scholarly journals Selection and Characterization of CSFV-Specific Single-Domain Antibodies and Their Application along with Immunomagnetic Nanobeads and Quantum Dots

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Shunli Yang ◽  
Li Yuan ◽  
Youjun Shang ◽  
Jinyan Wu ◽  
Xiangtao Liu ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Magnetic Beads ◽  
Molecular Probes ◽  
Single Domain ◽  
Molecular Probe ◽  
Economic Losses ◽  
Single Domain Antibody ◽  
Swine Industry ◽  
E2 Protein

Outbreak of classical swine fever (CSF) results in high mortality and thus causes severe economic losses in the swine industry. Single-domain antibody (sdAb) is the smallest antigen-binding molecule derived from camelid heavy-chain antibodies and has the potential to be used as a molecular probe for detection of CSF virus (CSFV). In this study, two sdAb fragments against the E2 antigen of CSFV were obtained, expressed in vitro. The functional characteristics analysis indicated that the recombinant sdAbE2-1 and sdAbE2-2 have excellent binding activity, specificity, and high affinity with equilibrium constant value of 3.34 × 10−7 and 1.35 × 10−8 M to E2 protein. Then, sdAbE2s were conjugated with quantum dots (QD)/AF488 to synthesize two molecular probes for imaging CSFV distribution in cells. The sdAbE2-1 was also labeled with carboxyl-magnetic beads to construct immunomagnetic nanobeads (IMNBs) able to capture CSFV virions and recombinant E2 protein. QD/AF455-sdAbE2s probes colocalised with CSFV virions in swine testis cells, and IMNBs were used as a detection template and proved to bind specifically with CSFV virions and E2 protein. The selected sdAb fragments and sdAb-based molecular probes may be used for the rapid identification of CSFV during field outbreaks and for research on CSFV and host interactions.

Production, characterization and in-vitro applications of single-domain antibody against thyroglobulin selected from novel T7 phage display library

2021 ◽  
Vol 492 ◽  
pp. 112990
Author(s):  
Jothivel Kumarasamy ◽  
Samar Kumar Ghorui ◽  
Chandrakala Gholve ◽  
Bharti Jain ◽  
Yogesh Dhekale ◽  
...  
Keyword(s):  
Phage Display ◽  
Phage Display Library ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
T7 Phage Display ◽  
T7 Phage

scholarly journals Aptamer-targeting of Aleutian mink disease virus (AMDV) can be an effective strategy to inhibit virus replication

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Taofeng Lu ◽  
Hui Zhang ◽  
Jie Zhou ◽  
Qin Ma ◽  
Wenzhuo Yan ◽  
...  
Keyword(s):  
Disease Virus ◽  
Magnetic Beads ◽  
Inhibitory Effect ◽  
Three Dimensions ◽  
Economic Losses ◽  
Effective Vaccine ◽  
Stem Loop ◽  
Aleutian Mink Disease Virus ◽  
Random Library

AbstractAleutian mink disease (AMD), which is caused by Aleutian mink disease virus (AMDV), is an important contagious disease for which no effective vaccine is yet available. AMD causes major economic losses for mink farmers globally and threatens some carnivores such as skunks, genets, foxes and raccoons. Aptamers have exciting potential for the diagnosis and/or treatment of infectious viral diseases, including AMD. Using a magnetic beads-based systemic evolution of ligands by exponential enrichment (SELEX) approach, we have developed aptamers with activity against AMDV after 10 rounds of selection. After incubation with the ADVa012 aptamer (4 μM) for 48 h, the concentration of AMDV in the supernatant of infected cells was 47% lower than in the supernatant of untreated cells, whereas a random library of aptamers has no effect. The half-life of ADVa012 was ~ 32 h, which is significantly longer than that of other aptamers. Sequences and three dimensions structural modeling of selected aptamers indicated that they fold into similar stem-loop structures, which may be a preferred structure for binding to the target protein. The ADVa012 aptamer was shown to have an effective and long-lasting inhibitory effect on viral production in vitro.

scholarly journals Streptococcus pluranimalium 2N12 Exerts an Antagonistic Effect Against the Swine Pathogen Actinobacillus pleuropneumoniae by Producing Hydrogen Peroxide

