Analytical biochemistry最新文献_第7页

ITGB3 as a promising non-invasive biomarker for type 2 diabetes and diabetic nephropathy ITGB3有望成为2型糖尿病和糖尿病肾病的无创生物标志物

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-08-25 DOI: 10.1016/j.ab.2025.115965

Seyed Amirhossein Hosseini , Parisa Ajorlou , Hasti Haddadian , Shahla Sohrabipour

The prevalence of Type 2 diabetes mellitus (T2DM) is increasing worldwide and represents a major risk factor for the development of diabetic nephropathy (DN), a severe microvascular complication. Chronic hyperglycemia activates inflammatory and fibrotic signaling pathways, which contribute to kidney damage. Integrins, as transmembrane adhesion receptors, play pivotal roles in regulating inflammation, immune cell trafficking, and insulin resistance. This research focused on identifying non-invasive biomarkers for T2DM and DN using PBMCs. Differentially expressed genes related to diabetes were identified through the analysis of multiple datasets retrieved from the Gene Expression Omnibus, including GSE95849, GSE9006, GSE25724, and GSE159984. ITGB3 was identified as a common gene across these datasets, and its expression in DN was further examined using the GSE142025 dataset. Real-time PCR analysis of PBMC samples revealed a significant upregulation of ITGB3 expression in individuals with DN and T2DM compared to healthy controls. The TF2DNA and miRNASNPv3 databases identified 10 transcription factors and 10 variants of ITGB3 involved in 60 miRNA interactions. Additionally, the DGIdb database revealed 15 drugs potentially regulating ITGB3 expression. These findings underscore the importance of integrin-related pathways in diabetes and suggest ITGB3 as a promising target for future research and therapeutic development.

2型糖尿病（T2DM）的患病率在全球范围内呈上升趋势，是糖尿病肾病（DN）发展的主要危险因素，是一种严重的微血管并发症。慢性高血糖会激活炎症和纤维化信号通路，从而导致肾脏损伤。整合素作为跨膜粘附受体，在调节炎症、免疫细胞运输和胰岛素抵抗中起关键作用。本研究的重点是利用pbmc识别T2DM和DN的非侵入性生物标志物。通过分析从Gene Expression Omnibus检索到的多个数据集，包括GSE95849、GSE9006、GSE25724和GSE159984，鉴定出与糖尿病相关的差异表达基因。ITGB3被鉴定为这些数据集中的共同基因，并使用GSE142025数据集进一步检测其在DN中的表达。PBMC样本的实时PCR分析显示，与健康对照相比，DN和T2DM患者的ITGB3表达显著上调。TF2DNA和miRNASNPv3数据库鉴定了参与60种miRNA相互作用的10个转录因子和10个ITGB3变体。此外，DGIdb数据库还发现了15种可能调节ITGB3表达的药物。这些发现强调了整合素相关通路在糖尿病中的重要性，并表明ITGB3是未来研究和治疗开发的有希望的靶点。

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引用次数: 0

A novel dual-function sustainable method for H2O2 determination and dye biomineralization utilizing peroxidase-like MnFe2O4@CrFe2O4 nanocomposite 利用过氧化物酶样MnFe2O4@CrFe2O4纳米复合材料的新型双功能可持续的H2O2测定和染料生物矿化方法

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-08-22 DOI: 10.1016/j.ab.2025.115962

Saeed Reza Hormozi Jangi , Masood Ranjoori , Anahita Barghi , Pardis Ahmadi

In this contribution, a novel dual-function sustainable green nanozyme-mediated method for highly sensitive and selective H₂O₂ quantification and reusable rhodamine B biomineralization utilizing highly active MnFe₂O₄@CrFe₂O₄ nanocomposite with synergistic peroxidase-like activity was designed and developed. This method also introduced a sustainable approach for probing the analyte instead of exploiting prevalent carcinogenic nanozyme-based analytical probes, making it absolutely more sustainable than the conventional methods. The hydrogen peroxide biosensor acquired a linear range of 1–100 μM and a very low detection limit of 0.6 μM, along with an inter-day %RSD of 2.74 % and a highly selective response against coexisting materials. Ultimately, the sensor was employed for H₂O₂ quantification in milk, revealing a recovery of 96.1–102.8 %, %RSD = 1.4–3.6 %. Besides, the effective factors on decolorization yield were optimized, providing a high biomineralization yield of 99.4 % at optimal experimental conditions within a short time of 35.0 min. The breakthrough volume, storage stability, and reusability of the nanozymes were assessed, revealing a breakthrough volume of 5.0–1000 mL, a shelf-life of 20 days, and 70 % yield saving after 10 cycles. The method was applied for dye degradation in real water media, including river water, pool water, and tap water, revealing a high yield of over 95.4–99.5 %, %RSD = 1.8–4.2 %.

