10.1016/j.ab.2026.116066
Kaoutar Aalilouch, Khalida Sabeur, Ikhlass El Berbri, Faouzi Kichou, Najet Safini, Mehdi Elharrak, Ouafaa Fassi Fihri
Inhibin is a dimeric glycoprotein hormone consisting of α and β subunits. Inhibin has gained significant interest as a biomarker for ovarian and testicular malignancies. While existing inhibin immunoassays have demonstrated efficacy, assays specifically targeting the free α subunit are not yet available. This study aimed to generate monoclonal antibodies against inhibin-α using hybridoma technology, targeting distinct epitopes within the N-region of the α subunit. These epitopes were chosen using bioinformatic analysis to ensure strong antigenicity and successful antibody production. To screen the antibodies, an ELISA was developed using plates coated with recombinant inhibin-α protein. This ELISA confirmed that the antibodies were reactive and could effectively detect inhibin-α in vitro. The antibodies were further evaluated for specificity and immunoreactivity using Western blotting, and IHC methods. Western blot analysis showed that the antibodies specifically recognized the free inhibin-α, demonstrating high specificity under both reducing and non-reducing conditions. IHC studies on normal mouse tissue sections further confirmed the localization of inhibin-α in granulosa, theca, Sertoli, and Leydig cells. The developed antibodies showed significant potential for use in vitro assays for cancer diagnosis. Further validation is needed to ensure their successful clinical application.
{"title":"Development and characterization of monoclonal antibodies against inhibin α subunit as a potential cancer biomarker.","authors":"Kaoutar Aalilouch, Khalida Sabeur, Ikhlass El Berbri, Faouzi Kichou, Najet Safini, Mehdi Elharrak, Ouafaa Fassi Fihri","doi":"10.1016/j.ab.2026.116066","DOIUrl":"10.1016/j.ab.2026.116066","url":null,"abstract":"<p><p>Inhibin is a dimeric glycoprotein hormone consisting of α and β subunits. Inhibin has gained significant interest as a biomarker for ovarian and testicular malignancies. While existing inhibin immunoassays have demonstrated efficacy, assays specifically targeting the free α subunit are not yet available. This study aimed to generate monoclonal antibodies against inhibin-α using hybridoma technology, targeting distinct epitopes within the N-region of the α subunit. These epitopes were chosen using bioinformatic analysis to ensure strong antigenicity and successful antibody production. To screen the antibodies, an ELISA was developed using plates coated with recombinant inhibin-α protein. This ELISA confirmed that the antibodies were reactive and could effectively detect inhibin-α in vitro. The antibodies were further evaluated for specificity and immunoreactivity using Western blotting, and IHC methods. Western blot analysis showed that the antibodies specifically recognized the free inhibin-α, demonstrating high specificity under both reducing and non-reducing conditions. IHC studies on normal mouse tissue sections further confirmed the localization of inhibin-α in granulosa, theca, Sertoli, and Leydig cells. The developed antibodies showed significant potential for use in vitro assays for cancer diagnosis. Further validation is needed to ensure their successful clinical application.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116066"},"PeriodicalIF":2.5,"publicationDate":"2026-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146083778","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-27DOI: 10.1016/j.ab.2026.116065
Ayushi Mishra , Suneel Kateriya
Various blue-light photoreceptor proteins have photo-responsive domains known as light, oxygen, voltage (LOV) domains, which are extensively distributed in plants, algae, fungi, and bacteria. When exposed to blue light, the flavin chromophore and a highly conserved cysteine residue form a covalent adduct on a microsecond time scale. LOV domains are common photosensory modules that can be applied to optogenetics, regulated synthesis of reactive oxygen species, and fluorescence microscopy. This review explores the photocycle kinetics and applications of various LOV domains, which have been explored for confocal microscopy, two-photon microscopy, and super-resolution microscopy. Many LOV domains have been derived and modulated for use in different types of microscopic applications. Molecular understanding, diversity of LOV domains, and versatile photo-physical characteristics of these proteins have immense potential for the development of useful probes for various microscopy tools. There is a great demand for perspective research on LOV domain proteins for harnessing their possible optobiotechnological applications.
