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

Wang, Rongzhi; Sui, Fang; Wu, Dinglong; Lian, Jiamei; Fan, Jue; Zhang, Yanfeng; Wang, Shihua; Lin, Wei

doi:10.1128/aem.00435-12

Cited by 34 publications

(39 citation statements)

References 43 publications

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“…What's more, the yield of functional scFv antibody is a limiting factor in its clinical application. To solve this problem, the co-expression vector of pACYC-Duet-1-skp was used to help the expression of scFv in cytoplasm of E. coli cells [27,37].…”

Section: Discussionmentioning

confidence: 99%

“…After 3 weeks, mice were given first booster dose using incomplete Freund's adjuvant [27]. After second booster dose, the anti-serum titer was detected by indirect enzyme-linked immunosorbent assay (ELISA) [30].…”

Section: Immunization and Anti-serum Titer Assaymentioning

confidence: 99%

“…To further investigate the interaction between scFv-FA7 and VP1694, western blot was performed as described [27]. First, the purified GST-VP1694 antigen was transferred from SDS-PAGE gel onto a polyvinylidene difluoride membrane, and the membrane was treated with the purified anti-VP1694 scFv and anti-His tag IgG.…”

Section: Western Blotmentioning

confidence: 99%

“…Hence, it is required to develop an effective antibody or inhibitor to enhance the blocking efficiency. Single-chain variablefragment antibody (scFv) generation is a versatile technology for getting antibody that is specific for a given antigen [27]. scFv plays a critical role in several human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent.…”

Section: Introductionmentioning

confidence: 99%

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Generation and Characterization of a scFv Antibody Against T3SS Needle of Vibrio parahaemolyticus

et al. 2013

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Vibrio parahaemolyticus, a halophilic gramnegative bacterium, is a food-borne pathogen that largely inhabits marine and estuarine environments, and poses a serious threat to human and animal health all over the world. The hollow ''needle'' channel, a specific assemble of T3SS which exists in most of gram-negative bacteria, plays a key role in the transition of virulence effectors to host cells. In this study, needle protein VP1694 was successfully expressed and purified, and the fusion protein Trx-VP1694 was used to immunize Balb/c mice. Subsequently, a phage single-chain fragment variable antibody (scFv) library was constructed, and a specific scFv against VP1694 named scFv-FA7 was screened by phage display panning. To further identify the characters of scFv, the soluble expression vector pACYC-scFv-skp was constructed and the soluble scFv was purified by Ni 2? affinity chromatography. ELISA analysis showed that the scFv-FA7 was specific to VP1694 antigen, and its affinity constant was 1.07 9 10 8 L/mol. These results offer a molecular basis to prevent and cure diseases by scFv, and also provide a new strategy for further research on virulence mechanism of T3SS in V. parahaemolyticus by scFv.

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Section: Discussionmentioning

confidence: 99%

Section: Immunization and Anti-serum Titer Assaymentioning

confidence: 99%

Section: Western Blotmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Generation and Characterization of a scFv Antibody Against T3SS Needle of Vibrio parahaemolyticus

et al. 2013

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“…T o date, antibodies are used for a very broad and still steadily expanding spectrum of applications, such as cancer therapy, disease diagnosis, and signal pathway investigation (1)(2)(3). Monoclonal antibodies (Abs) produced by hybridoma technology have been widely used to rapidly detect pathogens in food and raw seafood (4).…”

mentioning

confidence: 99%

Development of a Functional Antibody by Using a Green Fluorescent Protein Frame as the Template

Wang

Xiang

Zhang

et al. 2014

Appl Environ Microbiol

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Single-chain variable fragment (scFv) antibodies are widely used as diagnostic and therapeutic agents or biosensors for a majority of human disease. However, the limitations of the present scFv antibody in terms of stability, solubility, and affinity are challenging to produce by traditional antibody screening and expression formats. We describe here a feasible strategy for creating the green fluorescent protein (GFP)-based antibody. Complementarity-determining region 3 (CDR3), which retains the antigen binding activity, was introduced into the structural loops of superfolder GFP, and the result showed that CDR3-inserted GFP displayed almost the same fluorescence intensity as wild-type GFP, and the purified proteins of CDR3 insertion showed the similar binding activity to antigen as the corresponding scFv. Among of all of the CDRs, CDR3s are responsible for antigen recognition, and only the CDR3a insertion is the best format for producing GFP-based antibody binding to specific antigen. The wide versatility of this system was further verified by introducing CDR3 from other scFvs into loop 9 of GFP. We developed a feasible method for rapidly and effectively producing a high-affinity GFP-based antibody by inserting CDR3s into GFP loops. Further, the affinity can be enhanced by specific amino acids scanning and site-directed mutagenesis. Notably, this method had better versatility for creating antibodies to various antigens using GFP as the scaffold, suggesting that a GFP-based antibody with high affinity and specificity may be useful for disease diagnosis and therapy.

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Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display

Kühn

Fühner

Unkauf

et al. 2016

Proteomics Clinical Apps

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Antibodies are valuable molecules for the diagnostic and treatment of diseases caused by pathogens and toxins. Traditionally, these antibodies are generated by hybridoma technology. An alternative to hybridoma technology is the use of antibody phage display to generate recombinant antibodies. This in vitro technology circumvents the limitations of the immune system and allows-in theory-the generation of antibodies against all conceivable molecules. Phage display technology enables obtaining human antibodies from naïve antibody gene libraries when either patients are not available or immunization is not ethically feasible. On the other hand, if patients or immunized/infected animals are available, it is common to construct immune phage display libraries to select in vivo affinity-matured antibodies. Because the phage packaged DNA sequence encoding the antibodies is directly available, the antibodies can be smoothly engineered according to the requirements of the final application. In this review, an overview of phage display derived recombinant antibodies against bacterial, viral, and eukaryotic pathogens as well as toxins for diagnostics and therapy is given.

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

Cited by 34 publications

References 43 publications

Generation and Characterization of a scFv Antibody Against T3SS Needle of Vibrio parahaemolyticus

Generation and Characterization of a scFv Antibody Against T3SS Needle of Vibrio parahaemolyticus

Development of a Functional Antibody by Using a Green Fluorescent Protein Frame as the Template

Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display

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