本文设计并开发了一种新型的双功能可持续绿色纳米酶介导的方法，利用具有协同过氧化物酶样活性的高活性MnFe2O4@CrFe2O4纳米复合材料，用于高灵敏度和选择性的H2O2定量和可重复使用的罗丹明B生物矿化。该方法还引入了一种可持续的方法来探测分析物，而不是利用流行的致癌纳米酶分析探针，使其绝对比传统方法更具可持续性。过氧化氢生物传感器的线性范围为1 ~ 100 μM，检测限为0.6 μM，日间RSD为2.74%，对共存材料具有高选择性响应。最终将该传感器用于牛奶中H2O2的定量，回收率为96.1 ~ 102.8%,%RSD = 1.4 ~ 3.6%。此外，对影响脱色率的因素进行了优化，在最佳实验条件下，在35.0 min的短时间内，生物矿化率达到99.4%。对纳米酶的突破体积、储存稳定性和可重复使用性进行了评估，结果表明，纳米酶的突破体积为5.0-1000 mL，保质期为20天，10次循环后产量节约70%。将该方法应用于河流、池水、自来水等实际水介质中染料的降解，收率达95.4 ~ 99.5%以上，%RSD = 1.8 ~ 4.2%。

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引用次数: 0

Development of species-specific PCR for authentication of medicinal plant Bupleuri Radix and its common adulterants 药用植物柴胡及其常见掺假物种特异性PCR鉴定方法的建立

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-08-14 DOI: 10.1016/j.ab.2025.115963

Hui Tian , Xiuna Zhang , Chi Ma , Yidi Yang , Jing Fang , Fanna Qu , Lihong Yang

Bupleuri Radix is a widely used herbal plant, and its official source recognized by Chinese pharmacopoeia is the dried roots of Bupleurum chinense DC. or Bupleurum scorzonerifolium Willd. Although two species share core therapeutic functions, differences in the types of chemical components lead to different clinical applications. Currently, two species have not been distinguished in pharmacopeial standards. Therefore, we hypothesize prescribing them separately based on the pharmacological characteristics may provide better clinical efficacy. Additionally, Bupleurum marginatum Wall. ex DC. var stenophyllum (Wolff) Shan et Y. Li, and Bupleurum bicaule Helm were often misused as Bupleuri Radix. To address above issues, we developed species-specific PCR targeting ITS region. The observed fragments of 252bp, 183bp, 272bp and 165bp correspond to B. chinense, B. scorzonerifolium, B. marginatum var. stenophyllum, and B. bicaule respectively. The PCR exhibited strong discriminative capability, even when analyzing artificial adulterate samples. Furthermore, analysis of commercial samples validated the accuracy of the species information. In summary, the species-specific PCR enables accurate authentication of Bupleuri Radix and common adulterants in the market. It provides a practical tool for quality control of Bupleuri Radix and supports standardization of herbal medicine production.

