ANALYTICA CHIMICA ACTA
·
2022
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IF 6.911
Yarong Ren,Juan Wei,Ye Wang,Peng Wang,Yanwei Ji,Baoyuan Liu,Jianlong Wang,Gualberto González-Sapienza,Yanru Wang
Salmonella are major pathogens that cause foodborne diseases. In this work, a broad-spectrum Salmonella nanobody-01 (Nb-01) was isolated and applied in the development of a streptavidin-bridged sandwich ELISA (SAB-ELISA) for simultaneously identifying five Salmonella serovars , including Salmonella Enteritidis ( S. Enteritidis), Salmonella Typhimurium ( S. Typhimurium), Salmonella London (S. London), Salmonella Paratyphi B (S. Paratyphi B) and Salmonella Hadar (S. Hadar). In this work, streptavidin (SA) was utilized as a scaffold to directionally immobilize biotinylated nanobody (BiNb) and Salmonella was detected by phage-displayed nanobodies. The SAB-ELISA can be accomplished within 180 min with a limit of detection (LOD) of 6.31 × 10 3 colony forming units (CFU) mL −1 , 9.15 × 10 3 CFU mL −1 , 4.23 × 10 3 CFU mL −1 , 7.31 × 10 3 CFU mL −1 and 7.25 × 10 3 CFU mL −1 towards S. Typhimurium, S. Enteritidis, S. London, S. Paratyphi B and S. Hadar, respectively. In comparison of sandwich ELISA by passive immobilization of Nb-01 on polystyrene plate, the sensitivity was increased by around 6-fold, which confirmed the enhanced immobilization efficacy of SAB-ELISA. Furthermore, the feasibility of the assay for S. Typhimurium determination in actual samples was evaluated, showing excellent recovery, inter-day and intra-day precision.
研究领域:
微生物学
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免疫学
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食品科学
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分子生物学
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PubMed
高影响因子
MOLECULES
·
2024
·Vol. 29
·期 2
·366
·
IF 4.2
Zhuojian Lu,Rui Ge,Bin Zheng,Peng Zheng
Antibody arrays play a pivotal role in the detection and quantification of biomolecules, with their effectiveness largely dependent on efficient protein immobilization. Traditional methods often use heterobifunctional cross-linking reagents for attaching functional residues in proteins to corresponding chemical groups on the substrate surface. However, this method does not control the antibody’s anchoring point and orientation, potentially leading to reduced binding efficiency and overall performance. Another method using anti-antibodies as intermediate molecules to control the orientation can be used but it demonstrates lower efficiency. Here, we demonstrate a site-specific protein immobilization strategy utilizingOaAEP1 (asparaginyl endopeptidase) for building a nanobody array. Moreover, we used a nanobody-targeting enhanced green fluorescent protein (eGFP) as the model system to validate the protein immobilization method for building a nanobody array. Finally, by rapidly enriching eGFP, this method further highlights its potential for rapid diagnostic applications. This approach, characterized by its simplicity, high efficiency, and specificity, offers an advancement in the development of surface-modified protein arrays. It promises to enhance the sensitivity and accuracy of biomolecule detection, paving the way for broader applications in various research and diagnostic fields.Keywords:protein immobilization;enzymatic ligation;OaAEP1;nanobody
研究领域:
生物化学
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分子生物学
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诊断学
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PubMed
高影响因子
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
·
2024
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IF 2.8
Yiwen Zhang,Yang Guo,Liang Song,Wenshuai Liu,Rui Nian,Xiying Fan
This study introduces an efficient on-column refolding and purification method for preparing nanobodies (Nbs) expressed as inclusion bodies and fusion proteins. The HisTrap TM FF system was successfully employed for the purification of the fusion protein FN1-ΔI-CM-2D5. The intein ΔI-CM cleavage activity was activated at 42 °C, followed by incubation for 4 h. Leveraging the remarkable thermal stability of Nbs, 2D5 was further purified through heat treatment at 80 °C for 1h. This method yielded up to 107.2 mg of pure 2D5 with a purity of 99.2 % from just 1L of bacterial culture grown in a shaker flask. Furthermore, this approach successfully restored native secondary structure and affinity of 2D5. Additionally, the platform was effectively applied to the refolding and purification of a polystyrene-binding nanobody (B2), which exhibited limited expression in the periplasmic and cytoplasmic spaces of E. coli . This endeavor resulted in the isolation of 53.2 mg of pure B2 Nb with a purity exceeding 99.5 % from the same volume of bacterial culture. Significantly, this approach restored the native secondary structure of the Nbs, highlighting its potential for addressing challenges associated with expressing complex Nbs in E. coli . Overall, this innovative platform provides a scientifically rigorous and reproducible method for the efficient preparation of Nbs, offering a valuable tool for antibody research and development.
