scholarly journals Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers

2010 ◽  
Vol 17 (11) ◽  
pp. 1383-1390 ◽  
Author(s):  
Geoffrey K Feld ◽  
Katie L Thoren ◽  
Alexander F Kintzer ◽  
Harry J Sterling ◽  
Iok I Tang ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Structural Basis ◽  
Anthrax Lethal Factor

scholarly journals Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

2013 ◽  
Vol 20 (7) ◽  
pp. 986-997 ◽  
Author(s):  
Hang Lu ◽  
Jason Catania ◽  
Katalin Baranji ◽  
Jie Feng ◽  
Mili Gu ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Neutralization Assay ◽  
Dominant Factor ◽  
Anthrax Lethal Toxin ◽  
Serum Samples ◽  
Native Form ◽  
Anthrax Lethal Factor ◽  
Methionine Residues ◽  
Anthrax Vaccines

ABSTRACTThe cell-based anthrax toxin neutralization assay (TNA) is used to determine functional antibody titers of sera from animals and humans immunized with anthrax vaccines. The anthrax lethal toxin is a critical reagent of the TNA composed of protective antigen (PA) and lethal factor (LF), which are neutralization targets of serum antibodies. Cytotoxic potency of recombinant LF (rLF) lots can vary substantially, causing a challenge in producing a renewable supply of this reagent for validated TNAs. To address this issue, we characterized a more potent rLF variant (rLF-A) with the exact native LF amino acid sequence that lacks the additional N-terminal histidine and methionine residues present on the commonly used form of rLF (rLF-HMA) as a consequence of the expression vector. rLF-A can be used at 4 to 6 ng/ml (in contrast to 40 ng/ml rLF-HMA) with 50 ng/ml recombinant PA (rPA) to achieve 95 to 99% cytotoxicity. In the presence of 50 ng/ml rPA, both rLF-A and rLF-HMA allowed for similar potencies (50% effective dilution) among immune sera in the TNA. rPA, but not rLF, was the dominant factor in determining potency of serum samples containing anti-PA antibodies only or an excess of anti-PA relative to anti-rLF antibodies. Such anti-PA content is reflected in immune sera derived from most anthrax vaccines in development. These results support that 7- to 10-fold less rLF-A can be used in place of rLF-HMA without changing TNA serum dilution curve parameters, thus extending the use of a single rLF lot and a consistent, renewable supply.

scholarly journals Detection of Anthrax Toxin by an Ultrasensitive Immunoassay Using Europium Nanoparticles

2009 ◽  
Vol 16 (3) ◽  
pp. 408-413 ◽  
Author(s):  
Shixing Tang ◽  
Mahtab Moayeri ◽  
Zhaochun Chen ◽  
Harri Harma ◽  
Jiangqin Zhao ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Laboratory Research ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Serum Samples ◽  
Detection Range ◽  
Edema Factor ◽  
Anthrax Lethal Factor ◽  
Europium Nanoparticles

ABSTRACT We developed a europium nanoparticle-based immunoassay (ENIA) for the sensitive detection of anthrax protective antigen (PA). The ENIA exhibited a linear dose-dependent pattern within the detection range of 0.01 to 100 ng/ml and was approximately 100-fold more sensitive than enzyme-linked immunosorbent assay (ELISA). False-positive results were not observed with serum samples from healthy adults, mouse plasma without PA, or plasma samples collected from mice injected with anthrax lethal factor or edema factor alone. For the detection of plasma samples spiked with PA, the detection sensitivities for ENIA and ELISA were 100% (11/11 samples) and 36.4% (4/11 samples), respectively. The assay exhibited a linear but qualitative correlation between the PA injected and the PA detected in murine blood (r = 0.97731; P < 0.0001). Anthrax PA was also detected in the circulation of mice infected with spores from a toxigenic Sterne-like strain of Bacillus anthracis, but only in the later stages of infection. These results indicate that the universal labeling technology based on europium nanoparticles and its application may provide a rapid and sensitive testing platform for clinical diagnosis and laboratory research.

scholarly journals A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

2003 ◽  
Vol 100 (11) ◽  
pp. 6652-6657 ◽  
Author(s):  
N. Kushner ◽  
D. Zhang ◽  
N. Touzjian ◽  
M. Essex ◽  
J. Lieberman ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Lethal Factor

scholarly journals Development of Enzyme Linked Immunosorbent Assay for humoral immuneresponse and infection monitoring of anthrax

