Analytical biochemistry最新文献

{"title":"Dual-strategy qMSP reference optimization for Low-DNA clinical specimens","authors":"Shan Zhang ,&nbsp;Yixuan Tang ,&nbsp;Jiahui Jiang ,&nbsp;Ling Chen","doi":"10.1016/j.ab.2026.116068","DOIUrl":"10.1016/j.ab.2026.116068","url":null,"abstract":"<div><div>Detecting tumor DNA methylation in low DNA concentration samples at high qualification rates is a significant challenge in translating and conducting regulatory trials for tumor methylation detection kits. Existing internal reference systems frequently malfunction, resulting in the mislabelling of valuable clinical specimens as unusable. We developed a two-part framework for improving reference primer-probe sets, by combining a systematic improvement of existing <em>ACTB</em> assays with new primer designs that target previously unexplored, CpG-free genomic areas. A three-stage screening process involving SYBR Green qPCR, TaqMan probe-based qPCR, and validation across different biological samples led to the identification of two superior primer sets: an optimized combination (bisACTB_103_F2+R2+P2) and a novel set (bisProAB1_F2+R2+P1). Exhibited enhanced amplification efficiency and specificity in cell lines, tissues, blood cells, and plasma were observed in both. Notably, bisProAB1_F2+R2+P1 considerably enhanced amplification efficiency in low-concentration plasma cell-free DNA, resulting in a nearly 5 % increase in the detection success rate within a large clinical study group and allowing for reliable analysis of minimal-input samples. This work offers verified, high-performance reference systems to boost the reliability of DNA methylation-based diagnostics and make the most of precious biospecimens.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116068"},"PeriodicalIF":2.5,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146099521","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}

{"title":"Reference Plasma Proteome Map of Crotalus durissus terrificus","authors":"Isabela Tokuhara Makida ,&nbsp;Amanda Almeida Resende ,&nbsp;Letícia Gomes de Pontes ,&nbsp;Laudicéia Alves de Oliveira ,&nbsp;Leonardo Melo ,&nbsp;Francilene Capel Tavares de Carvalho ,&nbsp;Ivânio Teixeira Borba-Junior ,&nbsp;Regina Kiomi Takahira ,&nbsp;Rui Seabra Ferreira Junior ,&nbsp;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":"2026-05-01","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}

{"title":"Identity of protease-based biomarkers for viability and cytotoxicity revealed by CRISPR knockouts","authors":"Andrew Niles,&nbsp;Kennedy Kroening,&nbsp;Anthony Lauer,&nbsp;Adityarup Chakravorty","doi":"10.1016/j.ab.2026.116073","DOIUrl":"10.1016/j.ab.2026.116073","url":null,"abstract":"<div><div>A wide variety of assay chemistries are routinely employed to determine cell health within an in vitro test population. Each method relies on the consistent and robust measurement of specific biological surrogates for cell viability or cytotoxicity. Unfortunately, the precise cellular origin or identity of many of these biomarkers remain poorly characterized or unknown, and thus subject to a host of undetermined biological and chemical interferences. This work details efforts to pinpoint the enzymatic sources of a set of proteolytic activity profiles previously discovered in a phenotypic activity screen and measured in a multiplexed viability (“live cell”) and cytotoxicity (“dead cell”) assay. First, Clustered Regularly Interspaced Short Palindromic Repeats gene editing (CRISPR) was utilized to knockout (KO) genes encoding candidate enzymes in a human cell background to create clones for identity testing. Next, clones demonstrating discrete reduction of either the live or dead cell signals were further characterized by Western blot analysis for presence of immunogenic protein and by Sanger Sequencing of the targeted edit site. The KO data directed the sourcing of potent and selective inhibitors for orthogonal activity studies of the proteases in a parental population. Last, the utility of the multiplexed assay reagent was further explored in both non-human and human primary cell lines to characterize the universality of the application. Collectively, the positive identification of Cathepsin C (CatC) and Tripeptidyl peptidase II (TPP II) informs assay users about potential modulators of activity leading to possible interferences. Last, the work provides new information about assay performance in previously untested cell types.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116073"},"PeriodicalIF":2.5,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146117693","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}