研究领域:
生物技术
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蛋白质工程
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分子生物学
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抗体研究
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PubMed
高影响因子
Research
·
2022
·
IF 11.036
Song Liu,Guanghui Li,Lei Ding,Jin Ding,Qian Zhang,Dan Li,Xingguo Hou,Xiangxing Kong,Jing Zou,Shiming Zhang,Hongbin Han,Yakun Wan,Zhi Yang,Hua Zhu
Due to the rapid spread of coronavirus disease 2019 (COVID-19), there is an urgent requirement for the development of additional diagnostic tools for further analysis of the disease. The isolated nanobody Nb11-59 binds to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) with high affinity to neutralize the virus and block the angiotensin-converting enzyme 2- (ACE2-) RBD interaction. Here, we introduce a novel nanobody-based radiotracer named 68Ga-Nb1159. The radiotracer retained high affinity for the RBD and showed reliable radiochemical characteristics both in vitro and in vivo. Preclinical positron emission tomography (PET) studies of 68Ga-Nb1159 in mice revealed its rapid clearance from circulation and robust uptake into the renal and urinary systems. Fortunately, 68Ga-Nb1159 could specifically reveal the distribution of the RBD in mice. This study also helped to evaluate the pharmacodynamic effects of the neutralizing nanobody. Moreover, 68Ga-Nb1159 may be a promising tool to explore the distribution of the RBD and improve the understanding of the virus. In particular, this study identified a novel molecular radioagent and established a reliable evaluation method for specifically investigating the RBD through noninvasive and visual PET technology.
研究领域:
医学影像学
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病毒学
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药理学
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DOI
高影响因子
FOOD CONTROL
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2022
·
IF 6.652
Feng Wang,Yuan-Yuan Yang,De-Bin Wan,Jia-Dong Li,Yi-Fan Liang,Zhen-Feng Li,Yu-Dong Shen,Zhen-Lin Xu,Jin-Yi Yang,Hong Wang,Jan Gettemans,Bruce D. Hammock,Yuan-Ming Sun
The accurate analysis of chemical isomers plays an important role in the study of their different toxic effects and targeted detection of pollutant isomers in foods . The Alternaria mycotoxins tenuazonic acid (TeA) and iso-tenuazonic acid (ITeA) are two isomer mycotoxins with the lack of single analysis methods due to the similar structures. Antibody-based immunoassays exhibit high sensitivity and superior application in isomer-specific determination. Previously, various kinds of antibodies for TeA have been prepared in our group. Herein, highly specific nanobodies (Nbs) against ITeA mycotoxin were selected from immune nanobody phage display library, and one of Nbs, namely Nb(B3G3) exhibited excellent affinity, thermal stability as well as organic solvent tolerance. By molecular simulation and docking technology, it was found that stronger interaction between Nb(B3G3) and ITeA lead to higher affinity than that for its isomer TeA. Furthermore, a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was established with a limit of detection (LOD) of 0.09 ng/mL for ITeA mycotoxin. The recovery rate of ITeA in spiked samples was analyzed with 84.8%–89.5% for rice, 78.3%–96.3% for flour, and 79.5%–90.7% for bread. A conventional LC-MS/MS method was used to evaluate the accuracy of this proposed icELISA, which showed a satisfactory consistent correlation. Since the convenient strategy for nanobody generation by phage display technology, this study provide new biorecognition elements and sensitive immunoassay for analysis of ITeA in foods.
