scholarly journals Screening for a Single-Chain Variable-Fragment Antibody That Can Effectively Neutralize the Cytotoxicity of the Vibrio parahaemolyticus Thermolabile Hemolysin

2012 ◽  
Vol 78 (14) ◽  
pp. 4967-4975 ◽  
Author(s):  
Rongzhi Wang ◽  
Sui Fang ◽  
Dinglong Wu ◽  
Junwei Lian ◽  
Jue Fan ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Animal Health ◽  
Halophilic Bacterium ◽  
Cell Toxicity ◽  
Pathogenic Factor ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Content Type ◽  
High Affinity ◽  
Food Borne

ABSTRACTVibrio parahaemolyticusis a halophilic bacterium that is widely distributed in water resources. The bacterium causes lethal food-borne diseases and poses a serious threat to human and animal health all over the world. The major pathogenic factor ofV. parahaemolyticusis thermolabile hemolysin (TLH), encoded by thetlhgene, but its toxicity mechanisms are unknown. A high-affinity antibody that can neutralize TLH activity effectively is not available. In this study, we successfully expressed and purified the TLH antigen and discovered a high-affinity antibody to TLH, named scFv-LA3, by phage display screening. Cytotoxicity analysis showed that scFv-LA3 has strong neutralization effects on TLH-induced cell toxicity.

scholarly journals Long-Term Study of Vibrio parahaemolyticus Prevalence and Distribution in New Zealand Shellfish

2015 ◽  
Vol 81 (7) ◽  
pp. 2320-2327 ◽  
Author(s):  
C. D. Cruz ◽  
D. Hedderley ◽  
G. C. Fletcher
Keyword(s):  
New Zealand ◽  
Vibrio Parahaemolyticus ◽  
Most Probable Number ◽  
Pcr Analysis ◽  
Potential Hazard ◽  
Probable Number ◽  
Content Type ◽  
The North ◽  
Long Term Study ◽  
Food Borne

ABSTRACTThe food-borne pathogenVibrio parahaemolyticushas been reported as being present in New Zealand (NZ) seawaters, but there have been no reported outbreaks of food-borne infection from commercially grown NZ seafood. Our study determined the current incidence ofV. parahaemolyticusin NZ oysters and Greenshell mussels and the prevalence ofV. parahaemolyticustdhandtrhstrains. Pacific (235) and dredge (21) oyster samples and mussel samples (55) were obtained from commercial shellfish-growing areas between December 2009 and June 2012. TotalV. parahaemolyticusnumbers and the presence of pathogenic genestdhandtrhwere determined using the FDA most-probable-number (MPN) method and confirmed using PCR analysis. In samples from the North Island of NZ,V. parahaemolyticuswas detected in 81% of Pacific oysters and 34% of mussel samples, while the numbers ofV. parahaemolyticustdhandtrhstrains were low, with just 3/215 Pacific oyster samples carrying thetdhgene.V. parahaemolyticusorganisms carryingtdhandtrhwere not detected in South Island samples, andV. parahaemolyticuswas detected in just 1/21 dredge oyster and 2/16 mussel samples. Numbers ofV. parahaemolyticusorganisms increased when seawater temperatures were high, the season when most commercial shellfish-growing areas are not harvested. The numbers ofV. parahaemolyticusorganisms in samples exceeded 1,000 MPN/g only when the seawater temperatures exceeded 19°C, so this environmental parameter could be used as a trigger warning of potential hazard. There is some evidence that the totalV. parahaemolyticusnumbers increased compared with those reported from a previous 1981 to 1984 study, but the analytical methods differed significantly.

scholarly journals Characterization of a Lipopolysaccharide-Targeted Monoclonal Antibody and Its Variable Fragments as Candidates for Prophylaxis against the Obligate Intracellular Bacterial Pathogen Coxiella burnetii

2014 ◽  
Vol 82 (11) ◽  
pp. 4530-4541 ◽  
Author(s):  
Ying Peng ◽  
Laura Schoenlaub ◽  
Alexandra Elliott ◽  
William J. Mitchell ◽  
Guoquan Zhang
Keyword(s):  
Monoclonal Antibody ◽  
Coxiella Burnetii ◽  
Natural Infection ◽  
Bacterial Pathogen ◽  
Passive Transfer ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Fab Fragment ◽  
Content Type ◽  
Aerosol Infection