{"title":"Versatile applications of Light-Oxygen-Voltage (LOV) domain proteins in optical microscopy","authors":"Ayushi Mishra , Suneel Kateriya","doi":"10.1016/j.ab.2026.116065","DOIUrl":"10.1016/j.ab.2026.116065","url":null,"abstract":"<div><div>Various blue-light photoreceptor proteins have photo-responsive domains known as light, oxygen, voltage (LOV) domains, which are extensively distributed in plants, algae, fungi, and bacteria. When exposed to blue light, the flavin chromophore and a highly conserved cysteine residue form a covalent adduct on a microsecond time scale. LOV domains are common photosensory modules that can be applied to optogenetics, regulated synthesis of reactive oxygen species, and fluorescence microscopy. This review explores the photocycle kinetics and applications of various LOV domains, which have been explored for confocal microscopy, two-photon microscopy, and super-resolution microscopy. Many LOV domains have been derived and modulated for use in different types of microscopic applications. Molecular understanding, diversity of LOV domains, and versatile photo-physical characteristics of these proteins have immense potential for the development of useful probes for various microscopy tools. There is a great demand for perspective research on LOV domain proteins for harnessing their possible optobiotechnological applications.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116065"},"PeriodicalIF":2.5,"publicationDate":"2026-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146075775","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Background: Programmed death-ligand 1 (PD-L1) is one of the main immunoregulatory proteins. It binds to its receptor, PD-1, on the surface of T cells and suppresses their anti-tumor activity. Therefore, elevated levels of PD-L1 protein could predict a potential benefit from PD-L1/PD-1 checkpoint inhibitor immunotherapy. Immunohistochemistry (IHC) is the current standard technique for assessing PD-L1 expression in tumor tissues, and a sensitive and specific monoclonal antibody (mAb) to PD-L1 is a critical parameter for accurate results. Thus, in the current study, we aimed to produce new anti-PD-L1 mAbs with potential diagnostic applications in IHC.
Methods: By immunizing mice with human recombinant PD-L1 protein, specific mAbs were produced via the hybridoma method and then screened using enzyme-linked immunosorbent assay (ELISA) and IHC techniques in parallel. Furthermore, isotypes and affinity constants of the selected and purified mAbs were determined, and their specificities were evaluated in Western blot, flow cytometry, IHC, and immunocytochemistry (ICC) assays. Afterward, their reactivity was compared with commercially available anti-PD-L1 mAb in optimized IHC using human placenta tissue and 54 paraffin-embedded bladder cancer tissue samples, and the analytical sensitivity, specificity, and accuracy of homemade IHC were assessed.
Results: Among 30 clones that produce significant amounts of specific antibodies against PD-L1 in ELISA, three anti-PD-L1 mAbs (clones 1D6, 1D8, and 1F8) demonstrated high reactivity to their target antigen in human placental and cancerous tissues in IHC. Moreover, one of the selected mAbs, clone 1D6, was also able to specifically recognize its target by flow cytometry, western blotting, and ICC using a panel of human PD-L1 positive and negative tumor cell lines and placenta tissue. Compared to commercially available anti-PD-L1, the analytical sensitivity, specificity, and accuracy calculated for clone 1D6 were 94.87 %, 80 %, and 91.83 %, and for clone 1D8, they were 97.67 %, 81.81 %, and 94.4 %, respectively.
Conclusion: This study's results demonstrated that the novel anti-PD-L1 mAbs could recognize the target antigen with high specificity and sensitivity. Therefore, they might be appropriate tools for research and diagnosis.