研究领域:
食品安全
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免疫学
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分析化学
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DOI
PubMed
高影响因子
PHYSIOLOGIA PLANTARUM
·
2025
·Vol. 177
·期 2
·e70163
·
IF 3.6
Weixia Liu,Mengya Li,Xiangyu Zhang,Yongqiang Ma,Qing X. Li,Christophe Morisseau,Bruce D. Hammock,Ting Xu
Triclosan (TCS) is a broad-spectrum antimicrobial disinfectant widely used in pharmaceuticals and personal care products (PPCPs). Due to the extensive usage of PPCPs, TCS inevitably entered the environment and pose harmful effects on the ecosystem. Phytoremediation is an attractive approach to remove TCS from the environment. In this study, a gene encoding the anti-TCS nanobody was transformed into Arabidopsis thaliana ( A. thaliana ) to enhance the absorption capacity of TCS. Nanobodies are small antibody fragments (ca. 15 kDa) derived from the variable domain of camelid heavy-chain-only antibodies. We constructed two transgenic lines, the T-S-C line with nanobody expression throughout the plant and the T-S-P line with nanobody expression dominant in the roots, were constructed. The expression of nanobodies in A. thaliana alleviated the phytotoxicity of TCS. T-S-C and T-S-P exhibited significantly stronger tolerance to TCS toxicity than the wild type (WT), in either a solid medium system or a hydroponics system. Under the stress of TCS, the seedlings of both transgenic plants exhibited an increase in root length and fresh weight compared to those of WT. Moreover, in the presence of TCS, the activities of superoxide dismutase, peroxidase, catalase, and glutathione in transgenic plants were higher than those in WT. The concentration of TCS absorbed into the T-S-C and T-S-P plants from the solid medium increased by 50.0% and 24.1%, and from the hydroponics system increased by 55.6% and 38.0%, respectively, compared to those absorbed by WT. This study provides a proof of principle that transforming nanobodies into plants represents a novel technology to improve the efficiency of phytoremediation for environmental pollutants.
研究领域:
植物学
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环境科学
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分子生物学
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DOI
PubMed
高影响因子
JOURNAL OF HAZARDOUS MATERIALS
·
2019
·
IF 7.65
Zongwen Tang,Xing Liu,Benchao Su,Qi Chen,Hongmei Cao,Yonghuan Yun,Yang Xu,Bruce D. Hammock
Ochratoxin A (OTA) is a major concern for public health and the rapid detection of trace OTA in food is always a challenge. To minimize OTA exposure to consumers, a nanobody (Nb)-mediated förster resonance energy transfer (FRET)-based immunosensor using quantum dots (Nb-FRET immunosensor) was proposed for ultrasensitive, single-step and competitive detection of OTA in agro-products at present work. QDs of two sizes were covalently labeled with OTA and Nb, acting as the energy donor and acceptor, respectively. The free OTA competed with the donor to bind to acceptor, thus the FRET efficiency increased with the decrease of OTA concentration. The single-step assay could be finished in 5 min with a limit of detection of 5 pg/mL, which was attributed to the small size of Nb for shortening the effective FRET distance and improving the FRET efficiency. The Nb-FRET immunosensor exhibited high selectivity for OTA. Moreover, acceptable accuracy and precision were obtained in the analysis of cereals and confirmed by the liquid chromatography-tandem mass spectrometry. Thus the developed Nb-FRET immunosensor was demonstrated to be an efficient tool for ultrasensitive and rapid detection of OTA in cereals and provides a detection model for other toxic small molecules in food and environment.
