Jiangsu Nanobody Engineering and Research Center

Nantong, China

Jiangsu Nanobody Engineering and Research Center

Nantong, China
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Li H.,Nanjing Southeast University | Mu Y.,Nanjing Southeast University | Yan J.,Nanjing Southeast University | Cui D.,Nanjing Southeast University | And 4 more authors.
Analytical Chemistry | Year: 2015

Acute renal failure (ARF) represents a very important and potentially devastating disorder in clinical nephrology. Neutrophil gelatinase-associated lipocalin (NGAL) is an early biomarker for ARF in a wide range of different disease processes, which is frequently detected in clinical diagnosis. Herein, we present a label-free and sensitive photoelectrochemical (PEC) immunosensor for NGAL by utilizing a biotinylated anti-NGAL Nanobody (Nb) orientedly immobilized to streptavidin-coated cobalt 2,9,16,23-tetraaminophthalocyanine (CoPc)-sensitized TiO2 electrode. The Nb was biotinylated at the C-terminus, which is situated at the opposite site of the antigen binding region. Using highly oriented Nb as receptor molecules, a label-free PEC immunosensor for NGAL was developed by monitoring the changes in the photocurrent signals of the electrode resulting from immunoreaction. Immobilization of Nb to streptavidin-coated CoPc-sensitized TiO2 electrode surface provides high binding capacity to NGAL; thus, it can lead to a high sensitivity. The limit of detection (LOD) of the proposed immunosensor has been significantly lowered to 0.6 pg mL-1. This proposed immunosensor reveals high specificity to detect NGAL, with acceptable intra-assay precision and excellent stability. In addition, the present work provides a new approach to design Nb-based PEC immunosensor and increases versatility of Nbs. © 2015 American Chemical Society.


Wang P.,Nanjing Southeast University | Li G.,Nanjing Southeast University | Yan J.,Nanjing Southeast University | Hu Y.,Nanjing University | And 4 more authors.
Toxicon | Year: 2014

The variable domain of the heavy-chain-only antibody (VHH) or nanobody (Nb), derived from camelids, begins to play an important role on the detection of protein markers. In this study, we constructed a phage-displayed library of VHHs against Cry1Fa by immunizing a healthy Bactrian camel with Cry1Fa toxin. After a series of bio-panning and screening by phage display technology, three anti-Cry1Fa nanobodies (Nbs) with great difference in complementarity determining region 3 (CDR3) were obtained and they were highly specific to Cry1Fa as well as showed full of activity when exposed to 70°C for 3 h. Through modifying Nbs with Horseradish Peroxidase (HRP) and biotin, two Nbs which can recognize the different epitopes of Cry1Fa were determined and they were used to establish a novel sandwich immune ELISA based on biotin-SA interaction for Cry1Fa detection. The immunoassay exhibited a linear range from 1 to 100 ng/mL with a detection limit of 0.88 ng/mL. The recoveries from spiked corn and soybean samples were ranged from 83.33 to 117.17%, with a coefficient of variation (C.V) less than 6.0%. All together, the proposed immunoassay will be a promising way for sensitive and accurate determination of Cry1Fa toxin. © 2014 Elsevier Ltd. All rights reserved.


Liu H.,Nanjing Southeast University | Li G.,Nanjing Southeast University | Liu L.,Nanjing Southeast University | Wan Y.,Nanjing Southeast University | Wan Y.,Jiangsu Nanobody Engineering and Research Center
Bioscience Reports | Year: 2015