{"title":"Production and characterization of novel murine monoclonal antibodies against programmed death-ligand 1 with potential diagnostic application in immunohistochemistry.","authors":"Mansoure Mansouri, Mehdi Mohammadi, Fatemeh Montazer, Zahra Sadat Faghih, Mohsen Soltanshahi, Fazel Shokri, Mahmood Jeddi-Tehrani, Mahdi Shabani","doi":"10.1016/j.ab.2026.116067","DOIUrl":"10.1016/j.ab.2026.116067","url":null,"abstract":"<p><strong>Background: </strong>Programmed death-ligand 1 (PD-L1) is one of the main immunoregulatory proteins. It binds to its receptor, PD-1, on the surface of T cells and suppresses their anti-tumor activity. Therefore, elevated levels of PD-L1 protein could predict a potential benefit from PD-L1/PD-1 checkpoint inhibitor immunotherapy. Immunohistochemistry (IHC) is the current standard technique for assessing PD-L1 expression in tumor tissues, and a sensitive and specific monoclonal antibody (mAb) to PD-L1 is a critical parameter for accurate results. Thus, in the current study, we aimed to produce new anti-PD-L1 mAbs with potential diagnostic applications in IHC.</p><p><strong>Methods: </strong>By immunizing mice with human recombinant PD-L1 protein, specific mAbs were produced via the hybridoma method and then screened using enzyme-linked immunosorbent assay (ELISA) and IHC techniques in parallel. Furthermore, isotypes and affinity constants of the selected and purified mAbs were determined, and their specificities were evaluated in Western blot, flow cytometry, IHC, and immunocytochemistry (ICC) assays. Afterward, their reactivity was compared with commercially available anti-PD-L1 mAb in optimized IHC using human placenta tissue and 54 paraffin-embedded bladder cancer tissue samples, and the analytical sensitivity, specificity, and accuracy of homemade IHC were assessed.</p><p><strong>Results: </strong>Among 30 clones that produce significant amounts of specific antibodies against PD-L1 in ELISA, three anti-PD-L1 mAbs (clones 1D6, 1D8, and 1F8) demonstrated high reactivity to their target antigen in human placental and cancerous tissues in IHC. Moreover, one of the selected mAbs, clone 1D6, was also able to specifically recognize its target by flow cytometry, western blotting, and ICC using a panel of human PD-L1 positive and negative tumor cell lines and placenta tissue. Compared to commercially available anti-PD-L1, the analytical sensitivity, specificity, and accuracy calculated for clone 1D6 were 94.87 %, 80 %, and 91.83 %, and for clone 1D8, they were 97.67 %, 81.81 %, and 94.4 %, respectively.</p><p><strong>Conclusion: </strong>This study's results demonstrated that the novel anti-PD-L1 mAbs could recognize the target antigen with high specificity and sensitivity. Therefore, they might be appropriate tools for research and diagnosis.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116067"},"PeriodicalIF":2.5,"publicationDate":"2026-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146083687","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-21DOI: 10.1016/j.ab.2026.116062
Juan A Azcona, Anja S Wacker, Thomas M Jeitner, James M Kelly
Experimental and epidemiological studies indicate that significant reductions in dietary methionine intake profoundly impact both health and life spans. Methionine levels also decrease in the blood of mammals as they age. Here, we report that methionine uptake by four of the major methionine-consuming organs, the liver, kidneys, heart, and brain, is increased in old rats as compared to young adult rats, as measured by [11C]methionine uptake by these organs using positron emission tomography. This increased uptake was sustained for at least 30 min and resulted in 1.6- to 1.9-fold increases in methionine uptake by the major methionine-consuming organs in old rats, suggesting an age-associated acceleration of methionine metabolism and its associated biochemical pathways. By contrast, the uptake of [18F]fluorodeoxyglucose by the liver, kidneys, heart, and brain of older rats was reduced relative to that by their younger adult counterparts, indicating a decline in glucose metabolic activity with age. Remarkably, the uptake of 2-[18F]fluoropropionic acid by these organs remained essentially unchanged between young and aged adult rats, suggesting the presence of a stable biochemical equilibrium in propionate metabolism across the aging spectrum. These findings underscore the utility of positron emission tomography in revealing significant age-related alterations in organ-specific biochemical processes.