柴胡是一种应用广泛的中草药植物，其官方来源为中国药典认可的柴胡干根。或柴胡，野柴胡。虽然两种植物共享核心治疗功能，但化学成分类型的差异导致了不同的临床应用。目前，药典标准中未对两种进行区分。因此，我们假设根据其药理特点分别处方可能会有更好的临床疗效。此外，柴胡壁。交货。var stenophyllum (Wolff) Shan et Y. Li和Bupleurum bicaule Helm经常被误用为柴胡根。为了解决上述问题，我们开发了针对ITS区域的物种特异性PCR。252bp、183bp、272bp和165bp的片段分别对应于B. chinense、B. scorzonerifolium、B. marginatum vars . stenophyllum和B. bicaule。即使在分析人工掺假样品时，PCR也表现出较强的判别能力。此外，对商业样本的分析验证了物种信息的准确性。综上所述，物种特异性PCR能够准确鉴别柴胡和市场上常见的掺假物。为柴胡的质量控制提供了实用的工具，为中草药生产的标准化提供了支持。

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引用次数: 0

HCNS:A deep learning model for identifying essential proteins based on hypergraph convolution and sequence features HCNS：一种基于超图卷积和序列特征识别必需蛋白质的深度学习模型

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-08-07 DOI: 10.1016/j.ab.2025.115949

Jialong Tian , Pengli Lu , Huining Sha

Accurate identification of essential proteins is crucial in biomedical research. Traditional methods rely on Protein–Protein Interaction (PPI) networks and limited biological feature data, often neglecting the critical role of protein amino acid sequences. To address this, we propose the HCNS model, which integrates the Hypergraph Convolutional Network (HGCN) module, the Seq-CNN-MB-NAG feature extraction module, and the Multi-Layer Perceptron (MLP) recognition module, significantly enhancing the accuracy of essential protein identification. The HGCN module constructs a hypergraph from the PPI network and protein complex data, capturing the complex structural relationships between proteins. The Seq-CNN-MB-NAG module utilizes Convolutional Neural Networks (CNN), Multi-Head Self-Attention (MHSA), Bidirectional Long Short-Term Memory (Bi-LSTM), and NAG Transformer to process protein amino acid sequences and extract sequence features. The MLP module then fuses these two sets of features for precise recognition. Experimental results show that the HCNS model outperforms existing methods, achieving an accuracy of 93.38%, with an Area Under the Curve (AUC) of 98.33% and an Area Under the Precision–Recall Curve (AUPR) of 97.16%, demonstrating its potential in essential protein identification.

准确鉴定必需蛋白质在生物医学研究中至关重要。传统的方法依赖于蛋白质-蛋白质相互作用（PPI）网络和有限的生物学特征数据，往往忽略了蛋白质氨基酸序列的关键作用。为了解决这个问题，我们提出了HCNS模型，该模型集成了超图卷积网络（HGCN）模块、Seq-CNN-MB-NAG特征提取模块和多层感知器（MLP）识别模块，显著提高了必需蛋白质识别的准确性。HGCN模块利用PPI网络和蛋白质复合体数据构建超图，捕捉蛋白质之间复杂的结构关系。Seq-CNN-MB-NAG模块利用卷积神经网络（CNN）、多头自注意（MHSA）、双向长短期记忆（Bi-LSTM）和NAG Transformer处理蛋白质氨基酸序列并提取序列特征。然后，MLP模块融合这两组特征以进行精确识别。实验结果表明，HCNS模型的准确率为93.38%，曲线下面积（Area Under the Curve， AUC）为98.33%，精确召回曲线下面积（Area Under Precision-Recall Curve， AUPR）为97.16%，优于现有的方法，显示了其在必需蛋白鉴定中的潜力。

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引用次数: 0

K-SNOpred: Identification of protein S-nitrosylation sites through word embedding features and machine learning K-SNOpred：通过词嵌入特征和机器学习识别蛋白质s -亚硝基化位点。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-08-06 DOI: 10.1016/j.ab.2025.115952

Tasmin Karim , Md Shazzad Hossain Shaon , Md Mamun Ali , Sobhy M. Ibrahim , Mst Shapna Akter , Kawsar Ahmed , Francis M. Bui , Li Chen , Mohammad Ali Moni

