BackgroundNanobodies (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.MethodsWe 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.ResultsA 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.ConclusionThis 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.
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.
Alzheimer's disease (AD) biomarkers can reflect the neurochemical indicators used to estimate the risk in clinical nephrology. Apolipoprotein E (ApoE) is an early biomarker for AD in clinical diagnosis. In this research, through bactrian camel immunization, lymphocyte isolation, RNA extraction, and library construction, ApoE-specific Nbs with high affinity were successfully separated from an immune phage display nanobody library. Herein, a colorimetric immunosensor was developed for the point-of-care testing of ApoE by layer-by-layer nanoassembly techniques and novel nanobodies (Nbs). Using highly oriented Nbs as the capture and detection antibodies, an on-site immunosensor was developed by detecting the mean gray value of fade color due to the glutaraldehyde@3-aminopropyltrimethoxysilane oxidation by HO. The detection limit of AopE is 0.42 pg/mL, and the clinical analysis achieves a good performance. The novel easily operated immunosensor may have potential application in the clinical diagnosis and real-time monitoring for AD.
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.
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