{"title":"Aging differentially affects the uptake of methionine by select rat tissues.","authors":"Juan A Azcona, Anja S Wacker, Thomas M Jeitner, James M Kelly","doi":"10.1016/j.ab.2026.116062","DOIUrl":"10.1016/j.ab.2026.116062","url":null,"abstract":"<p><p>Experimental and epidemiological studies indicate that significant reductions in dietary methionine intake profoundly impact both health and life spans. Methionine levels also decrease in the blood of mammals as they age. Here, we report that methionine uptake by four of the major methionine-consuming organs, the liver, kidneys, heart, and brain, is increased in old rats as compared to young adult rats, as measured by [<sup>11</sup>C]methionine uptake by these organs using positron emission tomography. This increased uptake was sustained for at least 30 min and resulted in 1.6- to 1.9-fold increases in methionine uptake by the major methionine-consuming organs in old rats, suggesting an age-associated acceleration of methionine metabolism and its associated biochemical pathways. By contrast, the uptake of [<sup>18</sup>F]fluorodeoxyglucose by the liver, kidneys, heart, and brain of older rats was reduced relative to that by their younger adult counterparts, indicating a decline in glucose metabolic activity with age. Remarkably, the uptake of 2-[<sup>18</sup>F]fluoropropionic acid by these organs remained essentially unchanged between young and aged adult rats, suggesting the presence of a stable biochemical equilibrium in propionate metabolism across the aging spectrum. These findings underscore the utility of positron emission tomography in revealing significant age-related alterations in organ-specific biochemical processes.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"116062"},"PeriodicalIF":2.5,"publicationDate":"2026-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146040338","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-19DOI: 10.1016/j.ab.2026.116053
Un Na Koh , Si-Keun Lim
Digital PCR (dPCR) enables absolute nucleic acid quantification and has been widely adopted for quality control (QC) applications in cell therapy manufacturing. Ensuring patient safety during cell therapy manufacturing requires reliable detection of trace residual cells, such as undifferentiated induced pluripotent stem cells (iPSCs) and virus-producing cells. This study compared qPCR (CFX96 Opus System, Bio-Rad) with two dPCR platforms—the QX200 Droplet Digital PCR System (Bio-Rad) and the QIAcuity Digital PCR System (QIAGEN). For QC evaluation, iPSCs mixed with differentiated cardiomyocytes (CMs) or neural progenitor cells (NPCs) were analyzed using TDGF1, OCT4, and NANOG, while virus-producing 293T cells in CAR-T preparations were targeted using gag and VSVG sequences within the lentiviral packaging plasmid. Mixed samples were serially diluted from 1:1 to 1:106 to evaluate performance across a wide concentration range. Both dPCR platforms and qPCR showed comparable sensitivity and linearity across most dilution points. However, qPCR exhibited more frequent signal loss at low template concentrations. In contrast, dPCR showed reduced variability across dilution intervals, and lower coefficients of variation (CV), indicating more stable quantification at low target levels. Despite minor differences in absolute copy number, both dPCR systems demonstrated comparable analytical performance. These results indicate that, although overall sensitivity and linearity were similar between qPCR and dPCR, dPCR provides more consistent quantification across dilution ranges, supporting its suitability for detecting low-abundance residual cells in cell therapy manufacturing.
