MOLECULES
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2019
·Vol. 24
·期 11
·2119
·
IF 3.06
Lijun Zhang,Berlin Zang,Chundong Huang,Jun Ren,Lingyun Jia
Dialysis-related amyloidosis (DRA), which has been widely recognized to be associated with the accumulation of β2-microglobulin (β2-m) in blood, is one of the most common complications in patients receiving long-term dialysis treatment. The most significant side-effect of existing hemodialysis sorbents for the removal of β2-m from blood is the loss of vital proteins due to non-specific adsorptions. Although the traditional antibodies have the capability to specifically remove β2-m from blood, high cost limits their applications in clinics. Single domain antibodies derived from the Camelidae species serve as a superior choice in the preparation of immunoadsorbents due to their small size, high stability, amenability, simplicity of expression in microbes, and high affinity to recognize and interact with β2-m. In this study, we modified the anti-β2-m VHH by the formylglycine-generating enzyme (FGE), and then directly immobilized the aldehyde-modified VHH to the amino-activated beads. Notably, the fabrication is cost- and time-effective, since all the preparation steps were performed in the crude cell extract without rigorous purification. The accordingly prepared immunoadsorbent with VHHs as ligands exhibited the high capacity of β2-m (0.75 mg/mL). In conclusion, the VHH antibodies were successfully used as affinity ligands in the preparation of novel immunoadsorbents by the site-specific immobilization, and effectively adsorbed β2-m from blood, therefore opening a new avenue for efficient hemodialysis.Keywords:β2-microglobulin;VHH;formylglycine-generating enzyme;hemodialysis
研究领域:
生物医学工程
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分子生物学
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透析治疗
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DOI
PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
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2016
·Vol. 408
·期 22
·6019-6026
·
IF 3.125
Wang Yanru,Li Peiwu,Zhang Qi,Hu Xiaofeng,Zhang Wen
A toxin-free enzyme-linked immunosorbent assay (ELISA) for aflatoxins was developed using an anti-idiotype nanobody VHH 2–5 as surrogate standard. Anti-idiotype nanobody VHH 2–5 was generated by immunizing an alpaca with anti-aflatoxin monoclonal antibody 1C11. This assay was used to detect aflatoxins in agro-products after a simple extraction with 75 % methanol/H 2 O. Aflatoxin concentration was calculated by a two-step approach: the concentration of VHH 2–5 was first obtained by a four-parameter logistic regression from the detected absorbance value at 450 nm, and then converted to aflatoxin concentration by a linear equation. The assay exhibits a limit of detection (LOD) of 0.015 ng mL −1 , which is better than or comparable with conventional immunoassays. The performance of our VHH surrogate-based ELISA was further validated with a high-performance liquid chromatography (HPLC) method for total aflatoxins determination in 20 naturally contaminated peanut samples, displaying a good correlation ( R
2 = 0.988). In conclusion, the proposed assay represents a first example applying an anti-idiotype VHH antibody as a standard surrogate in ELISA. With the advantages of high stability and ease of production, the VHH antibody-based standard surrogate can be extended in the future to immunoassays for other highly toxic compounds.
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研究领域:
食品安全
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免疫学
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2018
·
IF 6.042
Yu-Qi Zhang,Zhen-Lin Xu,Feng Wang,Jun Cai,Jie-Xian Dong,Jin-Ru Zhang,Rui Si,Cheng-Long Wang,Yu Wang,Yu-Dong Shen,Yuanming Sun,Hong Wang
A heavy chain variable fragment of heavy chain only antibodies derived from camelids termed VHH shows beneficial characteristics for immunoassay in terms of high sensitivity, outstanding stability and ease in expression. In the present study, we isolated six VHHs from phage display library against parathion, which is a widely used organophosphorus pesticide with high toxicity and persistence. One of six selected VHHs named VHH9, showed highest specificity and superior thermo-stability. A VHH9-alkaline phosphatase (AP) fusion was constructed and used to establish a one-step direct competitive fluorescence enzyme immunoassay (dc-FEIA) with a half maximal inhibitory concentration (IC50) of 1.6 ng/mL and a limit of detection of 0.2 ng/mL which was 4-fold or 3-fold higher sensitivity than direct competitive enzyme-linked immunoassay (dc-ELISA) and indirect competitive enzyme-linked immunoassay (ic-ELISA) for parathion. Furthermore, our assay indicated a 50% reduction on operation time compared with the ic-ELISA method. The presented immunoassay was validated with spiked Chinese cabbage, cucumber, and lettuce samples, and confirmed by UPLC-MS/MS. The results indicated that the VHH-AP-based dc-FEIA is a reproducible detection assay for parathion residues in vegetable samples.