2021 ◽  
Vol 8 ◽  
Author(s):  
Katy Vaillancourt ◽  
Michel Frenette ◽  
Marcelo Gottschalk ◽  
Daniel Grenier
Keyword(s):  
Hydrogen Peroxide ◽  
Culture Supernatant ◽  
Actinobacillus Pleuropneumoniae ◽  
Antagonistic Effect ◽  
Economic Losses ◽  
Protective Mechanism ◽  
Upper Respiratory Tract ◽  
Lactate Oxidase ◽  
Swine Industry

Actinobacillus pleuropneumoniae is the causal agent of porcine pleuropneumonia, a highly contagious and often deadly respiratory disease that causes major economic losses in the swine industry worldwide. The aim of the present study was to investigate the hydrogen peroxide (H2O2)-dependent antagonistic activity of Streptococcus pluranimalium 2N12 (pig nasal isolate) against A. pleuropneumoniae. A fluorimetric assay showed that S. pluranimalium produces H2O2 dose- and time-dependently. The production of H2O2 increased in the presence of exogenous lactate, suggesting the involvement of lactate oxidase. All 20 strains of A. pleuropneumoniae tested, belonging to 18 different serovars, were susceptible to H2O2, with minimal inhibitory concentrations and minimal bactericidal concentrations ranging from 0.57 to 2.3 mM. H2O2, as well as a culture supernatant of S. pluranimalium, killed planktonic cells of A. pleuropneumoniae. Treating the culture supernatant with catalase abolished its bactericidal property. H2O2 was also active against a pre-formed biofilm-like structure of A. pleuropneumoniae albeit to a lesser extent. A checkerboard assay was used to show that there were antibacterial synergistic interactions between H2O2 and conventional antibiotics, more particularly ceftiofur. Based on our results and within the limitations of this in vitro study, the production of H2O2 by S. pluranimalium could be regarded as a potential protective mechanism of the upper respiratory tract against H2O2-sensitive pathogens such as A. pleuropneumoniae.

scholarly journals Conjugation of biotin-coated luminescent quantum dots with single domain antibody-rhizavidin fusions

2016 ◽  
Vol 10 ◽  
pp. 56-65 ◽  
Author(s):  
Jinny L. Liu ◽  
Scott A. Walper ◽  
Kendrick B. Turner ◽  
Audrey Brozozog Lee ◽  
Igor L. Medintz ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
Luminescent Quantum Dots

scholarly journals Inhibition of Tau seeding by targeting Tau nucleation core within neurons with a single domain antibody fragment

2021 ◽  
Author(s):  
Clément Danis ◽  
Elian Dupré ◽  
Orgeta Zejneli ◽  
Raphaëlle Caillierez ◽  
Alexis Arrial ◽  
...  
Keyword(s):  
Antibody Fragment ◽  
Optimal Strategies ◽  
Phage Display Library ◽  
Biochemical Properties ◽  
Single Domain ◽  
Tau Proteins ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
Intracellular Expression

Tau proteins aggregate into filaments in brain cells in Alzheimer's disease and related disorders referred to as tauopathies. Here, we used fragments of camelid heavy-chain-only antibodies (VHHs or single domain antibody fragments) targeting Tau as immuno-modulators of its pathologic seeding. A VHH issued from the screen against Tau of a synthetic phage-display library of humanized VHHs was selected for its capacity to bind Tau microtubule-binding domain, composing the core of Tau fibrils. This lead VHH was optimized to improve its biochemical properties and to act in the intracellular compartment, resulting in VHH Z70. VHH Z70 was more efficient than the lead to inhibit in vitro Tau aggregation in heparin-induced assays. Expression of VHH Z70 in a cellular model of Tau seeding also decreased the fluorescence-reported aggregation. Finally, intracellular expression of VHH Z70 in the brain of an established tauopathy mouse seeding model demonstrated its capacity to mitigate accumulation of pathological Tau. VHH Z70, by targeting Tau inside brain neurons, where most of the pathological Tau resides, provides a new tool to explore the optimal strategies of immunotherapy in tauopathies.