数字PCR （dPCR）能够实现绝对核酸定量，并已广泛应用于细胞治疗制造中的质量控制（QC）应用。在细胞治疗制造过程中确保患者安全需要可靠地检测微量残留细胞，如未分化的诱导多能干细胞（iPSCs）和产生病毒的细胞。本研究将qPCR （CFX96 Opus System, Bio-Rad）与两种dPCR平台——QX200液滴数字PCR系统（Bio-Rad）和QIAcuity数字PCR系统（QIAGEN）进行了比较。为了进行QC评估，我们使用TDGF1、OCT4和NANOG对混合分化心肌细胞（CMs）或神经祖细胞（npc）的iPSCs进行分析，同时使用慢病毒包装质粒中的gag和VSVG序列对CAR-T制剂中产生病毒的293T细胞进行靶向。混合样品从1:1稀释到1:106，以评估在广泛浓度范围内的性能。dPCR平台和qPCR在大多数稀释点上都显示出相当的灵敏度和线性。然而，在低模板浓度下，qPCR表现出更频繁的信号丢失。相比之下，dPCR在稀释间隔内的变异性较低，变异系数（CV）较低，表明在低目标水平下定量更稳定。尽管绝对拷贝数存在微小差异，但两种dPCR系统均表现出可比较的分析性能。这些结果表明，尽管qPCR和dPCR的总体灵敏度和线性相似，但dPCR在稀释范围内提供了更一致的定量，支持其在细胞治疗制造中检测低丰度残留细胞的适用性。
{"title":"Comparing digital and real-time PCR platforms for detecting residual iPSCs and virus-producing cells in manufacturing","authors":"Un Na Koh , Si-Keun Lim","doi":"10.1016/j.ab.2026.116053","DOIUrl":"10.1016/j.ab.2026.116053","url":null,"abstract":"<div><div>Digital PCR (dPCR) enables absolute nucleic acid quantification and has been widely adopted for quality control (QC) applications in cell therapy manufacturing. Ensuring patient safety during cell therapy manufacturing requires reliable detection of trace residual cells, such as undifferentiated induced pluripotent stem cells (iPSCs) and virus-producing cells. This study compared qPCR (CFX96 Opus System, Bio-Rad) with two dPCR platforms—the QX200 Droplet Digital PCR System (Bio-Rad) and the QIAcuity Digital PCR System (QIAGEN). For QC evaluation, iPSCs mixed with differentiated cardiomyocytes (CMs) or neural progenitor cells (NPCs) were analyzed using <em>TDGF1</em>, <em>OCT4</em>, and <em>NANOG</em>, while virus-producing 293T cells in CAR-T preparations were targeted using <em>gag</em> and VSVG sequences within the lentiviral packaging plasmid. Mixed samples were serially diluted from 1:1 to 1:10<sup>6</sup> to evaluate performance across a wide concentration range. Both dPCR platforms and qPCR showed comparable sensitivity and linearity across most dilution points. However, qPCR exhibited more frequent signal loss at low template concentrations. In contrast, dPCR showed reduced variability across dilution intervals, and lower coefficients of variation (CV), indicating more stable quantification at low target levels. Despite minor differences in absolute copy number, both dPCR systems demonstrated comparable analytical performance. These results indicate that, although overall sensitivity and linearity were similar between qPCR and dPCR, dPCR provides more consistent quantification across dilution ranges, supporting its suitability for detecting low-abundance residual cells in cell therapy manufacturing.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116053"},"PeriodicalIF":2.5,"publicationDate":"2026-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146016843","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-17DOI: 10.1016/j.ab.2026.116052
Mitsuki Nakamura, Masafumi Sakono
Proteases play essential roles in diverse biological processes and are closely associated with various diseases, making them important targets for diagnostics and therapeutic development. Conventional protease activity assays often rely on chromogenic or fluorogenic substrates that require organic synthesis, increasing production costs and limiting accessibility. To address these, we developed a genetically encoded, bioluminescence resonance energy transfer (BRET)-based protease substrate composed of NanoLuc (NLuc) and green fluorescent protein (GFP) fused to either end of a cleavable peptide sequence. In the intact fusion protein, NLuc luminescence excites GFP via BRET, resulting in green emission. Proteolytic cleavage disrupts BRET, reducing GFP fluorescence. Using tobacco etch virus (TEV) protease as a model, we demonstrated that introducing glycine–serine linkers flanking the cleavage site (TEVcs) enhances proteolytic accessibility and signal responsiveness. The optimized substrate enabled quantitative detection of TEV protease activity with a detection limit of 0.0426 μM. Furthermore, substituting the TEVcs with a caspase-3-specific sequence allowed sensitive detection of caspase-3, with a limit of 0.62 nM. This system offers a cost-effective and broadly applicable platform for real-time protease activity measurement using standard Escherichia coli expression systems, eliminating the need for chemically synthesized substrates.
