Escherichia coliSkp Chaperone Coexpression Improves Solubility and Phage Display of Single-Chain Antibody Fragments

Hayhurst, Andrew; Harris, William J.

doi:10.1006/prep.1999.1035

Cited by 126 publications

(87 citation statements)

References 32 publications

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“…Briefly, the M18 murine scFv (19) was isolated from an error-prone library derived from the anti-PA 14B7 murine monoclonal antibody (29) using a new bacterial display technology referred to as anchored periplasmic expression (APEx) (19 to produce a fusion between a human kappa chain and the heavy-chain variable domain of the scFv. The resulting construct is referred to as a scAb (21). The purified M18 scAb fragment binds PA with an equilibrium dissociation constant (K D ) of 35 pM (19).…”

Section: Resultsmentioning

confidence: 99%

Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

Mabry

Brasky

Geiger

et al. 2006

Clin Vaccine Immunol

115

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Several strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. The assays utilize as capture agents an engineered high-affinity antibody to PA, a soluble form of the extracellular domain of the anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwich immunoassays were used to detect and quantify PA and LF in animals infected with the Ames or Vollum strains of anthrax spores. PA and LF were detected before and after signs of toxemia were observed, with increasing levels reported in the late stages of the infection. These results represent the detection of free PA and LF by ELISA in the systemic circulation of two animal models exposed to either of the two fully virulent strains of anthrax. Simple anthrax toxin detection ELISAs could prove useful in the evaluation of potential therapies and possibly as a clinical diagnostic to complement other strategies for the rapid identification of B. anthracis infection.The gram-positive Bacillus anthracis bacterium is the causative agent of anthrax. During inhalational anthrax, spores are inhaled into the lungs, leading to germination followed by the emergence of vegetative anthrax bacteria in circulation. Key anthrax virulence factors include the three components of the anthrax exotoxin, protective antigen (PA), lethal factor (LF), and edema factor (EF), which are secreted into the host's system (11). The PA and LF toxins combined are often referred to as anthrax lethal toxin (LeTx) and operate analogous to other A-B toxin systems (18).The PA component is secreted as an 83-kDa protein that is cleaved by a host furin-like protease to yield a 63-kDa fragment (5, 24) that readily heptamerizes. The PA 63 heptamer is bound to the host cell surface receptor ANTXR1 (ATR/ TEM8) (45) or ANTXR2 (CMG2) (7) with relatively high affinity (170 pM) (47). The LF component binds to the PA heptamer and gains entry into the cell through clathrin-dependent endocytosis (1). A drop in endosomal pH leads to a conformational change in PA and transport of LF into the host cell cytosol (17, 26). In the cytosol, LF toxin acts as a zinc metalloprotease, cleaving the N termini of mitogen-activated protein kinase kinases (9, 13), leading to a number of signs associated with toxemia and ultimately death (2, 32).In the rodent model, signs of anthrax infection are rapidly followed by death within a few hours, resulting in a very small, if existent, window for postexposure therapy after observed toxemia. However, in the nonhuman primate model and in human disease, the infection is characterized by delayed death up to 10 days following initial symptom onset (8, 10). It is theref...

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Section: Resultsmentioning

confidence: 99%

Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

Mabry

Brasky

Geiger

et al. 2006

Clin Vaccine Immunol

115

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“…The M18 scFv gene (16) was cloned via terminal SfiI sites into pMoPac16 (18), a modified version of the pAk4000 vector (26) that carries an scFv gene with the human light chain to create a single-chain antibody (scAb) (18,34). The pMoPac16 plasmid also carries a gene for the coexpression of the skp periplasmic chaperone (17,18). To create a protein suitable for PEG conjugation, the M18 scAb gene was amplified with a C-terminal primer that incorporated a Cys residue downstream of the C-terminal His 6 purification tag.…”

Section: Methodsmentioning

confidence: 99%

Passive Protection against Anthrax by Using a High-Affinity Antitoxin Antibody Fragment Lacking an Fc Region

et al. 2005

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Passive immunization has been successfully employed for protection against bacterial and viral infections for over 100 years. Immunoglobulin Fc regions play a critical role in the clearance of bacterial pathogens by mediating antibody-dependent and complement-dependent cytotoxicity. Here we show that antibody fragments engineered to recognize the protective antigen component of the B. anthracis exotoxin with high affinity and conjugated to polyethylene glycol (PEG) for prolonged circulation half-life confer significant protection against inhalation anthrax despite their lack of Fc regions. The speed and lower manufacturing cost of bacterially expressed PEGylated antibody fragments could provide decisive advantages for anthrax prophylaxis. Importantly, our results suggest that PEGylated antibody fragments may represent a unique approach for mounting a rapid therapeutic response to emerging pathogen infections.

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“…Six unique free antigen binders were identified and named HSL-1 to HSL-6. These six clones were converted into soluble single-chain antibody (scAb) format by cloning into the expression vector pIMS147 (27).…”

Section: Resultsmentioning

confidence: 99%

High-Sensitivity Monoclonal Antibodies Specific for Homoserine Lactones Protect Mice from Lethal Pseudomonas aeruginosa Infections

Palliyil

Downham

Broadbent

et al. 2014

Appl Environ Microbiol

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cA number of bacteria, including pathogens like Pseudomonas aeruginosa, utilize homoserine lactones (HSLs) as quorum sensing (QS) signaling compounds and engage in cell-to-cell communication to coordinate their behavior. Blocking this bacterial communication may be an attractive strategy for infection control as QS takes a central role in P. aeruginosa biology. In this study, immunomodulation of HSL molecules by monoclonal antibodies (MAbs) was used as a novel approach to prevent P. aeruginosa infections and as tools to detect HSLs in bodily fluids as a possible first clue to an undiagnosed Gram-negative infection. Using sheep immunization and recombinant antibody technology, a panel of sheep-mouse chimeric MAbs were generated which recognized HSL compounds with high sensitivity (nanomolar range) and cross-reactivity. These MAbs retained their nanomolar sensitivity in complex matrices and were able to recognize HSLs in P. aeruginosa cultures grown in the presence of urine. In a nematode slow-killing assay, HSL MAbs significantly increased the survival of worms fed on the antibiotic-resistant strain PA058. The therapeutic benefit of these MAbs was further studied using a mouse model of Pseudomonas infection in which groups of mice treated with HSL-2 and HSL-4 MAbs survived, 7 days after pathogen challenge, in significantly greater numbers (83 and 67%, respectively) compared with the control groups. This body of work has provided early proof-of-concept data to demonstrate the potential of HSL-specific, monoclonal antibodies as theranostic clinical leads suitable for the diagnosis, prevention, and treatment of life-threatening bacterial infections.

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Escherichia coliSkp Chaperone Coexpression Improves Solubility and Phage Display of Single-Chain Antibody Fragments

Cited by 126 publications

References 32 publications

Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

Passive Protection against Anthrax by Using a High-Affinity Antitoxin Antibody Fragment Lacking an Fc Region

High-Sensitivity Monoclonal Antibodies Specific for Homoserine Lactones Protect Mice from Lethal Pseudomonas aeruginosa Infections

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