A tetravalent single-chain variable fragment antibody for the detection of staphylococcal enterotoxin A

The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10−8 mol·L−1, its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10−7 mol·L−1. The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L−1.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01441-10 ◽

2010 ◽

Vol 76 (24) ◽

pp. 8184-8191 ◽

Cited By ~ 31

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.

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Efficient silkworm expression of a single-chain variable fragment antibody against ginsenoside Re and its application in ELISA for quality control of total ginsenosides in various ginseng

Planta Medica ◽

10.1055/s-0030-1264815 ◽

2010 ◽

Vol 76 (12) ◽

Author(s):

S Sakamoto ◽

B Pongkitwitoon ◽

S Nakamura ◽

K Maenaka ◽

H Tanaka ◽

...

Keyword(s):

Quality Control ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Ginsenoside Re

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Immunofluorescence Assay Using Monoclonal and Polyclonal Antibodies for Detection of Staphylococcal Enterotoxins A in Milk

The Open Biotechnology Journal ◽

10.2174/187407070190130137 ◽

2019 ◽

Vol 13 (1) ◽

pp. 137-145 ◽

Cited By ~ 1

Author(s):

Tzonka Godjevargova ◽

Zlatina Becheva ◽

Yavor Ivanov ◽

Andrey Tchorbanov

Keyword(s):

Monoclonal Antibody ◽

Magnetic Nanoparticles ◽

Polyclonal Antibody ◽

Polyclonal Antibodies ◽

Food Poisoning ◽

Staphylococcal Enterotoxin ◽

Staphylococcal Enterotoxin A ◽

Fluorescence Immunoassay ◽

Magnetic Separator ◽

Milk Samples

Objectives: Staphylococcus aureus is a Gram-positive microorganism. S. aureus can grow in various foods and cause food poisoning by secreting enterotoxins. The most common enterotoxins involved in food poisoning are staphylococcal enterotoxin A and staphylococcal enterotoxin B, but Staphylococcal Enterotoxin A (SEA) is predominant. The main types of food contaminated with SEs are meat and meat products, poultry and eggs, milk and dairy products. The aim of this study was to develop a rapid and sensitive fluorescence immunoassay for detection of staphylococcal enterotoxin A in milk. Methods: Monoclonal and polyclonal antibodies for SEA were produced and characterized. Competitive fluorescence immunoassay based on Magnetic Nanoparticles (MNPs) was performed and optimized. MNPs were used as a solid carrier of the antibodies. The first step of the assay was immunoreaction between the immobilized antibody onto MNPs and SEA in milk sample. Then the fluorescein-SEA conjugate was added to the sample. Thus, competitive immunoreaction between MNP-mAb/MNP-pAb with SEA and SEA-FITC was performed. These immuno-complexes were separated by a magnetic separator and the obtained supernatants were analyzed. The fluorescent signal from the excess of conjugated SEA was proportional to the SEA contained in the milk. The assay duration was only 30 min. Results: The fluorescence immunoassays performed with polyclonal antibody had linear ranges from 5 pg/mL to 100 ng/mL SEA in a buffer, and from 50 pg/mL to 50 ng/mL SEA in spiked milk samples. While the same assays performed with monoclonal antibody had linear ranges from 1 pg/mL to 20 ng/mL SEA in buffer, and from 10 pg/mL to 10 ng/mL SEA in spiked milk samples. The detection limits of the developed immunoassays performed in milk were: 48 pg/mL with polyclonal antibody and 9 pg/mL with monoclonal antibody. Conclusion: A rapid and sensitive fluorescence immunoassay based on magnetic nanoparticles with a polyclonal and monoclonal antibody for determination of staphylococcal enterotoxin A in milk was developed.

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Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.64.12.4862-4869.1998 ◽

1998 ◽

Vol 64 (12) ◽

pp. 4862-4869 ◽

Cited By ~ 39

Author(s):

Jörg F. Rippmann ◽

Michaela Klein ◽

Christian Hoischen ◽

Bodo Brocks ◽

Wolfgang J. Rettig ◽

...

Keyword(s):

Escherichia Coli ◽

Proteus Mirabilis ◽

Binding Activity ◽

Host Cells ◽

Heterologous Proteins ◽

Single Chain Variable Fragment ◽

Single Chain ◽

E Coli ◽

Active Product ◽

Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

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Development of a bispecific immune engager using a recombinant malaria protein

Cell Death and Disease ◽

10.1038/s41419-021-03611-0 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Mie A. Nordmaj ◽

Morgan E. Roberts ◽

Emilie S. Sachse ◽

Robert Dagil ◽

Anne Poder Andersen ◽

...

Keyword(s):

Cancer Cells ◽

Immune Evasion ◽

Blood Cells ◽

Xenograft Model ◽

Cancer Targeting ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Conjugation System ◽

Immune Mediated

AbstractAs an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolved a protein named VAR2CSA. This protein mediates sequestration of infected red blood cells in the placenta through the interaction with a unique carbohydrate abundantly and exclusively present in the placenta. Cancer cells were found to share the same expression of this distinct carbohydrate, termed oncofetal chondroitin sulfate on their surface. In this study we have used a protein conjugation system to produce a bispecific immune engager, V-aCD3, based on recombinant VAR2CSA as the cancer targeting moiety and an anti-CD3 single-chain variable fragment linked to a single-chain Fc as the immune engager. Conjugation of these two proteins resulted in a single functional moiety that induced immune mediated killing of a broad range of cancer cells in vitro and facilitated tumor arrest in an orthotopic bladder cancer xenograft model.

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Single Chain Variable Fragment Fused to Maltose Binding Protein: A Modular Nanocarrier Platform for the Targeted Delivery of Antitumorals

Biomaterials Science ◽

10.1039/d0bm01903h ◽

2021 ◽

Author(s):

Francisco J. Reche-Perez ◽

Simona Plesselova ◽

Eduardo De los Reyes-Berbel ◽

Mariano Ortega-Muñoz ◽

F. Javier Lopez-Jaramillo ◽

...

Keyword(s):

Monoclonal Antibody ◽

Targeted Delivery ◽

Specific Binding ◽

Protein A ◽

Antibody Fragments ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Binding Properties ◽

Single Chain Variable Fragments ◽

Selective Delivery

The use of the specific binding properties of monoclonal antibody fragments such as single-chain variable fragments (ScFv) for the selective delivery of antitumor therapeutics for cancer cells is attractive due...

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