{"title":"Engineering GFP–NanoLuc fusion substrates for sensitive, quantitative detection of protease activity via BRET","authors":"Mitsuki Nakamura, Masafumi Sakono","doi":"10.1016/j.ab.2026.116052","DOIUrl":"10.1016/j.ab.2026.116052","url":null,"abstract":"<div><div>Proteases play essential roles in diverse biological processes and are closely associated with various diseases, making them important targets for diagnostics and therapeutic development. Conventional protease activity assays often rely on chromogenic or fluorogenic substrates that require organic synthesis, increasing production costs and limiting accessibility. To address these, we developed a genetically encoded, bioluminescence resonance energy transfer (BRET)-based protease substrate composed of NanoLuc (NLuc) and green fluorescent protein (GFP) fused to either end of a cleavable peptide sequence. In the intact fusion protein, NLuc luminescence excites GFP via BRET, resulting in green emission. Proteolytic cleavage disrupts BRET, reducing GFP fluorescence. Using tobacco etch virus (TEV) protease as a model, we demonstrated that introducing glycine–serine linkers flanking the cleavage site (TEVcs) enhances proteolytic accessibility and signal responsiveness. The optimized substrate enabled quantitative detection of TEV protease activity with a detection limit of 0.0426 μM. Furthermore, substituting the TEVcs with a caspase-3-specific sequence allowed sensitive detection of caspase-3, with a limit of 0.62 nM. This system offers a cost-effective and broadly applicable platform for real-time protease activity measurement using standard <em>Escherichia coli</em> expression systems, eliminating the need for chemically synthesized substrates.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116052"},"PeriodicalIF":2.5,"publicationDate":"2026-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145996378","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-13DOI: 10.1016/j.ab.2026.116051
Isabela Tokuhara Makida , Amanda Almeida Resende , Letícia Gomes de Pontes , Laudicéia Alves de Oliveira , Leonardo Melo , Francilene Capel Tavares de Carvalho , Ivânio Teixeira Borba-Junior , Regina Kiomi Takahira , Rui Seabra Ferreira Junior , Lucilene Delazari dos Santos
Snakebite envenoming remains a major public health concern worldwide, while the maintenance of captive snakes is essential for conservation, venom production, and biomedical research. However, little is known about the plasma proteome of snakes, a gap that limits physiological assessments and the development of species-specific reference protein profile. This study aimed to characterize the plasma proteome of healthy adult Crotalus durissus terrificus snakes to establish a molecular baseline that supports biological evaluation and future diagnostic research. Blood samples were collected from 19 specimens kept in capitivity, followed by hematological, biochemical, and proteomic analyses. Hematology confirmed preserved cell morphology and values within established reptilian reference ranges, indicating good health status. Biochemical analyses showed albumin, total protein, and aspartate aminotransferase levels consistent with previous reports, while uric acid values were comparatively lower, reflecting reptilian renal physiology. Proteomic profiling using 2D electrophoresis and LC-MS/MS identified 301 protein spots, of which 273 were successfully annotated, covering approximately 90% of the detectable plasma proteome. Functional classification revealed proteins associated with immune function, transport, metabolism, and anti-hemorrhagic activity. The plasma proteomic baseline generated here contributes to the understanding of snake systemic physiology and provides foundational data to support future diagnostic, veterinary and biotechnological investigations, with direct benefits for animal welfare, biosafety, and reproducibility in venom-based biotechnological applications.