ABSTRACTOur previous study demonstrated that treatment ofCoxiella burnetiiwith the phase I lipopolysaccharide (PI-LPS)-targeted monoclonal antibody (MAb) 1E4 significantly inhibitedC. burnetiiinfection in mice, suggesting that 1E4 is a protective MAb. To determine whether passive transfer of antibodies (Abs) can provide protection againstC. burnetiinatural infection, we examined if passive transfer of 1E4 would protect SCID mice againstC. burnetiiaerosol infection. The results indicated that 1E4 conferred significant protection against aerosolizedC. burnetii, suggesting that 1E4 may be useful for preventingC. burnetiinatural infection. To further understand the mechanisms of 1E4-mediated protection and to test the possibility of using humanized 1E4 to preventC. burnetiiinfection, we examined whether the Fab fragment of 1E4 (Fab1E4), a recombinant murine single-chain variable fragment (muscFv1E4), and a humanized single-chain variable fragment (huscFv1E4) retained the ability of 1E4 to inhibitC. burnetiiinfection. The results indicated that Fab1E4, muscFv1E4, and huscFv1E4 were able to inhibitC. burnetiiinfection in mice but that their ability to inhibitC. burnetiiinfection was lower than that of 1E4. In addition, treatment ofC. burnetiiwith Fab1E4, muscFv1E4, or huscFv1E4 can blockC. burnetiiinfection of macrophages. Interestingly, treatment ofC. burnetiiwith huscFv1E4 can significantly reduceC. burnetiiinfectivity in human macrophages. This report provides the first evidence to demonstrate that the humanized variable fragments of an LPS-specific MAb can neutralizeC. burnetiiinfection and appears to be a promising step toward the potential use of a humanized MAb as emergency prophylaxis againstC. burnetiiexposure.

scholarly journals The intrabody targeting of hTERT attenuates the immortality of cancer cells

2010 ◽  
Vol 15 (1) ◽  
Author(s):  
Xiangying Zhu ◽  
Nan Yang ◽  
Jianguo Cai ◽  
Guimei Yang ◽  
Shenghua Liang ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Malignant Tumors ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Human Telomerase Reverse Transcriptase ◽  
High Affinity ◽  
Promising Tool ◽  
Cell Immortalization ◽  
Synthesis Activity ◽  
Human Telomerase

AbstracthTERT (human telomerase reverse transcriptase) plays a key role in the process of cell immortalization. Overexpression of hTERT has been implicated in 85% of malignant tumors and offers a specific target for cancer therapy. In this paper, we describe an effective approach using a single-chain variable fragment (scFv) intrabody derived from monoclonal hybridoma directed against hTERT to attenuate the immortalization of human uterine cervix and hepatoma cells. The scFv we constructed had a high affinity to hTERT, and specifically neutralized over 70% of telomere synthesis activity, thereby inhibiting the viability and proliferation of the cancer cells. Our results indicate that this anti-hTERT intrabody is a promising tool to target hTERT and intervene in the immortalization process of cancer cells.

scholarly journals Lactobacillus helveticus MIMLh5-Specific Antibodies for Detection of S-Layer Protein in Grana Padano Protected-Designation-of-Origin Cheese

2013 ◽  
Vol 80 (2) ◽  
pp. 694-703 ◽  
Author(s):  
Milda Stuknytė ◽  
Eeva-Christine Brockmann ◽  
Tuomas Huovinen ◽  
Simone Guglielmetti ◽  
Diego Mora ◽  
...  
Keyword(s):  
Western Blotting ◽  
Lactobacillus Helveticus ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Content Type ◽  
Bacterial Surface ◽  
Protected Designation Of Origin ◽  
Antibody Libraries ◽  
Grana Padano ◽  
Detection And Localization

ABSTRACTSingle-chain variable-fragment antibodies (scFvs) have considerable potential in immunological detection and localization of bacterial surface structures. In this study, synthetic phage-displayed antibody libraries were used to select scFvs against immunologically active S-layer protein ofLactobacillus helveticusMIMLh5. After three rounds of panning, five relevant phage clones were obtained, of which four were specific for the S-layer protein ofL. helveticusMIMLh5 and one was also capable of binding to the S-layer protein ofL. helveticusATCC 15009. All five anti-S-layer scFvs were expressed inEscherichia coliXL1-Blue, and their specificity profiles were characterized by Western blotting. The anti-S-layer scFv PolyH4, with the highest specificity for the S-layer protein ofL. helveticusMIMLh5, was used to detect the S-layer protein in Grana Padano protected-designation-of-origin (PDO) cheese extracts by Western blotting. These results showed promising applications of this monoclonal antibody for the detection of immunomodulatory S-layer protein in dairy (and dairy-based) foods.

scholarly journals Direct RNA Sequencing Unfolds the Complex Transcriptome of Vibrio parahaemolyticus

mSystems ◽  
2021 ◽  
Author(s):  
Mohamad Al kadi ◽  
Eiji Ishii ◽  
Dang Tat Truong ◽  
Daisuke Motooka ◽  
Shigeaki Matsuda ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Marine Environment ◽  
Vibrio Parahaemolyticus ◽  
Halophilic Bacterium ◽  
Content Type ◽  
The Past

Vibrio parahaemolyticus is a halophilic bacterium found in the marine environment. Outbreaks of gastroenteritis resulting from seafood poisoning by these pathogens have risen over the past 2 decades.

scholarly journals Construction of a Single-Chain Variable-Fragment Antibody against the Superantigen Staphylococcal Enterotoxin B

