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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.

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.

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.

Li M.,Nanjing Southeast University | Zhu M.,Nanjing Southeast University | Zhang C.,Jiangsu Academy of Agricultural Sciences | Liu X.,Jiangsu Academy of Agricultural Sciences | And 2 more authors.
Toxins | Year: 2014

Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies) sandwich-ELISA (DAS-ELISA) assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac) were selected as capture antibody (Nb61) and detection antibody (Nb44). The capture antibody (Nb61) was biotinylated in vivo for directional immobilization on wells coated with streptavidin matrix. Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 μg·mL−1 and a working range 0.010–1.0 μg·mL−1. The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system. © 2014 by the authors; licensee MDPI, Basel, Switzerland.

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