Delivery of single-domain antibodies into neurons using a chimeric toxin–based platform is therapeutic in mouse models of botulism

2021 ◽  
Vol 13 (575) ◽  
pp. eaaz4197
Author(s):  
Shin-Ichiro Miyashita ◽  
Jie Zhang ◽  
Sicai Zhang ◽  
Charles B. Shoemaker ◽  
Min Dong
Keyword(s):  
Motor Neurons ◽  
Therapeutic Protein ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Exposure Treatment ◽  
Post Exposure ◽  
In Vitro Characterization ◽  
Endosomal Membranes ◽  
Delivery Platform

Efficient penetration of cell membranes and specific targeting of a cell type represent major challenges for developing therapeutics toward intracellular targets. One example facing these hurdles is to develop post-exposure treatment for botulinum neurotoxins (BoNTs), a group of bacterial toxins (BoNT/A to BoNT/G) that are major potential bioterrorism agents. BoNTs enter motor neurons, block neurotransmitter release, and cause a paralytic disease botulism. Members of BoNTs such as BoNT/A exhibit extremely long half-life within neurons, resulting in persistent paralysis for months, yet there are no therapeutics that can inhibit BoNTs once they enter neurons. Here, we developed a chimeric toxin–based delivery platform by fusing the receptor-binding domain of a BoNT, which targets neurons, with the membrane translocation domain and inactivated protease domain of the recently discovered BoNT-like toxin BoNT/X, which can deliver cargoes across endosomal membranes into the cytosol. A therapeutic protein was then created by fusing a single-domain antibody (nanobody) against BoNT/A with the delivery platform. In vitro characterization demonstrated that nanobodies were delivered into cultured neurons and neutralized BoNT/A in neurons. Administration of this protein in mice shortened duration of local muscle paralysis, restoring muscle function within hours, and rescued mice from systemic toxicity of lethal doses of BoNT/A. Fusion of two nanobodies, one against BoNT/A and the other against BoNT/B, created a multivalent therapeutic protein able to neutralize both BoNT/A and BoNT/B in mice. These studies provide an effective post-exposure treatment for botulism and establish a platform for intracellular delivery of therapeutics targeting cytosolic proteins and processes.

scholarly journals Amentoflavone AmelioratesStreptococcus suis-Induced InfectionIn VitroandIn Vivo

2018 ◽  
Vol 84 (24) ◽  
Author(s):  
Xue Shen ◽  
Xiaodi Niu ◽  
Gen Li ◽  
Xuming Deng ◽  
Jianfeng Wang
Keyword(s):  
Bacterial Resistance ◽  
Cell Model ◽  
Streptococcus Suis ◽  
Chinese Herbs ◽  
Economic Losses ◽  
Swine Industry ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Anti Inflammatory

ABSTRACTStreptococcus suis, an important zoonotic pathogen, has caused considerable economic losses in the swine industry and severe public health issues worldwide. The development of a novel effective strategy for the prevention and therapy ofS. suisis urgently needed. Here, amentoflavone, a natural biflavonoid compound isolated from Chinese herbs that has negligible anti-S. suisactivity, was identified as a potent antagonist of suilysin (SLY)-mediated hemolysis without interfering with the expression of SLY. Amentoflavone effectively inhibited SLY oligomerization, which is critical for its pore-forming activity. The treatment with amentoflavone reducedS. suis-induced cytotoxicity in macrophages (J774 cells). Furthermore,S. suis-infected mice that received amentoflavone exhibited lower mortality and bacterial burden. Additionally, amentoflavone significantly decreased the production of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 in anS. suis-infected cell model. Analyses of signaling pathways demonstrated that amentoflavone reducedS. suis-induced inflammation inS. suisserotype 2 (SS2)-infected cells by regulating the p38, Jun N-terminal protein kinase 1 and 2 (JNK1/2), and NF-κB pathways. The antivirulence and anti-inflammatory properties of amentoflavone againstS. suisinfection provide the possibility for future pharmaceutical application of amentoflavone in the treatment ofS. suisinfection.IMPORTANCEThe widespread use of antibiotics in therapy and in the prevention ofStreptococcus suisinfection in the swine industry raises concerns for the emergence of a resistant strain. The use of antivirulence agents has potential benefits, mainly because of the reduced selective pressure for the development of bacterial resistance. In this study, we found that amentoflavone is an effective agent againstS. suisserotype 2 (SS2) infection bothin vitroandin vivo. Our results demonstrated that amentoflavone is a promising anti-infective therapeutic forS. suisinfections, due to its antivirulence and anti-inflammatory effects without antibacterial activity, with fewer side effects than conventional antibacterial agents.

scholarly journals NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Sandrine Moutel ◽  
Nicolas Bery ◽  
Virginie Bernard ◽  
Laura Keller ◽  
Emilie Lemesre ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Rho Gtpase ◽  
Fluorescent Proteins ◽  
Phage Display Library ◽  
Specific Protein ◽  
Single Domain ◽  
Direct Selection ◽  
Single Domain Antibody ◽  
Domain Antibody

In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.

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