2010 ◽  
Vol 76 (24) ◽  
pp. 8184-8191 ◽  
Author(s):  
Pawan Kumar Singh ◽  
Ranu Agrawal ◽  
Dev Vrat Kamboj ◽  
Garima Gupta ◽  
M. Boopathi ◽  
...  
Keyword(s):  
Phage Display ◽  
Staphylococcal Enterotoxin ◽  
Scfv Antibody ◽  
Affinity Constant ◽  
Enzyme Linked Immunosorbent Assay ◽  
Staphylococcal Enterotoxin B ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
High Affinity ◽  
Enterotoxin B

ABSTRACT Staphylococcal food poisoning (SFP) is one of the most prevalent causes of food-borne illness throughout the world. SFP is caused by 21 different types of staphylococcal enterotoxins produced by Staphylococcus aureus. Among these, staphylococcal enterotoxin B (SEB) is the most potent toxin and is a listed biological warfare (BW) agent. Therefore, development of immunological reagents for detection of SEB is of the utmost importance. High-affinity and specific monoclonal antibodies are being used for detection of SEB, but hybridoma clones tend to lose their antibody-secreting ability over time. This problem can be overcome by the use of recombinant antibodies produced in a bacterial system. In the present investigation, genes from a hybridoma clone encoding monoclonal antibody against SEB were immortalized using antibody phage display technology. A murine phage display library containing single-chain variable-fragment (ScFv) antibody genes was constructed in a pCANTAB 5E phagemid vector. Phage particles displaying ScFv were rescued by reinfection of helper phage followed by four rounds of biopanning for selection of SEB binding ScFv antibody fragments by using phage enzyme-linked immunosorbent assay (ELISA). Soluble SEB-ScFv antibodies were characterized from one of the clones showing high affinity for SEB. The anti-SEB ScFv antibody was highly specific, and its affinity constant was 3.16 nM as determined by surface plasmon resonance (SPR). These results demonstrate that the recombinant antibody constructed by immortalizing the antibody genes from a hybridoma clone is useful for immunodetection of SEB.

scholarly journals A genetically encoded probe for imaging HA-tagged protein translation, localization, and dynamics in living cells and animals

2018 ◽  
Author(s):  
Ning Zhao ◽  
Kouta Kamijo ◽  
Philip D. Fox ◽  
Haruka Oda ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mrna Translation ◽  
Protein Translation ◽  
Cell Types ◽  
Living Cells ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
High Affinity ◽  
Single Molecule Studies

ABSTRACTTo expand the toolbox of imaging in living cells, we have engineered a new single chain variable fragment (scFv) that binds the classic linear HA epitope with high affinity and specificity in vivo. The resulting probe, which we call the HA frankenbody, is capable of lighting up in multiple colors HA-tagged nuclear, cytoplasmic, and membrane proteins in diverse living cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments, which we demonstrate by tracking single mRNA translation dynamics in living U2OS cells and neurons. In combination with the SunTag, we track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful new tool for imaging protein dynamics in vivo.One-sentence summaryA genetically encodable intracellular single-chain variable fragment that selectively binds the HA epitope (YPYDVPDYA) with high affinity in living cells and organisms can be used to quantify HA-tagged protein translation, localization, and dynamics.

scholarly journals High-Affinity, Human Antibody-Like Antibody Fragment (Single-Chain Variable Fragment) Neutralizing the Lethal Factor (LF) of Bacillus anthracis by Inhibiting Protective Antigen-LF Complex Formation

2007 ◽  
Vol 51 (8) ◽  
pp. 2758-2764 ◽  
Author(s):  
Thibaut Pelat ◽  
Michael Hust ◽  
Emmanuelle Laffly ◽  
Florence Condemine ◽  
Chantal Bottex ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Germ Line ◽  
Lethal Factor ◽  
Antibody Fragment ◽  
Human Antibody ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Vitro Assay ◽  
High Affinity

ABSTRACT The anthrax lethal toxin (LT) consists of two subunits, the protective antigen (PA) and the lethal factor (LF), and is essential for anthrax pathogenesis. Several recombinant antibodies directed against PA and intended for medical use have been obtained, but none against LF, despite the recommendations of anthrax experts. Here we describe an anti-LF single-chain variable fragment (scFv) that originated from an immunized macaque (Macaca fascicularis) and was obtained by phage display. Panning of the library of 1.8 × 108 clones allowed the isolation of 2LF, a high-affinity (equilibrium dissociation constant, 1.02 nM) scFv, which is highly neutralizing in the standardized in vitro assay (50% inhibitory concentration, 1.20 ± 0.06 nM) and in an in vivo assay. The scFv neutralizes anthrax LT by inhibiting the formation of the LF-PA complex. The genes encoding 2LF are very similar to those of human immunoglobulin germ line genes, sharing substantial (84.2%) identity with their most similar, germinally encoded counterparts; this feature favors medical applications. These results, and others formerly published, demonstrate that our approach can generate antibody fragments suitable for prophylaxis and therapeutics.

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