{"title":"Reference Plasma Proteome Map of Crotalus durissus terrificus","authors":"Isabela Tokuhara Makida , Amanda Almeida Resende , Letícia Gomes de Pontes , Laudicéia Alves de Oliveira , Leonardo Melo , Francilene Capel Tavares de Carvalho , Ivânio Teixeira Borba-Junior , Regina Kiomi Takahira , Rui Seabra Ferreira Junior , Lucilene Delazari dos Santos","doi":"10.1016/j.ab.2026.116051","DOIUrl":"10.1016/j.ab.2026.116051","url":null,"abstract":"<div><div>Snakebite envenoming remains a major public health concern worldwide, while the maintenance of captive snakes is essential for conservation, venom production, and biomedical research. However, little is known about the plasma proteome of snakes, a gap that limits physiological assessments and the development of species-specific reference protein profile. This study aimed to characterize the plasma proteome of healthy adult <em>Crotalus durissus terrificus</em> snakes to establish a molecular baseline that supports biological evaluation and future diagnostic research. Blood samples were collected from 19 specimens kept in capitivity, followed by hematological, biochemical, and proteomic analyses. Hematology confirmed preserved cell morphology and values within established reptilian reference ranges, indicating good health status. Biochemical analyses showed albumin, total protein, and aspartate aminotransferase levels consistent with previous reports, while uric acid values were comparatively lower, reflecting reptilian renal physiology. Proteomic profiling using 2D electrophoresis and LC-MS/MS identified 301 protein spots, of which 273 were successfully annotated, covering approximately 90% of the detectable plasma proteome. Functional classification revealed proteins associated with immune function, transport, metabolism, and anti-hemorrhagic activity. The plasma proteomic baseline generated here contributes to the understanding of snake systemic physiology and provides foundational data to support future diagnostic, veterinary and biotechnological investigations, with direct benefits for animal welfare, biosafety, and reproducibility in venom-based biotechnological applications.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116051"},"PeriodicalIF":2.5,"publicationDate":"2026-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145987600","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-10DOI: 10.1016/j.ab.2026.116048
Jan Voldřich , Hana Zemková , Markéta Šmídková , Marika Matoušová , Eva Kudová , Helena Mertlíková-Kaiserová
GABAA receptor modulating steroid antagonists (GAMSA) represent a promising therapeutic class by selectively inhibiting the effects of positive allosteric modulators (PAMs) at GABAA receptors without affecting GABA-evoked currents alone. This mechanism avoids the risk of overexcitation and seizures associated with direct GABAA receptor blockers, making GAMSA a safer alternative for conditions linked to elevated levels of endogenous PAMs such as allopregnanolone (ALLO), including compulsive disorders and hepatic encephalopathy. Despite growing interest, a high-throughput screening (HTS) method for the identification of novel GAMSA has been lacking.
Here, we introduce a fluorescence-based membrane potential assay in CHO cells stably expressing GABAA (α1β2γ2) receptors for rapid identification of compounds with GAMSA activity. This robust three-component assay, previously unused in this context, yielded a Z-factor of 0.45 and a minimum significant difference (MSD) of 4.90 %. A library of 108 neurosteroids (10 μM) was subsequently tested in the presence of GABA and ALLO. Isoallopregnanolone (IsoALLO), a known GAMSA, was the only compound that significantly reduced ALLO-potentiated GABA responses (to 42 ± 3 %) without affecting GABA-evoked responses alone.
The inhibitory effect of IsoALLO was confirmed using manual patch-clamp recordings in primary rat anterior pituitary cells, which endogenously express GABAA receptors, where it reduced ALLO-potentiated GABA-induced currents by 32 ± 4 %.
This study validates the first cell-based HTS method for GAMSA identification and demonstrates its utility in distinguishing GAMSA from direct channel blockers or negative allosteric modulators (NAMs) using standard laboratory equipment.