Protein S-nitrosylation (SNO) is a process involving the covalent modification of cysteine residues by nitric oxide (NO) and its derivatives. Numerous studies have demonstrated that SNO is significantly involved in cell function and pathophysiology. The identification of SNO sites is significant in clarifying their function in cellular physiology, disease processes, and potential treatment strategies, rendering it of paramount importance in medical science. This study developed a machine learning (ML) model named “K-SNOpred” and found notable performance in identifying SNO sites using the Latent Semantic Analysis (LSA) feature embedding system. After collecting dbSNO and RecSNO datasets from the literature search, we applied three feature embedding systems: Doc2vec, FastText, and LSA on each dataset. The study employed various ML models and assessed their performance using multiple evaluation metrics through independent testing and 10-fold cross-validation. The evaluation's outcomes demonstrate that the proposed model achieved an accuracy of 87.56 % and an AUC score of 95.06 %, outperforming existing state-of-the-art (SOTA) models by nearly 10 % in accuracy and 6 % in AUC. Furthermore, the model demonstrated balanced sensitivity and specificity, indicating its ability to detect both positive and negative SNO sites accurately. The outstanding performance of the K-SNOpred model demonstrates its high potential for clinical use and its applicability in the biotechnology field.

蛋白质s -亚硝基化（SNO）是一氧化氮（NO）及其衍生物对半胱氨酸残基进行共价修饰的过程。大量研究表明，SNO在细胞功能和病理生理中起着重要作用。SNO位点的鉴定对于阐明其在细胞生理学、疾病过程和潜在治疗策略中的功能具有重要意义，因此在医学上具有至关重要的意义。本研究开发了一个名为“K-SNOpred”的机器学习（ML）模型，并发现使用潜在语义分析（LSA）特征嵌入系统在识别SNO位点方面具有显着的性能。在从文献检索中收集dbSNO和RecSNO数据集之后，我们在每个数据集上应用了三种特征嵌入系统：Doc2vec、FastText和LSA。本研究采用了多种机器学习模型，并通过独立测试和10倍交叉验证使用多种评估指标评估其性能。评估结果表明，该模型的准确率为87.56%，AUC分数为95.06%，比现有的最先进（SOTA）模型的准确率和AUC分数分别高出近10%和6%。此外，该模型表现出平衡的敏感性和特异性，表明其能够准确检测阳性和阴性SNO位点。K-SNOpred模型的卓越性能表明其在临床应用和生物技术领域的适用性方面具有很高的潜力。

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引用次数: 0

A sensitive split luciferase assay for detecting olfactory receptor response to odorants 一种灵敏的荧光素酶分裂试验，用于检测嗅觉受体对气味的反应。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-08-05 DOI: 10.1016/j.ab.2025.115954

Natsumi Tanazawa , Sho Obayashi , Shuji Hinuma , Shun'ichi Kuroda

In this study, olfactory receptors (ORs) and Golf proteins fused with split luciferase were examined to develop a sensitive assay for identifying OR ligands. Although specific luminescence was observed from HEK293T cells expressing both fusion proteins (OR1A1-LgBiT and SmBiT-Mini-Golf) in response to an OR1A1 ligand, its intensity was low. To improve it, a fusion protein (V1N-G4S3-SmBiT-G4S3-Mini-Golf), was created where V1N contained the palmitoylation site necessary for localizing Mini-Golf to the cell membrane. When OR1A1-LgBiT and V1N-G4S3-SmBiT-G4S3-Mini-Golf were co-expressed in HEK293T cells, specific luminescence by ligand stimulation was considerably increased. The cell surface expression of OR1A1-LgBiT was markedly lower than that of OR1A1. Co-expression of receptor transporting protein 1 short form (RTP1S) promoted the cell surface expression of OR1A1-LgBiT more efficiently than OR1A1. Prediction using AlphaFold3 suggested that the N-terminal domain of RTP1S interacts with LgBiT in OR1A1-LgBiT. Co-expression of OR1A1-LgBiT, V1N-G4S3-SmBiT-G4S3-Mini-Golf, and RTP1S in HEK293T cells enhanced the sensitivity for detecting at least some ligands by 1000- to 3000-fold compared to the conventional cAMP assay. These findings suggest that optimizing the assembly of OR1A1-LgBiT and SmBiT-Mini-Golf at the cell membrane is essential for developing a sensitive assay. The OR response-detection system employing split luciferase holds significant potential for investigating ligands of orphan ORs.