研究领域:
食品安全
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生物技术
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分析化学
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DOI
高影响因子
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
·
2025
·
IF 2.8
Xiao Ding,Shuo Wang,Wei Wang,Jingjing Sun,Chengcheng Jiang,Jianhua Hao
Recent studies on immunoaffinity chromatography (IAC) columns for aflatoxin B 1 (AFB 1 ) have not provided a comprehensive preparation procedure or explored their capacity, reusability, and stability. In this study, we outline a preparation process for an AFB 1 immune affinity column filler (AFB 1 -IAC) for extracting AFB 1 from food sources like rice, peanuts, and soybeans. Using commercially available IAC is often limited due to high costs, low capacity, slow flow rates, and poor reusability. To overcome these issues, we developed succinic anhydride (SA)-modified agarose pre-crosslinked microspheres (SA-A) activated with 1,1′‑carbonyldiimidazole (CDI). The activated microspheres were coupled with the anti-AFB 1 nanobody Nb02, creating an immunoaffinity packing called CDI-SA-A-Nb02 (D1-IAC). After verification, the results indicated that the column capacity of D1-IAC was about 4.67 times that of several commercial IAC brands. Additionally, the maximum flow rate at a pressure of 0.04 MPa was 25 % greater than specific commercial IAC, and the recovery rate remained above 80 % after being reused five times. This method is considered effective for extracting and detecting AFB 1 in food products. The preparation process for D1-IAC shows great consistency, and the column's performance is better than that of available immunoaffinity columns. This study explores using an IAC to extract and purify AFB 1 , which is then quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).
研究领域:
食品安全
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生物化学
·
分析化学
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PubMed
高影响因子
JOURNAL OF CHROMATOGRAPHY A
·
2024
·
IF 3.8
Yuxiang Zhang,Yao Zhang,Luyao Zhang,Yongdong Liu
The use of nanobodies (Nbs) in affinity chromatography for biomacromolecule purification is gaining popularity. However, high-performance Nb-based affinity resins are not readily available, mainly due to the lack of suitable immobilization methods. In this study, we explored an autocatalytic coupling strategy based on the SpyCatcher/SpyTag chemistry to achieve oriented immobilization of Nb ligands. To facilitate this approach, a variant cSpyCatcher003 (cSC003) was coupled onto agarose microspheres, providing a specific attachment site for SpyTagged nanobody ligands. The cSC003 easily purified from Escherichia coli through a two-step procedure, exhibits exceptional alkali resistance and structural recovery capability, highlighting its robustness as a linker in the coupling strategy. To validate the effectiveness of cSC003-derivatized support, we employed VHSA, a nanobody against human serum albumin (HSA), as the model ligand. Notably, the immobilization of SpyTagged VHSA onto the cSC003-derivatized support was achieved with a coupling efficiency of 90 %, significantly higher than that of traditional thiol-based coupling method. This improvement directly correlated to the preservation of the native conformation of nanobodies during the coupling process. In addition, the Spy-immobilized resin demonstrated better performance in the binding capacity, with a 3-fold improvement in capture efficiency, underscoring the advantages of the Spy immobilization strategy for oriented immobilization of VHSA ligands. Moreover, online purification and immobilization of SpyTagged VHSA from crude bacterial lysate was achieved using the cSC003-derivatized support. The resulting resin exhibited high binding specificity towards HSA, yielding a purity above 95 % directly from human serum, and maintained good stability throughout multiple purification cycles. These findings highlight the potential of the Spy immobilization strategy for developing Nb-based affinity chromatographic materials, with significant implications for biopharmaceutical downstream processes.