{"title":"Fluorescence-based assay for rapid screening of GABAA receptor modulating steroid antagonists (GAMSA)","authors":"Jan Voldřich , Hana Zemková , Markéta Šmídková , Marika Matoušová , Eva Kudová , Helena Mertlíková-Kaiserová","doi":"10.1016/j.ab.2026.116048","DOIUrl":"10.1016/j.ab.2026.116048","url":null,"abstract":"<div><div>GABA<sub>A</sub> receptor modulating steroid antagonists (GAMSA) represent a promising therapeutic class by selectively inhibiting the effects of positive allosteric modulators (PAMs) at GABA<sub>A</sub> receptors without affecting GABA-evoked currents alone. This mechanism avoids the risk of overexcitation and seizures associated with direct GABA<sub>A</sub> receptor blockers, making GAMSA a safer alternative for conditions linked to elevated levels of endogenous PAMs such as allopregnanolone (ALLO), including compulsive disorders and hepatic encephalopathy. Despite growing interest, a high-throughput screening (HTS) method for the identification of novel GAMSA has been lacking.</div><div>Here, we introduce a fluorescence-based membrane potential assay in CHO cells stably expressing GABA<sub>A</sub> (α1β2γ2) receptors for rapid identification of compounds with GAMSA activity. This robust three-component assay, previously unused in this context, yielded a Z-factor of 0.45 and a minimum significant difference (MSD) of 4.90 %. A library of 108 neurosteroids (10 μM) was subsequently tested in the presence of GABA and ALLO. Isoallopregnanolone (IsoALLO), a known GAMSA, was the only compound that significantly reduced ALLO-potentiated GABA responses (to 42 ± 3 %) without affecting GABA-evoked responses alone.</div><div>The inhibitory effect of IsoALLO was confirmed using manual patch-clamp recordings in primary rat anterior pituitary cells, which endogenously express GABA<sub>A</sub> receptors, where it reduced ALLO-potentiated GABA-induced currents by 32 ± 4 %.</div><div>This study validates the first cell-based HTS method for GAMSA identification and demonstrates its utility in distinguishing GAMSA from direct channel blockers or negative allosteric modulators (NAMs) using standard laboratory equipment.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116048"},"PeriodicalIF":2.5,"publicationDate":"2026-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145958470","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-10DOI: 10.1016/j.ab.2026.116050
Bruna Garlet Rossato , Rafael Noal Moresco
Immunoturbidimetric assays are widely used to quantify immunoglobulins and complement proteins but are susceptible to interference. This study evaluated the effects of oxidative agents, including the Fenton reaction and hypochlorous acid (HOCl), on these assays. Serum pools were oxidized in vitro, and IgA, IgG, IgM, C3, and C4 were measured. Oxidative conditions increased measured concentrations of IgA, IgM, C3, and C4, whereas IgG was unaffected by HOCl. Increased oxidative stress index values and reduced thiol levels confirmed oxidative modification, indicating potential impairment of assay accuracy under oxidative stress.
{"title":"Oxidative interference in immunoturbidimetric assays for the quantification of immunoglobulins and complement proteins","authors":"Bruna Garlet Rossato , Rafael Noal Moresco","doi":"10.1016/j.ab.2026.116050","DOIUrl":"10.1016/j.ab.2026.116050","url":null,"abstract":"<div><div>Immunoturbidimetric assays are widely used to quantify immunoglobulins and complement proteins but are susceptible to interference. This study evaluated the effects of oxidative agents, including the Fenton reaction and hypochlorous acid (HOCl), on these assays. Serum pools were oxidized <em>in vitro</em>, and IgA, IgG, IgM, C3, and C4 were measured. Oxidative conditions increased measured concentrations of IgA, IgM, C3, and C4, whereas IgG was unaffected by HOCl. Increased oxidative stress index values and reduced thiol levels confirmed oxidative modification, indicating potential impairment of assay accuracy under oxidative stress.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116050"},"PeriodicalIF":2.5,"publicationDate":"2026-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145951142","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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