在这项研究中，研究嗅觉受体（ORs）和与分裂荧光素酶融合的Golf蛋白，以建立一种识别OR配体的敏感试验。虽然在表达OR1A1- lgbit和SmBiT-Mini-Golf两种融合蛋白的HEK293T细胞中观察到对OR1A1配体的特异性发光，但其强度很低。为了改进它，一个融合蛋白（V1N- g4s3 - smbit - g4s3 -Mini-Golf）被创造出来，其中V1N含有将Mini-Golf定位到细胞膜所必需的棕榈酰化位点。当OR1A1-LgBiT和V1N-G4S3-SmBiT-G4S3-Mini-Golf在HEK293T细胞中共表达时，配体刺激的特异性发光显著增加。OR1A1- lgbit的细胞表面表达量明显低于OR1A1。受体转运蛋白1短链（RTP1S）的共表达比OR1A1更有效地促进OR1A1- lgbit的细胞表面表达。利用AlphaFold3进行预测表明，RTP1S的n端结构域与OR1A1-LgBiT中的LgBiT相互作用。OR1A1-LgBiT、V1N-G4S3-SmBiT-G4S3-Mini-Golf和RTP1S在HEK293T细胞中的共表达，与传统的cAMP检测相比，至少可以将检测某些配体的灵敏度提高1000至3000倍。这些发现表明，优化OR1A1-LgBiT和SmBiT-Mini-Golf的组装对于建立灵敏的检测方法至关重要。采用分裂荧光素酶的OR反应检测系统在研究孤儿OR配体方面具有重要的潜力。

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引用次数: 0

Characterization and in vitro antioxidant activity of C-phycocyanin from the cyanobacterium Spirulina subsalsa HKAR-19 下螺旋藻HKAR-19中c -藻蓝蛋白的表征及体外抗氧化活性研究

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-08-04 DOI: 10.1016/j.ab.2025.115953

Jyoti Jaiswal, Ashish P. Singh, Rajeshwar P. Sinha

C-phycocyanin is a natural blue-colored pigment-protein complex existing as phycobiliprotein in cyanobacteria. C-phycocyanin is made up of two different subunits of α- and -β monomers. These subunits assemble to form a cylindrical structure known as phycocyanobilin chromophore. In the present study, C-phycocyanin was purified from the cyanobacterium Spirulina subsalsa HKAR-19 using ammonium sulfate precipitation followed by sucrose density gradient ultracentrifugation methods and characterized by various spectroscopic techniques. The purified C-phycocyanin showed an absorption peak at 615 nm. A characteristic fluorescence emission peak at 642 nm when excited over 615 nm, indicates the integrity and functionality of protein structure. The scavenging activity against free radicals of DPPH, ABTS, and superoxide (O₂·⁻) was evaluated by using free radical scavenging assays. C-phycocyanin showed dose-dependent in vitro antioxidative properties towards free radicals. It possesses higher antioxidant activity toward ABTS^•+ and DPPH^•. Due to its high antioxidant activity, it can be used as a colorant and therapeutic agent against oxidative stress.

c -藻蓝蛋白是一种天然的蓝色色素蛋白复合物，以藻胆蛋白的形式存在于蓝藻细菌中。c -藻蓝蛋白由α-和-β单体的两个不同亚基组成。这些亚基组合形成一个圆柱形结构，称为藻蓝胆素发色团。本研究采用硫酸铵沉淀-蔗糖密度梯度超离心的方法从藻蓝杆菌下螺旋藻HKAR-19中纯化出c -藻蓝蛋白，并用各种光谱技术对其进行了表征。纯化后的c -藻蓝蛋白在615 nm处有吸收峰。在615 nm以上激发时，在642 nm处有一个特征荧光发射峰，表明蛋白质结构的完整性和功能性。通过自由基清除试验评估其对DPPH、ABTS和超氧化物（O2·−）自由基的清除活性。c -藻蓝蛋白对自由基的体外抗氧化性能表现出剂量依赖性。对ABTS•+和DPPH•具有较高的抗氧化活性。由于其高抗氧化活性，它可以用作着色剂和抗氧化应激治疗剂。

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引用次数: 0

Advances in aptamer technology and its updated applications in gastric cancer 适体技术及其在胃癌中的最新应用进展