{"title":"Production and characterization of novel murine monoclonal antibodies against programmed death-ligand 1 with potential diagnostic application in immunohistochemistry","authors":"Mansoure Mansouri ,&nbsp;Mehdi Mohammadi ,&nbsp;Fatemeh Montazer ,&nbsp;Zahra Sadat Faghih ,&nbsp;Mohsen Soltanshahi ,&nbsp;Fazel Shokri ,&nbsp;Mahmood Jeddi-Tehrani ,&nbsp;Mahdi Shabani","doi":"10.1016/j.ab.2026.116067","DOIUrl":"10.1016/j.ab.2026.116067","url":null,"abstract":"<div><h3>Background</h3><div>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.</div></div><div><h3>Methods</h3><div>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.</div></div><div><h3>Results</h3><div>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.</div></div><div><h3>Conclusion</h3><div>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.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"712 ","pages":"Article 116067"},"PeriodicalIF":2.5,"publicationDate":"2026-05-01","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}

{"title":"A high-performance PdO@SWCNTs/NiO nanocomposite-based electrochemical sensor for sensitive and selective detection of hydroquinone","authors":"M. Sabbir Hossain ,&nbsp;M. Hafizur Rahman ,&nbsp;Md. A. Rashed ,&nbsp;Umme Salma ,&nbsp;Jahir Ahmed ,&nbsp;M. Faisal ,&nbsp;Jari S. Algethami ,&nbsp;Farid A. Harraz","doi":"10.1016/j.ab.2026.116046","DOIUrl":"10.1016/j.ab.2026.116046","url":null,"abstract":"<div><div>In this study, a highly sensitive electrochemical sensor with low cost is designed for determining Hydroquinone (HQ) by modifying glassy carbon (GC) electrodes with PdO@SWCNTs/NiO nanocomposite. Herein, a semiconductor metal oxide (NiO) is decorated with single-walled carbon-nanotubes (SWCNTs) and palladium oxide (PdO), using a simple ultrasonication procedure, followed by a photo-reduction approach to achieve the final PdO@SWCNTs/NiO nanocomposite. The synthesized nanocomposites' morphological and structural characteristics were effectively investigated by High-Resolution Transmission Electron Microscope (HR-TEM), Field Emission Scanning Electron Microscope (FE-SEM), X-ray Diffraction (XRD), and X-ray Photoelectron Spectroscopy (XPS) techniques. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were employed to evaluate the electro-catalytic performance. For the sensing studies, Differential Pulse Voltammetry (DPV) and amperometric (<em>i-t</em>) techniques were successfully employed, where DPV demonstrated superior sensitivity with 0.74 μAμM⁻¹cm⁻² and a low limit of detection (LOD) of 0.29 μM within the concentration range of 10 μM–495 μM of HQ. Moreover, in the amperometric technique, the designed sensor revealed superb results with a sensing concentration ranging from 60 μM to 1920 μM and a sensitivity of 0.35 μA <span><math><mrow><mi>μ</mi></mrow></math></span> M⁻¹cm⁻² with a detection limit of 0.62 μM. Furthermore, the developed sensor exhibits acceptable reproducibility, repeatability, and stability. Under experimental conditions, the examined sensor showed outstanding selectivity towards HQ in the presence of both inorganic and organic interference components. Moreover, in real-world analysis of samples, the developed sensors recover HQ with a reliable Relative Standard Deviation (%RSD) value.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116046"},"PeriodicalIF":2.5,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145905294","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}

{"title":"FTIR spectroscopy combined with machine learning reveals molecular signatures distinguishing three phenotypes of endometriosis","authors":"Piotr Olcha ,&nbsp;Wiesław Paja ,&nbsp;Michał Kępski ,&nbsp;Krzysztof Pancerz ,&nbsp;Bartosz Klebowski ,&nbsp;Łukasz Nowakowski ,&nbsp;Krzysztof Gałczyński ,&nbsp;Joanna Depciuch","doi":"10.1016/j.ab.2026.116047","DOIUrl":"10.1016/j.ab.2026.116047","url":null,"abstract":"<div><div>Endometriosis is a chronic inflammatory disorder in which endometrial tissue grows outside the uterus, leading to pelvic pain and infertility. It remains a major challenge in women's health due to delayed diagnosis and limited therapeutic outcomes. The disease is heterogeneous and manifests in three main phenotypes: superficial peritoneal lesions, ovarian endometriomas, and deep infiltrating endometriosis, the latter still poorly understood at the molecular level. In this study, Fourier-transform infrared (FTIR) spectroscopy combined with machine learning was applied to explore biochemical differences among these phenotypes. Spectral analysis demonstrated that lipid- and carbohydrate-associated vibrations, progressively increased from ovarian to bowel and were most pronounced in peritoneal endometriosis. These findings suggest phenotype-specific alterations in lipid and carbohydrate composition. Using the Boruta feature selection algorithm, discriminative spectral intervals were identified for pairwise classification. Key signals involved protein, lipid, and carbohydrate vibrations, including C<img>O and C–O stretching and N–H bending. Machine learning models: Deep Learning (DL), Support Vector Machine (SVM), and Extreme Gradient Boosting (XGBoost), were trained on both full spectra and Boruta-reduced datasets. Extending to three-class classification, Boruta-selected features enabled robust discrimination across all phenotypes. A decision tree revealed the 1752 cm⁻¹ ester C<img>O band as a critical marker for phenotype differentiation. Overall, FTIR spectroscopy combined with machine learning provides valuable molecular insights into endometriosis and represents a powerful tool for distinguishing its clinical phenotypes.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116047"},"PeriodicalIF":2.5,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145916549","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}

