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 Generation of Fab Fragment-Like Molecular Recognition Proteins against Staphylococcal Enterotoxin B by Phage Display Technology

2010 ◽  
Vol 17 (11) ◽  
pp. 1708-1717 ◽  
Author(s):  
Yuji Urushibata ◽  
Kunihiko Itoh ◽  
Motohiro Ohshima ◽  
Yasuo Seto
Keyword(s):  
Phage Display ◽  
Dissociation Constants ◽  
Staphylococcal Enterotoxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Staphylococcal Enterotoxin B ◽  
Single Chain ◽  
Fab Fragment ◽  
Phage Display Technology ◽  
Display Technology ◽  
Enterotoxin B

ABSTRACT Antigen-binding fragments (Fab fragments) and single-chain variable fragments (scFv) against staphylococcal enterotoxin B (SEB) were produced by phage display technology. SEB epitopes were first identified by phage display approach using the commercial anti-SEB monoclonal antibody ab53981 as the target. Heptamer and dodecamer mimotope peptides recognized by ab53981 were screened from Ph.D-7 or Ph.D-12 random peptide phage libraries expressed in Escherichia coli. The isolated 7-mer and 12-mer mimotopes were shown to share a sequence homologous to 8PDELHK14S in the amino acid sequence of SEB. The N-terminal 15-mer peptide of SEB was determined to be an epitope of ab53981. After immunization of mice with maltose-binding protein-tagged N-terminal 15-mer peptide, a phage display Fab library was constructed using cDNA prepared from the mRNAs of spleen cells. Three phage clones displaying the Fab molecule which recognized SEB were isolated through three rounds of panning. Only one of them produced a soluble Fab fragment from the transformed cells, and the fragment fused with a histidine tag sequence was produced in E. coli cells and converted into scFv. Surface plasmon resonance analysis showed that the dissociation constants of these proteins with SEB were (4.1 ± 1.1) × 10−9 M and (8.4 ± 2.3) × 10−10 M, respectively. The produced molecule was applied to the determination of SEB by enzyme-linked immunosorbent assay and Western blot analysis.

Effect of Thermal Processing during Yogurt Production upon the Detection of Staphylococcal Enterotoxin B

2009 ◽  
Vol 72 (10) ◽  
pp. 2212-2216 ◽  
Author(s):  
MARYANN PRINCIPATO ◽  
THOMAS BOYLE ◽  
JOYCE NJOROGE ◽  
ROBERT L. JONES ◽  
MICHAEL O'DONNELL
Keyword(s):  
Storage Period ◽  
Staphylococcal Enterotoxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Staphylococcal Enterotoxin B ◽  
Type I ◽  
Type Ii ◽  
Biochemical Characteristics ◽  
Enterotoxin B ◽  
Study Type ◽  
Physical Changes

This research was conducted to examine the inherent properties of yogurt contaminated with staphylococcal enterotoxin B (SEB). Two types of yogurts were produced for this study. Type I yogurts were produced by adding SEB at the start of yogurt production, and type II yogurts were produced by adding SEB after the milk base had been boiled. Biochemical characteristics inherent to yogurt, including pH, lactic acid and acetaldehyde concentrations, were analyzed weekly for each batch beginning at a time just after production and throughout a storage period of at least 4 weeks. The presence of toxin during yogurt production did not result in any significant biochemical or physical changes in yogurt. However, we were unable to detect SEB toxin in type I yogurt using a commercially available enzyme-linked immunosorbent assay (ELISA). In contrast, SEB was easily detectable by our ELISA in type II yogurt samples. Higher levels of SEB were recovered from type II yogurt that had been stored for 1 week than from type II yogurt that had been stored for any other length of time. These results indicate that the biochemical characteristics of yogurt did not change significantly (relative to control yogurt) in the presence of either thermally processed SEB or native SEB. However, the ability to detect SEB by ELISA was dependent on whether the toxin had been processed.

Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli

2014 ◽  
Vol 60 (11) ◽  
pp. 737-743 ◽  
Author(s):  
Weifeng Chen ◽  
Li Hu ◽  
Aiping Liu ◽  
Jinquan Li ◽  
Fusheng Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Food Poisoning ◽  
Staphylococcal Enterotoxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Staphylococcal Enterotoxin A ◽  
Single Chain Variable Fragment ◽  
Single Chain

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.

scholarly journals Measurement of Staphylococcal Enterotoxin B in Serum and Culture Supernatant with a Capture Enzyme-Linked Immunosorbent Assay

2007 ◽  
Vol 14 (9) ◽  
pp. 1094-1101 ◽  
Author(s):  
E. Cook ◽  
X. Wang ◽  
N. Robiou ◽  
B. C. Fries
Keyword(s):  
Immunosorbent Assay ◽  
Staphylococcal Enterotoxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Staphylococcal Enterotoxin B ◽  
Low Concentrations ◽  
Life Threatening ◽  
Nasal Inhalation ◽  
Enterotoxin B ◽  
Rapid Binding

ABSTRACT Staphylococcal enterotoxin B (SEB) is a select agent because it is a potent mitogen that elicits life-threatening polyclonal T-cell proliferation and cytokine production at very low concentrations. Efforts are in progress to develop therapeutic reagents and vaccines that neutralize or prevent the devastating effects of this toxin. Because of its rapid binding to in vivo receptors, this toxin is difficult to detect in serum. This rapid binding also constitutes a major challenge for the development of effective therapeutic reagents that can neutralize the effects of the toxin in vivo. We have developed a highly sensitive capture enzyme-linked immunosorbent assay that detects SEB in body fluids at very low levels. With this assay, the peak levels of SEB in serum and renal clearance can be measured in mice. After either oral ingestion or nasal inhalation of SEB by mice, this assay documents the transcytosis of SEB across the mucosal membranes into serum within 2 h. Furthermore, this assay was used to compare the SEB levels in different murine models for SEB-induced lethal shock and demonstrated that the coadministration of toxin-enhancing chemicals, such as d-galactosamine and lipopolysaccharide, can alter the peak serum SEB levels. Hence, this assay is a potentially useful tool for the study of the pharmacokinetics of SEB and the effects of potential therapeutic reagents on serum SEB levels.

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