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-08-03 DOI: 10.1016/j.ab.2025.115950

Sayedeh Azimeh Hosseini , Arghavan Rakhshani Nejad , Hassan Valadbeigi , Mohammad Hossein Haddadi , Monireh Bazdar

Gastric cancer (GC) is one of the leading causes of cancer-related mortality worldwide, with late-stage diagnosis and limited treatment efficacy. The major challenges in chemotherapy and radiotherapy for GC include drug resistance, systemic toxicity, limited tumor selectivity, adverse side effects, tumor heterogeneity, and the development of radioresistance, all of which hinder treatment efficacy and patient outcomes. Aptamers, single-stranded DNA or RNA molecules with high specificity and affinity for molecular targets, offer a promising alternative to traditional diagnostic and therapeutic approaches. Due to their high specificity and low immunogenicity, aptamers improve non-invasive diagnostic techniques, enabling early GC detection and targeted therapeutic applications. Therapeutically, aptamers facilitate precise drug targeting, reducing systemic toxicity and improving treatment outcomes, especially for drug-resistant and metastatic cases. Integration with nanotechnology further enhances their potential, enabling theranostic applications that combine diagnosis and therapy. Despite these advancements, challenges such as physiological stability, renal clearance, and tissue permeability remain, necessitating further research into chemical modifications and nanocarrier-based delivery systems. This study provides a comprehensive review of recent advancements in aptamer technology and its evolving applications in GC, with a particular emphasis on diagnostic and therapeutic strategies. The first section explores aptamer development strategies, including emerging selection techniques and molecular modifications to enhance their specificity and stability. The subsequent section examines the role of aptamers in GC diagnostics and treatment, with a focus on targeted chemotherapy and immunotherapy. Lastly, we discuss the current challenges in aptamer-based approaches and outline future perspectives for their clinical translation.

胃癌（GC）是全球癌症相关死亡的主要原因之一，诊断较晚，治疗效果有限。胃癌化疗和放疗面临的主要挑战包括耐药、全身毒性、有限的肿瘤选择性、不良副作用、肿瘤异质性和放射耐药的发展，这些都阻碍了治疗效果和患者预后。适配体是一种单链DNA或RNA分子，对分子靶点具有高特异性和亲和力，为传统的诊断和治疗方法提供了一种有希望的替代方法。由于其高特异性和低免疫原性，适体改善了非侵入性诊断技术，使早期GC检测和靶向治疗应用成为可能。在治疗上，适体促进精确的药物靶向，减少全身毒性和改善治疗结果，特别是对耐药和转移病例。与纳米技术的结合进一步增强了它们的潜力，使诊断和治疗相结合的治疗应用成为可能。尽管取得了这些进展，但生理稳定性、肾脏清除率和组织渗透性等方面的挑战仍然存在，因此需要进一步研究化学修饰和基于纳米载体的递送系统。本研究全面回顾了适体技术的最新进展及其在GC中的应用，特别强调了诊断和治疗策略。第一部分探讨适体发展策略，包括新兴的选择技术和分子修饰，以提高其特异性和稳定性。下一节探讨适体在胃癌诊断和治疗中的作用，重点是靶向化疗和免疫治疗。最后，我们讨论了目前基于适配体的方法所面临的挑战，并概述了其临床翻译的未来前景。

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引用次数: 0

A modular and universal platform for neutralizing-antibody profiling by quantifying antigen-receptor interactions via qPCR 通过qPCR定量抗原-受体相互作用的中和抗体分析的模块化和通用平台

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-07-30 DOI: 10.1016/j.ab.2025.115951

Xia Li , Qi Chen , Yating Zhu , Haofei Hong , Zhifang Zhou , Zhimeng Wu , Jie Shi