{"title":"Towards canine immunotherapy models: Monoclonal antibodies with redox regulated epitopes targeting TIM3 attenuate Galectin-9 binding","authors":"Jack Brydon ,&nbsp;Radovan Krejcir ,&nbsp;Mariana Grima ,&nbsp;Filip Zavadil-Kokas ,&nbsp;Zuzana Kuncova ,&nbsp;Sousan Sousan ,&nbsp;Robin L. Pflughaupt ,&nbsp;Jennifer R. Thomson ,&nbsp;Paul Davies ,&nbsp;Ella Senior ,&nbsp;Geoffrey A. Wood ,&nbsp;Kathryn L. Ball ,&nbsp;David Saliba ,&nbsp;David J. Argyle ,&nbsp;Borivoj Vojtesek ,&nbsp;Ted Hupp ,&nbsp;Maciej Parys","doi":"10.1016/j.ab.2025.116033","DOIUrl":"10.1016/j.ab.2025.116033","url":null,"abstract":"<div><div>The TIM3 receptor acts as an immune checkpoint protein. Canine cancers exhibit higher pan-cancer penetrance of TIM3 compared to PD1, highlighting the potential of TIM3 as a compelling target in comparative immuno-oncology. We have used a highly diverse naïve canine scFv phage library to isolate antibodies to canine TIM3. Alternating rounds of biopanning were performed using either Fc or GST tagged canine TIM3 synthesized in mammalian cells. scFv sequences were identified using colony screening and next generation deep sequencing (NGS). The NGS protocol identified lower abundant frequency clones, demonstrating the enhanced depth of repertoire discovery possible using sequence-tag based tracing. Three representative scFv were expressed as mouse-canine chimeric scFv-Fc fusions or as full-length chimeric IgG. These antibodies all bound to the TIM3 receptor using either ELISA or immunoblotting. All the antibodies displayed sensitivity to reducing agents, which indicates the existence of disulfide-stabilized conformational epitopes. Epitope mapping using pepscan libraries suggested that the antibodies recognize a shared structural motif within the IgV domain of TIM3, within β-sheets fixed by disulfide bonds which would form the conformational epitope. Such conformational epitopes might be functional because they overlap with ligand-binding interfaces. Consistent with this, the antibodies attenuated TIM3 binding to Galectin-9. These data affirm that naïve canine scFv antibody libraries can yield self-antigen reactive antibodies to immune blockade receptor antigens. The data also emphasize the value in using native, folded, mammalian expressed receptor antigens to increase the probability of acquiring conformationally sensitive antibodies with potential therapeutic applications in veterinary and human medicine.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116033"},"PeriodicalIF":2.5,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145772941","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}