研究领域:
生物化学
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分子生物学
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生物工程
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2025
·
IF 6
Zhihao Xu,Man Liu,Qiyi He,Wenkai Li,Junkang Pan,El-Sayed A. El-Sheikh,Wenzhi Liu,Bruce D. Hammock,Dongyang Li
Aquatic viruses cause devastating diseases in aquaculture, severely limiting production and resulting in significant economic losses. The giant salamander iridovirus (GSIV), a member of the genus Ranavirus , is the only virus reported to infect giant salamanders, posing a severe threat to the farming industry. Currently, there are no effective strategies available for its control. In this context, a reliable diagnostic tool for the rapid detection of GSIV is crucial to mitigate its impact. This study developed a nanobody-based immunoassay for the rapid and reliable detection of GSIV. GSIV was cultured and used to immunize an alpaca, A phage library with an original diversity of 1.89 × 10 8 PFU was established, and six nanobodies were identified after three rounds of panning. Among them, HC-2 exhibited superior performance and was used to develop a highly sensitive ELISA method employing streptavidin-PolyHRP (SA-PolyHRP) as a signal amplification strategy. The assay achieved a detection limit of 3.3 × 10 5 PFU/mL and demonstrated high specificity without cross-reactivity. Practical application was validated in infected giant salamander samples, underscoring its diagnostic potential. This work provides a robust tool for GSIV diagnosis and showcases the potential of nanobodies in advancing aquaculture diagnostics.
研究领域:
水产养殖学
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病毒学
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诊断学
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DOI
PubMed
高影响因子
TALANTA
·
2024
·
IF 5.6
Wanmei Guo,Yongsen Yu,Chao Xin,Guangyong Jin
In this study, we present a novel biomarker detection platform employing a modified S-tapered fiber coated with gold nanoparticle/graphene oxide (GNP/GO) for quantifying human epidermal growth factor receptor-2 (HER2) concentrations, using antibodies as sensing elements. The fabrication of this device involves implementing an in-situ layer-by-layer technique coupled with a chemical adsorption step to achieve the self-assembly of GNP, GO, and antibodies on the STF surface. The detection mechanism relies on monitoring the refractive index changes induced by the adsorption of HER2 onto the immobilized antibodies. For comparative analysis, both monoclonal antibody (mAb) and the novel nanobody (Nb) were employed in constructing the STF immunosensor, referred to as the mAb immunosensor and Nb immunosensor, respectively. Spectral analysis results highlight that the Nb immunosensor exhibits twice the sensitivity of the mAb immunosensor. This enhanced sensitivity is attributed to the small size, high antigen affinity, strong specificity, and structural stability of Nb. The Nb immunosensor demonstrated an impressive detection limit of 0.001 nM for HER2, surpassing the detection limit of the mAb immunosensor. These findings underscore the potential of the proposed Nb immunosensor as a promising and sensitive tool for HER2 detection, contributing to the diagnosis and prognosis of breast cancer. Furthermore, the simplicity of production and excellent optical performance position the Nb immunosensor as a prospective real-time biosensor with minimal cytotoxicity.
研究领域:
生物医学工程
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分子生物学
·
癌症研究
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DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Junjie Zeng,Yanping Li,Qinghua He,Ji Xia,Jiebiao Guo,Zhui Tu
Phage display-mediated immuno-polymerase chain reaction (PD-IPCR) combines the strengths of phage display technology and immuno-PCR. The link between genotype and phenotype has been established by displaying antibody fragments on the surface of phage particles, allowing signal transmission. In this study, we utilized aflatoxin B 1 (AFB 1 ) as a model system to investigate the efficacy of PD-IPCR. Signal amplification was achieved using both real-time fluorescence quantitative PCR (qPCR) and a custom-made droplet digital PCR (dPCR) system. Under optimized conditions, the assay using dPCR achieved a limit of detection of 0.015 ng/mL, a 6-fold improvement over qPCR. The validation of our method using major agricultural produce, such as corn, flour, and soy sauce, shows excellent recoveries ranging from 85.77 % to 108.28 %, with a total assay time of less than 2 h. Furthermore, we compared our results with a high-performance liquid chromatography (HPLC) method for AFB 1 content in corn and flour, revealing consistent outcomes (r = 0.9847). These results demonstrate the potential of nanobody-based PD-IPCR as a reliable tool for detecting mycotoxins.
研究领域:
食品安全
·
分子生物学
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）