Rapid and accurate detection of neutralizing antibody (nAb) is essential for assessing vaccine efficacy and immune protection against infectious diseases. In the present study, we developed a neutralization-antibody detection qPCR platform (NAD-qPCR) that quantifies nAb potency by integrating antigen-receptor binding specificity with qPCR sensitivity. Using the SARS-CoV-2 nAb as a proof-of-concept target, this system comprised a hybrid probe with the viral RBD as an antigen module covalently conjugated to a reporter DNA, and magnetic beads functionalized with the ACE2 mimic mini-protein as a receptor module, thereby recapitulating the natural antigen-receptor interactions. With this design, the neutralizing capability of nAb against the antigen was transformed into a competitive reduction of the probe binding and DNA amplification signal by qPCR. The NAD-qPCR demonstrated robust performance in quantifying commercial SARS-CoV-2 nAb with a LOD of 4 ng/μL. Furthermore, it was demonstrated to be capable of effectively profiling a large number of vaccinated donor serum with broad neutralization activity. Notably, the hybrid probe and the capture beads were designed modularly, which allows for straightforward adaptation to other pathogens by replacing the module component. This platform offers a rapid, high-throughput, and universally applicable approach for nAb detection in vaccine development and immune monitoring.

快速、准确地检测中和抗体（nAb）对于评估疫苗效力和对传染病的免疫保护至关重要。在本研究中，我们开发了一种中和抗体检测qPCR平台（NAD-qPCR），通过整合抗原受体结合特异性和qPCR敏感性来定量nAb的效力。该系统以SARS-CoV-2 nAb作为概念验证靶点，包括以病毒RBD作为抗原模块共价偶联到报告DNA的杂交探针，以及与ACE2模拟迷你蛋白功能化的磁珠作为受体模块，从而概括了天然抗原-受体相互作用。通过这种设计，将nAb对抗原的中和能力转化为通过qPCR竞争性地减少探针结合和DNA扩增信号。NAD-qPCR在商业SARS-CoV-2 nAb定量中表现出稳健的性能，LOD为4 ng/μL。此外，它被证明能够有效地分析大量具有广泛中和活性的接种供体血清。值得注意的是，杂交探针和捕获珠是模块化设计的，通过替换模块组件，可以直接适应其他病原体。该平台为疫苗开发和免疫监测中的nAb检测提供了一种快速、高通量和普遍适用的方法。

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引用次数: 0

Colorimetric assays for the detection of C4- and C2-epimerization of NDP-glucose to NDP-galactose and NDP-mannose 检测ndp -葡萄糖向ndp -半乳糖和ndp -甘露糖的C4-和c2 -外映体化的比色法

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-07-18 DOI: 10.1016/j.ab.2025.115947

Ulrike Vogel, Renger Dijkstra, Koen Beerens, Tom Desmet

CDP-tyvelose 2-epimerase-like enzymes and UDP-glucose 4-epimerases belong to the Nucleotide Sugar active Short-Chain Dehydrogenase/Reductase (NS-SDR) superfamily and convert NDP-Glc to NDP-Man and NDP-Gal, respectively. The product detection is well established using HPLC-based methods, capillary electrophoresis and thin layer chromatography for both epimerization reactions. However, the lack of a reliable colorimetric assays and poor substrate availability slow down the screening process of these enzymes, for example during enzyme engineering projects. After testing 16 phenolic compounds with different aldohexoses in concentrated sulfuric acid, thymol was found suitable for the detection of (NDP-) mannose and 1-naphthol for the detection of (NDP-) galactose in the presence of (NDP-) glucose. As proof-of-concept, the optimized assay was used for the biochemical characterization of the CDP-tyvelose 2-epimerase from Ardenticatenia bacterium.

CDP-tyvelose 2- epimase样酶和UDP-glucose 4- epimase属于核苷酸糖活性短链脱氢酶/还原酶（NS-SDR）超家族，分别将NDP-Glc转化为NDP-Man和NDP-Gal。采用基于高效液相色谱的方法，毛细管电泳和薄层色谱对这两种外映反应进行了很好的检测。然而，缺乏可靠的比色测定和较差的底物可用性减慢了这些酶的筛选过程，例如在酶工程项目中。在浓硫酸中检测了16种不同醛己糖的酚类化合物，发现百里香酚适用于（NDP-）甘露糖的检测，1-萘酚适用于（NDP-）葡萄糖存在下（NDP-）半乳糖的检测。作为概念验证，优化后的检测方法被用于Ardenticatenia细菌的CDP-tyvelose 2- epimase的生化表征。

引用次数: 0