{"title":"Mining bacterial (meta)genomes for enzymes active in aerobic, mesophilic conditions","authors":"Rodrigo Campos-Silva ,&nbsp;Fahimeh Rahimi ,&nbsp;Jaya Joshi ,&nbsp;Cătălin Voiniciuc ,&nbsp;Juannan Zhou ,&nbsp;Andrew D. Hanson","doi":"10.1016/j.ab.2026.116045","DOIUrl":"10.1016/j.ab.2026.116045","url":null,"abstract":"<div><div>Enzyme biochemistry can now draw on hundreds of thousands of prokaryotic genomes and metagenomes to identify orthologous genes for research, biocatalysis, and metabolic engineering. In many applications, adaptation to O₂ and mild temperatures are essential. But as organism lifestyle information can be poor or absent (especially for metagenomes), it is challenging to avoid orthologous genes from anaerobes and extremophiles. Taking bacterial sulfide-dependent THI4 thiazole synthases as test-cases, we built computational pipelines that use only DNA sequence inputs to explore (i) the average oxidation state of carbon (Z_C) in orthologous enzymes and (ii) the presence of O₂-metabolism genes in the corresponding (meta)genomes. Z_C has been proposed to be highest (least negative) in proteins of organisms from O₂-rich, mesophilic environments. We found that Z_C values of 2300 THI4s ranged from −0.107 (relatively oxidized) to −0.302 (strongly reduced). As predicted, genes specifying cytochrome <em>c</em> or <em>o</em> oxidases (supporting respiration at relatively high O₂ levels) and, to a lesser extent, cytochrome <em>bd</em> oxidases (which can function to scavenge O₂) were more frequent in genomes encoding THI4s with high Z_C values. Eight THI4s with Z_C values in the top 5 % and from (meta)genomes having cytochrome oxidases were tested for ability to complement a <em>THI4</em>Δ yeast strain in aerobic conditions. Three THI4 genes from a metagenome with cytochrome <em>c</em>/<em>o</em> oxidase (but without cytochrome <em>bd</em>) were active. These results support the feasibility of combining Z_C and cytochrome oxidase profiles to identify bacterial orthologous enzymes that work in aerobic, mild temperature conditions.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116045"},"PeriodicalIF":2.5,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145905422","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}

{"title":"Fast quantification of methylated nucleosides in urine by triple quadrupole mass spectrometry","authors":"Carmen Mena-Iglesias,&nbsp;Myriam Bustamante-Rangel,&nbsp;Diego García-Gómez,&nbsp;M. Teresa Fernández-del-Campo-García,&nbsp;Encarnación Rodríguez-Gonzalo,&nbsp;José Luis Pérez Pavón","doi":"10.1016/j.ab.2025.116036","DOIUrl":"10.1016/j.ab.2025.116036","url":null,"abstract":"<div><div>Modified nucleosides and nucleobases have raised great scientific interest due to their possible use as diagnostic and prognostic markers for various diseases. One of the most common types of modification is methylation. Sensitive and accurate methods are essential for the quantification of these compounds in biological samples. In this work we propose, for the first time, a rapid non-separative method for the determination of methylated nucleosides in urine by direct injection on electrospray ionization triple quadrupole mass spectrometer (ESI-MS/MS). The proposed method has been applied to the simultaneous determination of five methylated nucleosides in human urine samples. A separative method based on HILIC-MS/MS has been developed in parallel with the same instrumental setup to confirm the results obtained by the non-separative method. Both methods were validated in terms of linearity, matrix effect, limits of quantification, precision and recovery. The proposed method was successfully applied to the determination of the selected analytes in the urine of healthy volunteers and was compared with the results obtained by the separative method. The proposed non-separative method considerably reduced the required analysis time and simplified the sample treatment, which is a remarkable advantage over the chromatographic methods described. Therefore, this method is suitable for rapid, simple, and reliable determination of endogenous biomarkers in urine samples for routine or high-throughput analysis. The separative method is recommended as a complementary tool for confirmation in complex situations or in cases of abnormally high biomarker levels, maintaining specificity and robustness without compromising the practical advantages of the rapid method.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"711 ","pages":"Article 116036"},"PeriodicalIF":2.5,"publicationDate":"2026-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145832985","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}

{"title":"Fluorescence-based assay for rapid screening of GABAA receptor modulating steroid antagonists (GAMSA)","authors":"Jan Voldřich ,&nbsp;Hana Zemková ,&nbsp;Markéta Šmídková ,&nbsp;Marika Matoušová ,&nbsp;Eva Kudová ,&nbsp;Helena Mertlíková-Kaiserová","doi":"10.1016/j.ab.2026.116048","DOIUrl":"10.1016/j.ab.2026.116048","url":null,"abstract":"<div><div>GABA_A receptor modulating steroid antagonists (GAMSA) represent a promising therapeutic class by selectively inhibiting the effects of positive allosteric modulators (PAMs) at GABA_A receptors without affecting GABA-evoked currents alone. This mechanism avoids the risk of overexcitation and seizures associated with direct GABA_A 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_A (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_A 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":"2026-04-01","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}