2020 ◽  
Vol 71 (1) ◽  
pp. 1935
Author(s):  
H. R. NOURI ◽  
H. RAZZAZ ◽  
M. TAGHDIRI ◽  
K. TADAYON ◽  
S. R. BANIHASHEMI
Keyword(s):  
Protective Antigen ◽  
Critical Role ◽  
Lethal Factor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibody ◽  
Elisa Kit ◽  
Gram Positive Bacteria ◽  
Anthrax Lethal Factor ◽  
Antibody Titers ◽  
Immune Assays

Immune assays were taken into consideration to diagnose and quantify metabolites such as antigen and antibody. Enzyme-Linked Immunosorbent Assays (ELISAs), which are used to detect antigens and antibodies, generated several periods of infectious and vaccination conditions. There is an extensive range of commercial infectious disease ELISA kits useful for the detection of human and animal IgG, IgA, IgM antibodies and microorganism antigens. Anthrax is one of the serious infectious diseases caused by rod-shaped, gram-positive bacteria known as Bacillus anthracis. Subunit or attenuated vaccines applied against anthrax disease increase the antibody against the Protective Antigen (PA) which has a critical role as a toxin of B. anthracis. Herein, the ELISA was developed using PA domain 4 and anthrax Lethal Factor to detect IgG antibody in serum. Besides, the level of anti-LF antibodies were determined as a complementary test to measure variance in antibody titers associated with vaccination or infection that leads to detection of anthrax in livestock. The results show that we developed high-quality ELISA kit that can be used to test immunogenicity of vaccines and infections in mice. We tried to develop the Anti- PA4 ELISA kit and conduct the validation studies to evaluate the fluctuation level of the antibody in the anthrax vaccine and distinction between disease and vaccination in mice.

scholarly journals Structural Determinants for the Binding of Anthrax Lethal Factor to Oligomeric Protective Antigen

2005 ◽  
Vol 281 (3) ◽  
pp. 1630-1635 ◽  
Author(s):  
Roman A. Melnyk ◽  
Krissi M. Hewitt ◽  
D. Borden Lacy ◽  
Henry C. Lin ◽  
Chris R. Gessner ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Structural Determinants ◽  
Anthrax Lethal Factor

scholarly journals Clindamycin Protects Nonhuman Primates Against Inhalational Anthrax But Does Not Enhance Reduction of Circulating Toxin Levels When Combined With Ciprofloxacin

2020 ◽  
Author(s):  
Nicholas J Vietri ◽  
Steven A Tobery ◽  
Donald J Chabot ◽  
Susham Ingavale ◽  
Brandon C Somerville ◽  
...  
Keyword(s):  
Nonhuman Primates ◽  
Protective Antigen ◽  
Rhesus Macaques ◽  
Lethal Factor ◽  
Toxin Production ◽  
Protein Synthesis Inhibitors ◽  
Anthrax Lethal Factor ◽  
Bacillus Anthracis Spores ◽  
Combination Antibiotics ◽  
First Time

Abstract Background Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking. Methods Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics. Results In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached. Conclusion Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels.

scholarly journals Involvement of Domain II in Toxicity of Anthrax Lethal Factor

2004 ◽  
Vol 279 (50) ◽  
pp. 52473-52478 ◽  
Author(s):  
Xudong Liang ◽  
John J. Young ◽  
Sherrie A. Boone ◽  
David S. Waugh ◽  
Nicholas S. Duesbery
Keyword(s):  
Proteolytic Activity ◽  
Active Site ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Site Directed Mutagenesis ◽  
Mitogen Activated Protein Kinase ◽  
Anthrax Lethal Factor ◽  
Fold Reduction ◽  
Mitogen Activated Protein

Anthrax lethal factor (LF) is a Zn2+-metalloprotease that cleaves and inactivates mitogen-activated protein kinase kinases (MEKs). We have used site-directed mutagenesis to identify a cluster of residues in domain II of LF that lie outside the active site and are required for cellular proteolytic activity toward MEKs. Alanine substituted for Leu293, Lys294, Leu514, Asn516, or Arg491caused a 10–50-fold reduction in LF toxicity. Further, whereas pairwise substitution of alanine for Leu514and either Leu293, Lys294, or Arg491completely abrogated LF toxicity, pairwise mutation of Leu514and Asn516resulted in toxicity comparable with N516A alone. The introduction of these mutations reduced LF-mediated cleavage of MEK2 in cell-based assays but altered neither the ability of LF to bind protective antigen nor its ability to translocate across a membrane. Interestingly, directin vitromeasurement of LF activity indicated that decreased toxicity was not always accompanied by reduced proteolytic activity. However, mutations in this region significantly reduced the ability of LF to competitively inhibit B-Raf phosphorylation of MEK. These results provide evidence that elements of domain II are involved in the association of LF into productive complex with MEKs.

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