研究领域:
生物化学
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分析化学
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环境科学
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农业科学
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DOI
PubMed
高影响因子
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
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2015
·
IF 2.729
Zhui Tu,Yang Xu,Jinheng Fu,Zhibing Huang,Yao Wang,Bin Liu,Yong Tao
This work aimed to evaluate novel affinity resin used to purify immunoglobulin G (IgG) with a variable domain of the heavy chain of the heavy-chain antibody (VHH) as an affinity ligand. The VHH, isolated from a naïve camelid single-domain phage display library, exhibits not only affinity to the fragment crystallizable (Fc) region of IgG but also high thermal stability . This anti-Fc VHH (AFV) was expressed as a soluble protein in Escherichia coli and purified using a simple heat treatment procedure. The effects of pH and NaCl concentrations on the capacity of AFV resin were also investigated. Results showed a robust property of the AFV resin. It could bind IgGs at various pH conditions (from 6.0 to 9.0) and NaCl concentrations. The static binding capacities of AFV resin ranged from 3.40 ± 0.53 mg/ml to 15.04 ± 0.37 mg/ml measured using rabbit, mouse, and human IgGs. The bound IgGs can be efficiently eluted at pH 5.0, which is conducive to acid-sensitive IgGs and prevents the aggregation of IgGs. After 10 purification cycles or a 7-day period of storage at 37 °C, recovery did not decrease. These findings suggested that VHHs from non-immunized library could also be robust and functional reagent as an affinity purification ligand.
研究领域:
生物技术
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生物化学
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分子生物学
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蛋白质工程
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2026
·
IF 6.1
Congcong Jia,Min Zhu,Yingtung Lo,Meixiang Yu,Xinyi Xia,Weiwei Ji,Xiang Cao,Yakun Wan,Jianzhong Lu,Hai Zhang
Background Early screening for venous thromboembolism and the precise use of anticoagulant drugs are crucial, and further basic innovations are urgently needed to advance related diagnostic and therapeutic practices. This study aimed to develop an innovative antibody for detecting D-dimer and to verify its performance. The core aim was to screen a pair of variable domains of the heavy chain of heavy-chain antibodies (VHH) that can specifically recognize unique epitopes on the D-dimer molecule. This study is expected to promote technological advancements in the diagnosis, risk assessment, and efficacy monitoring of thrombotic diseases. Results D-dimer-positive clones underwent positive and negative screening across seven rounds of phage biopanning, yielding a pair of VHHs. The superior thermal stability of the sandwich VHH pair was demonstrated after continuous heating at 80 °C for up to 1 h by comparison with a commercial monoclonal antibody. Subsequently, by integrating the reliable and convenient Luminex platform with these novel VHHs, a new detection method for D-dimer was established under optimal conditions, with a wide quantitative range of 0.5 to 1000 ng/mL and detection limit as low as 0.25 ng/mL. The recovery rate of spiked samples ranged from 99.3% to 106.0%, with a relative standard deviation of 1.1% to 3.2%. Furthermore, this newly developed VHH-based Luminex assay was utilized to examine clinical D-dimer samples, and its performance was evaluated by correlation analysis with the clinical INNOVANCE® D-Dimer Assay, resulting in a Pearson correlation coefficient of 0.99086 (P < 0.0001). Significance This study identified a novel VHH pair targeting D-dimer, demonstrating good binding activity and exceptional thermal stability, making it highly effective for bioanalysis experiments. Utilizing the reliable and efficient Luminex platform, a detection method for D-dimer was developed, exhibiting substantial potential for applications in venous thrombus screening, anticoagulant efficacy assessment, and related fundamental research.
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DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
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2021
·
IF 4.821
Chong Cai,Qi Zhang,Seyni Nidiaye,Honglin Yan,Wen Zhang,Xiaoqian Tang,Peiwu Li
Anti-idiotypic antibodies (AIds) display stable protein conformation, offering an alternative to conventional antigens in immunoassays. In this work, an anti-idiotypic variable domain of heavy-chain antibody (VHH) or nanobody named VHH C4 binding to monoclonal antibody (mAb) 2C9 against aflatoxin M 1 (AFM 1 ) was screened out from an alpaca immunized phage-displayed library. VHH C4 shows outstanding features in specificity and sensitivity, the IC 50 is 0.25 ng/mL and exhibit almost no cross-reactivity with other aflatoxin analogs. As our knowledge, it is the first report for developing an anti-idiotypic nanobody for AFM 1 . An environment-friendly competitive ELISA was developed using VHH C4 as a coating antigen. The results revealed a linear range from 0.10 ng/mL to 0.60 ng/mL, with a limit of detection (LOD) of 0.05 ng/mL. The recovery of spiked samples (pure milk, yogurt, and milk powder) ranged from 84.1% to 119.1%. The ELISA results of AFM 1 content in naturally contaminated samples were in good agreement with those determined by high performance liquid chromatography (HPLC). The results demonstrated that the nanobody VHH C4 is a promising alternative antigen in developing immunological methods for the detection of AFM 1 in dairy products.
研究领域:
免疫学
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食品科学
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分析化学
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产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）