Chromatin structure is implicated in regulating gene transcription in stress response. Transcription factors, transferases and deacetylases, such as multicopy suppressor of SNF1 protein 2 (Msn2), SET domain-containing protein 1 (Set1) and sucrose NonFermenting protein 1 (Snf1), have been identified as key regulators in stress response. In the present study, we reported the dynamics of nucleosome occupancy, Histone Two A Z1 (Htz1) deposition and histone H3 lysine 4 dimethylation (H3K4me2) and histone H3 lysine 79 trimethylation (H3K79me3) in Saccharomyces cerevisiae under oleate stress. Our results indicated that citrate cycle-associated genes are enhanced and ribosome genes are repressed during the glucose-oleate shift. Importantly, Htz1 acts as a sensor for oleate stress. High-throughput ChIP-chip analysis showed that Htz1 has redistributed across the genome during oleate stress. The number of Htz1-bound genes increases with stress and the number of Htz1-bound ribosome genes decreases with stress. The dynamics of Htz1 and H3K79me3 around transcription factor-binding sites correlate with transcriptional changes. Moreover, we found that nucleosome dynamics are coupled with Htz1 binding changes upon stress. In unstressed conditions (2% glucose), nucleosome occupancy is comparable between Htz1-bound genes and Htz1-depleted genes; in stressed conditions (0.2% oleate for 8 h), the nucleosome occupancy of Htz1-depleted genes is significantly lower than that of Htz1-bound genes. We also found that Msn2 acts an important role in response to the oleate stress and Htz1 is dynamic in Msn2-target genes. Htz1 senses the oleate stress and undergoes a global redistribution and this change couples dynamics of nucleosome occupancy. Our analysis suggests that Htz1 and nucleosome dynamics change in response to oleate stress. © 2015 Authors.


Li G.,CAS Shanghai Institute of Materia Medica | Zhu M.,CAS Shanghai Institute of Materia Medica | Ma L.,Nanjing Southeast University | Yan J.,Nanjing Southeast University | And 4 more authors.
ACS Applied Materials and Interfaces | Year: 2016

A phage display library of variable domain of the heavy chain only antibody or nanobody (Nb) was constructed after immunizing a bactrian camel with testosterone. With the smaller molecular size (15 kDa), improved solubility, good stability, high affinity, specificity, and lower immunogenicity, Nbs are a promising tool in the next generation of diagnosis and medical applications. Testosterone is a reproductive hormone, playing an important role in normal cardiac function and being the highly predictive marker for many diseases. Herein, a simple and sensitive immunosensor based on electrochemical impedance spectroscopy (EIS) and Nbs was successfully developed for the determination of testosterone. We successfully isolated the antitestosterone Nbs from an immune phage display library. Moreover, one of the Nbs was biotinylated according to in vivo BirA system, which showed the highest production yield and the most stable case. Further, the EIS immunosensor was set up for testosterone detection by applying the biotinylated antitestosterone Nb. As a result, the biosensor exhibited a linear working range from 0.05 to 5 ng mL-1 with a detection limit of 0.045 ng mL-1. In addition, the proposed immunosensor was successfully applied in determining testosterone in serum samples. In conclusion, the proposed immunosensor revealed high specificity of testosterone detection and showed as a potential approach for sensitive and accurate diagnosis of testosterone. © 2016 American Chemical Society.


Li H.,Nanjing Southeast University | Yan J.,Nanjing Southeast University | Ou W.,Jiangsu Nanobody Engineering and Research Center | Liu H.,Plexera LLCWA | And 3 more authors.
Biosensors and Bioelectronics | Year: 2015

Nanobodies (Nbs), also known as the variable domain of the heavy-chain-only antibody (VHH), are single-domain antigen-binding fragments derived from heavy-chain antibodies that occur naturally in sera of camelids. Due to their unique properties of small size (15kD), intrinsic stability, high affinity and specificity, Nbs are suitable for detecting clinical relevant antigens. Apolipoprotein B-100 (ApoB-100) is a highly predictive marker for coronary artery disease (CAD), which is frequently detected in clinical diagnosis. Herein, we successfully obtained anti-ApoB-100 Nbs for the first time and further fabricated a label-free and sensitive immunosensor for ApoB-100 based on isolated anti-ApoB-100 nanobody (Nb) using the electrochemical impedance spectroscopy (EIS) technique. We have generated an immunized phage display library against ApoB-100 and isolated four anti-ApoB-100 Nbs with high affinity and stability. The Nb with the highest affinity was biotinylated based on in vivo BirA system. Further, we developed a label-free electrochemical impedance immunosensor for ApoB-100 using this anti-ApoB-100 Nb. The attachment of ApoB-100 onto the anti-ApoB-100 Nb-immobilized sensing layer led to the increased electron-transfer resistance, which was proportional to ApoB-100 concentration in the range from 0.05 to 5ngmL-1 with a detection limit of 0.03ngmL-1. This proposed immunosensor revealed high specificity to detect ApoB-100, acceptable intra-assay precision and good stability, functioning as a feasible technique for CAD diagnosis. © 2014 Elsevier B.V.


