Anthrax toxin can enter living cells and the toxin enzymes, Lethal Factor (LF), and Edema Factor (EF) make known changes.  Because of this activity, anthrax toxins are valuable tools to investigate cell processes.  Some of the work currently accomplished with these toxins can be described by the following selected references: 

• Work with anthrax toxins is providing a better understanding of cellular processes. Protectative Antigen (PA) binds specifically to two toxin receptors, tumor endothelial marker 8 (TEM8, also called ANTXR1) and capillary morphogenesis 2 (CMG2, or ANTXR2).  Several other factors are involved in the internalization of anthrax toxin. 

Abrami L, Bischofberger M, Kunz B, Groux R, van der Goot FG (2010) Endocytosis of the Anthrax Toxin Is Mediated by Clathrin, Actin and Unconventional Adaptors. PLoS Pathog 6(3): e1000792. PMID:  20221438 

• Anthrax vaccines are currently under development and demonstration that antibodies will neutralize anthrax toxin is essential. 

Laws TR, Kuchuloria T, Chitadze N, et al (2016) A Comparison of the Adaptive Immune Response between Recovered Anthrax Patients and Individuals Receiving Three Different Anthrax Vaccines. PLoS One 11(3):e0148713. Published 2016 Mar 23. doi:10.1371/journal.pone.0148713 PMID: 27007118 

• PA could potentially deliver polypeptides and compounds to the cell cytoplasm. In the two studies described in the following papers, PA was used as a tool to deliver biochemicals to the cytoplasm of eukaryotic cells. 

Dyer PDR, Shepherd TR, Gollings AS et al (2015) Disarmed anthrax toxin delivers antisense oligonucleotides and siRNA with high efficiency and low toxicity. J. Control. Release 2015, 220, 316–328. PMID: 26546271 

Rabideau, AE, Liao XL, Akcay G, Pentelute BL (2015) Translocation of Non-Canonical Polypeptides into Cells Using Protective Antigen. Sci Rep 5: 11944, PMID:26178180, PMCID: PMC 4503955 

• PA has been used to target cancer cells overexpressing TEM8. This receptor has been shown to be upregulated during tumor angiogenesis and provides a convenient target for anti-angiogenic therapy.  

Chen KH, Liu S, Bankston LA, Liddington RC, Leppla SH (2007) Selection of anthrax toxin protective antigen variants that discriminate between the cellular receptors TEM8 and CMG2 and achieve targeting of tumor cells. J Biol Chem 282: 9834–9845. PMID: 17251181, PMCID: PMC2530824 

Chaudhary A, Hilton MB, Seaman S, et al (2012) TEM8/ANTXR1 blockade inhibits pathological angiogenesis and potentiates tumoricidal responses against multiple cancer types. Cancer Cell 21:212–226. PMID: 22340594 PMCID: PMC3289547 

To learn more about how our highly purified Anthrax Toxins were used in research, check out our over 200 Anthrax Toxin citations. 

By: Nancy Shine, PhD, Director of R&D, List Labs

A fast, sensitive, specific and accurate detection method to determine active infection by Bacillus anthracis in plasma has been developed at List Biological Laboratories.

Bacillus anthracis is regarded as a major biological warfare threat. The inhalation form of Bacillus anthracis infection can kill quickly.  While antibiotic treatment can clear the bacterium from the host, if diagnosis is delayed, the toxin, which is rapidly produced, may already be present in lethal amounts. There is a critical need for a rapid, accurate, sensitive and simple assay to determine whether infection has occurred thereby allowing immediate treatment.

Anthrax Detection Method

Anthrax lethal factor (LF), an endopeptidase, is present in blood in the early stages of the infection.  The use of peptidic substrates in plasma is problematic due to the presence of other proteases and the likelihood of nonspecific cleavage of the substrate.  A fluorescently labeled peptide substrate, MAPKKide Plus, Prod #532, which is not cleaved by plasma proteases and thus is specific for LF has been designed. The LF is enriched by capture from plasma using an LF antibody-coated microtiter plate, and the captured LF is then exposed to the fluorescent substrate.  The amount of cleaved peptide substrate is determined by HPLC with fluorescence detection. Concentration of the LF using the antibody-coated plates allows for the detection of 5 pg LF/ml of neat plasma after 2 hours of incubation.  Alternately, the MAPKKide Plus may be added directly to diluted plasma and cleavage monitored by an increase in fluorescence as a function of time using a fluorescent microplate reader.  The limit of detection by this simpler method is 1 ng LF/ml of plasma after 5 hours of digestion.  Both methods can be confirmed by analysis of the reaction as a function of time.  These methods are described in the poster Sensitive Detection of Anthrax Lethal Factor in Plasma Using a Specific Biotinylated Fluorogenic Substrate.

What’s Next for Anthrax Detection Method

We are currently working with a biotinylated form of MAPKKide Plus to enhance the sensitivity of the simpler method using the fluorescent plate reader rather than HPLC.

You can see the poster here. To see a complete list of all of List Labs’ posters check out this blog post.

Interested in learning more about this List Labs patented peptide substrate? Contact us!

By: Nancy Shine, Ph.D., Director of Research & Development

List Biological Laboratories, Inc. has designed a fluorescently labeled substrate for specific and quantitative detection of anthrax lethal factor in plasma.

Bacillus anthracis is regarded as a major biological warfare threat. The inhalation form of Bacillus anthracis infection can kill quickly. While antibiotic treatment can clear the bacterium from the host, if diagnosis is delayed, the toxin, which is rapidly produced, may already be present in lethal amounts. There is a critical need for a rapid, accurate, sensitive and simple assay to determine whether infection has occurred thereby allowing immediate treatment.

MAPKKide® Plus for Anthrax Detection in Plasma

The use of MAPKKide® Plus allows for a fast, sensitive, specific and accurate method to detect active infection by Bacillus anthracis in plasma. Anthrax lethal factor (LF), an endopeptidase, is present in blood early in the infection. The use of peptidic substrates in plasma is problematic due to the presence of other proteases and the likelihood of nonspecific cleavage of the substrate. MAPKKide® Plus is a fluorescently labeled peptide substrate which is not cleaved by plasma proteases and thus is specific for LF.

Methods for Using MAPKKide® Plus

There are two methods to use MAPKKide® Plus for anthrax detection. One method involves the enrichment of LF by capture from plasma using an LF antibody-coated microtiter plate, and the captured LF is then exposed to MAPKKide® Plus. The amount of cleaved peptide substrate is determined by HPLC with fluorescence detection. Concentration of the LF using the antibody-coated plates allows for the detection of 5 pg LF/ml of neat plasma after 2 hours of incubation. Alternately the MAPKKide® Plus may be added directly to diluted plasma and cleavage monitored by an increase in fluorescence as a function of time using a fluorescent microplate reader. The limit of detection by this simpler method is 1 ng LF/ml of plasma after 5 hours of digestion. Both methods can be confirmed by analysis of the reaction as a function of time.

MAPKKide® Plus details are as follows:

• Product: #532
• Price per vial: $250
• Vial size: 100 nmoles
• Not a Select Agent or a controlled export item
• A patent application has been filed for this peptide substrate.

MAPKKide® Plus is in its final stages of release and will be available from stock early next month. We are accepting orders now. Orders may be placed online on the product detail page.

Please do not hesitate to contact us with any other questions about MAPKKide® Plus. More information about all List Labs products and potential future products can be found on the following pages:

• Seach all Research Reagent Products
• Product Pipeline