Ma L.,Nanjing Southeast University | Sun Y.,Nanjing Southeast University | Kang X.,Nanjing Southeast University | Wan Y.,Nanjing Southeast University | Wan Y.,Jiangsu Nanobody Engineering and Research Center
Biosensors and Bioelectronics | Year: 2014

Nanobodies, derived from camelid heavy-chain antibodies, have novel and impactful applications in clinical diagnostics. Our objective is to develop a nanobody-based chemiluminescence immunoassay for sensitive detection of human prealbumin (PA). In this context, a phage display nanobody library is constructed via immunizing dromedary camel with human prealbumin. Three nanobodies have been identified by five successive bio-panning steps. Based on their high expression level and good affinity, two out of three are chosen for further study. Magnetic beads (MBs) were functionalized with PEI by acylamide bond formed between the carboxyl group on the surface of the MB. Then, an anti-PA nanobody (Nb1) can be effectively immobilized onto the surface of the functionalized MB using glutaradehyde as the link. The modified MBs with Nb1 can specifically capture the target PA and reacted with silica nanoparticles with co-immobilized HRP and anti-PA nanobody (Nb2). The concentration of PA was detected by flow injection chemiluminescence. When using MB/PEI as the carrier of anti-PA Nb1, the CL signal significantly increased to 4-fold compared with the signal using MB without PEI modification. The CL signal was further amplified to 5-fold when Si/Nb2 was used as the signal probe. Under optimized conditions, the present immunoassay exhibited a wide quantitative range from 0.05 to 1000μgL-1 with a detection limit of 0.01μgL-1. The sensitivity of the proposed immunoassay offers great promises in providing a sensitive, specific, time saving, and potential method for detecting PA in clinical settings. © 2014 Elsevier B.V.


Li H.,Nanjing Southeast University | Sun Y.,Nanjing Southeast University | Elseviers J.,Nanobody Service Facility | Muyldermans S.,Vrije Universiteit Brussel | And 4 more authors.
Analyst | Year: 2014

The development of a nanobody-based electrochemiluminescent immunosensor for procalcitonin quantification is described. A highly specific and enhanced sensitivity of target detection was achieved by CdTe quantum dot encapsulated silica nanoparticle-assisted signal amplification. This journal is © the Partner Organisations 2014.


Yan J.,Nanjing Southeast University | Li G.,Nanjing Southeast University | Hu Y.,Nanjing University of Technology | Ou W.,Jiangsu Nanobody Engineering and Research Center | And 2 more authors.
Journal of Translational Medicine | Year: 2014

Nanobodies (Nbs) have proved their great value as therapeutic molecules and clinical diagnostic tools. Although the routine procedure to obtain Nbs is to immunize camels with antigens, it is unavailable to immunize a camel when the antigens are highly toxic, pathogenic or nonimmunogenic. A synthetic phage display library is an alternative to generate Nbs against such targets, besides all the other ones. Methods: We constructed a large and diverse synthetic phage display Nanobody (Nb) library based on the conserved camel single-domain antibody fragment (VHH) framework of cAbBCII10. Diversity was introduced in the complementarity-determining region 3 (CDR3) by means of randomization of synthetic oligonucleotides. Then human prealbumin (PA) and neutrophil gelatinase-associated lipocalin (NGAL) were used to select specific Nbs from this library. Furthermore, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect PA based on horseradish peroxidase (HRP)-conjugated anti-PA Nb isolated from this study and another biotinylated anti-PA Nb obtained from an immune library, in our previous study. Results: A large and diverse synthetic phage display Nb library with CDR3 regions randomized by trinucleotide cassettes was constructed. The library size was 1.65×109 CFU/mL and the correct insertion ratio was nearly 100%. A Nb against human PA and against NGAL was successfully isolated from the synthetic library. The obtained anti-PA Nb was effectively used to develop a sandwich ELISA for PA detection and it demonstrated a working range from 50 to 1000 ng/mL, with a limit of detection (LOD) of 27.1 ng/mL. Conclusion: This proposed novel synthetic library was a good source for obtaining some antigen-specific Nbs. This approach could provide crucial support to an immune library and a naïve library in the acquisition of specific Nbs, potentially functioning as a great resource for medical diagnostic applications. In addition, we have successfully developed a novel sandwich ELISA to detect PA, which could provide great assistance for clinical PA detection. © Yan et al.


PubMed | Jiangsu Nanobody Engineering and Research Center, Nanjing University of Technology, 302 Military Hospital of China and Nanjing Southeast University
Type: Journal Article | Journal: Nanoscale research letters | Year: 2014

Our objective is to develop a rapid and sensitive assay based on magnetic beads to detect the concentration of influenza H3N2. The possibility of using variable domain heavy-chain antibodies (nanobody) as diagnostic tools for influenza H3N2 was investigated. A healthy camel was immunized with inactivated influenza H3N2. A nanobody library of 810(8) clones was constructed and phage displayed. After three successive biopanning steps, H3N2-specific nanobodies were successfully isolated, expressed in Escherichia coli, and purified. Sequence analysis of the nanobodies revealed that we possessed four classes of nanobodies against H3N2. Two nanobodies were further used to prepare our rapid diagnostic kit. Biotinylated nanobody was effectively immobilized onto the surface of streptavidin magnetic beads. The modified magnetic beads with nanobody capture specifically influenza H3N2 and can still be recognized by nanobodies conjugated to horseradish peroxidase (HRP) conjugates. Under optimized conditions, the present immunoassay exhibited a relatively high sensitive detection with a limit of 50 ng/mL. In conclusion, by combining magnetic beads with specific nanobodies, this assay provides a promising influenza detection assay to develop a potential rapid, sensitive, and low-cost diagnostic tool to screen for influenza infections.


PubMed | Jiangsu Nanobody Engineering and Research Center, Nanjing University of Technology and Nanjing Southeast University
Type: | Journal: Journal of translational medicine | Year: 2015

Nanobodies (Nbs) have proved their great value as therapeutic molecules and clinical diagnostic tools. Although the routine procedure to obtain Nbs is to immunize camels with antigens, it is unavailable to immunize a camel when the antigens are highly toxic, pathogenic or nonimmunogenic. A synthetic phage display library is an alternative to generate Nbs against such targets, besides all the other ones.We constructed a large and diverse synthetic phage display Nanobody (Nb) library based on the conserved camel single-domain antibody fragment (VHH) framework of cAbBCII10. Diversity was introduced in the complementarity-determining region 3 (CDR3) by means of randomization of synthetic oligonucleotides. Then human prealbumin (PA) and neutrophil gelatinase-associated lipocalin (NGAL) were used to select specific Nbs from this library. Furthermore, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect PA based on horseradish peroxidase (HRP)-conjugated anti-PA Nb isolated from this study and another biotinylated anti-PA Nb obtained from an immune library, in our previous study.A large and diverse synthetic phage display Nb library with CDR3 regions randomized by trinucleotide cassettes was constructed. The library size was 1.6510(9)CFU/mL and the correct insertion ratio was nearly 100%. A Nb against human PA and against NGAL was successfully isolated from the synthetic library. The obtained anti-PA Nb was effectively used to develop a sandwich ELISA for PA detection and it demonstrated a working range from 50 to 1000ng/mL, with a limit of detection (LOD) of 27.1ng/mL.This proposed novel synthetic library was a good source for obtaining some antigen-specific Nbs. This approach could provide crucial support to an immune library and a nave library in the acquisition of specific Nbs, potentially functioning as a great resource for medical diagnostic applications. In addition, we have successfully developed a novel sandwich ELISA to detect PA, which could provide great assistance for clinical PA detection.

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