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

 

By: Suzanne Canada, Ph.D.
Tanager Medical Writing

 

Three anthrax toxin components—Protective antigen (PA), edema factor (EF) and lethal factor (LF) are available for research purposes from LIST Biological Laboratories, separately at a high level of purification. At least two out of three of these components are necessary to enter a mammalian cell and exert a toxic effect.

With the aim of developing antitoxin therapies, scientists have been investigating the structure of PA, EF, and LF, and the complexes that they form with mammalian cell surface receptors, in hopes of finding the best way to disrupt or block the toxicity. Previously, NIAID-supported scientists have shown that protective antigen can bind edema factor and lethal factor at the same time, forming a greater variety of toxin complexes than were formerly known.¹ They also had produced a three-dimensional molecular structure of the anthrax protective antigen protein bound to one of the receptors (CMG2) it uses to enter cells.² More recently, a group of students in Kansas used Jmol and 3D printing technology to model and Anthrax toxin heterotrimer (PA, EF and LF) which forms a pore in the mammalian cell surface.

In an in vitro disease model, researchers constructed an artificial membrane bilayer using lipid and demonstrated that the blood of animals carrying anthrax infections was able to disrupt this membrane, a model of the cell membrane.  Membrane disruption requires acidification, and therefore the membrane remains intact until the pH is lowered.  When the pH is lowered to the required level for toxin complex binding, the membrane is disrupted by the anthrax toxin already embedded in it.⁴

Anthrax researchers have explored ways to protect healthcare workers and others who may have been exposed or are likely to be infected. One group of scientists has investigated the feasibility of RNA silencing technology (siRNA) to block expression of the anthrax toxin PA receptors on the cell surface, two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2).  Blocking expression of the receptors was reported to provide almost complete protection against the LF intoxication in mice, and also protected against LF effects in human kidney cells as well as macrophage-like cells.⁵ 

Methods of vaccination have been under investigation for some time, as one of the most likely methods to provide lasting protection against anthrax infection.  In another 2014 publication, researchers at the University of Texas have reported success in vaccinating guinea pigs against anthrax infection using vaccines based on DNA-protein antigen components as well as another based on recombinant protein components.  After immunization, the animals were challenged with a lethal dose of B. cereus G9241 aerosol.  Complete protection against lethal challenge was observed in all guinea pigs that had a detectable pre-challenge serum titer of toxin neutralizing antibodies.⁶

List Biological Laboratories, Inc. offers EF (Product number 167A), LF (176), and PA (171) as well as the antibodies for their detection (773, 769 and 772, and 771, respectively). Refer to the website: https://listlabs.com/product-information/anthrax-toxins/ for more information.

 

References

1. NIAID website: http://www.niaid.nih.gov/topics/anthrax/pages/default.aspx
2. CDC website: http://www.cdc.gov/anthrax/
3. Andrews J, Chick A, Chao T, Chogada V, Douglas A, Florack A, McCormick S, Kessler E, Tuel K, Whalen J and Fisher M (2014) The Anthrax Toxin Heterotrimer: Explorations of the Protective Antigen and Edema and Lethal Factors (LB98). FASEB J 28:LB98 
4. Nablo BJ, Panchal RG, Bavari S, Nguyen TL, Gussio R, Ribot W, Friedlander A, Chabot D, Reiner JE, Robertson JW, Balijepalli A, Halverson KM, Kasianowicz JJ (2013) Anthrax toxin-induced rupture of artificial lipid bilayer membranes J Chem Phys 139(6):065101. PMID: 23947891
5. Arévalo MT, Navarro A, Arico CD, Li J, Alkhatib O, Chen S, Diaz-Arévalo D, Zeng M  (2014) Targeted silencing of anthrax toxin receptors protects against anthrax toxins.  J Biol Chem 289(22):15730-8. PMID: 24742682
6. Palmer J, Bell M, Darko C, Barnewall R, Keane-Myers A. (2014) Protein- and DNA-based anthrax toxin vaccines confer protection in guinea pigs against inhalational challenge with Bacillus cereus G9241. Pathog Dis 72(2):138-42. PMID: 25044336

What Are Toxin Neutralization Assays & How Do They Work?

For clinical detection or vaccine testing, it is hard to beat a toxin neutralization assay.  Toxin neutralization assays (TNA) assess the ability of antibodies to protect cells in culture from the cytotoxic affect of the specific toxins.  Interestingly, these assays may be used for sensitive and reliable testing for disease states where toxins are involved, as well as for development of vaccines to treat infectious disease.  In TNA testing, potential sources of toxin and antibodies are combined and applied to cell culture in a series of dilutions.  Excess toxin in the sample, not neutralized by the antibody, will kill the cells, the amount of excess toxin determined by the dilution of the sample which will cause a defined amount of cell death.  The end point in such assays is cell viability, and this may be visualized by several different methods.  A commonly used method is to visualize viable cells through metabolism of a staining reagent; the intensity of the developed color is directly proportional to the percent of remaining cell viability.  TNA assays can also be used as a definitive identification of the causal agent of the disease.

TNA Assays for Clostridium Difficile Diagnosis

Cytotoxin neutralization (CTN or TNA) assays have great value in the specific diagnosis of C. difficile.  Laboratory diagnosis is described by Alfa and Sepehri (Alfa, 2013).  These assays can progress through a stepwise process starting with testing for glutamate dehydrogenase (GDH) in stool from potential C. difficile infected (CDI) patients.  C. difficile GDH (cdGDH) is a highly active enzyme which can be readily detected and correlates well with C. difficile infections.  Test results that are negative for GDH can identify samples in which C. difficile is highly unlikely, whereas tests positive for this enzyme can be used to identify potential C. difficile infections.  However, since GDH is also produced by other inhabitants of the digestive tract, the presence of GDH is not conclusive evidence of C. difficile.  To take diagnosis a step farther, immunological assays for C. difficile toxins A and B are used and when positive, identify C. difficile infections.  Low sensitivity of these assays produce false negative results when only a small amount of toxin is present; this is when a TNA assay on highly sensitive cells comes into play.  Depending on the type of cell culture, it is possible to detect C. difficile toxin B at a concentration of picograms per ml.  Because cell cultures may be killed by a variety of components in a test sample, specific identity of the toxin relies on the use of standard neutralizing antibodies directed uniquely to C. difficile toxin A or B.  When the antibody protects the cells from toxin directed death, the presence of, for example, the C. difficile toxin B is shown; this is a positive indication of a CDI patient.  Toxin neutralization is a valuable assay in identifying patients infected with C. difficile and List Labs products are used in the development of these assays and subsequent testing to detect the toxins in samples.  Products available from List Lab are C. difficile toxin A, C. difficile toxin B, our new product C. difficile GDH as well as antibodies directed to these three proteins, all of which are used to perform TNA.

TNA Assays Used to Evaluate Potential Vaccines

Another equally important use of toxin neutralization is in testing for the evaluation of potential vaccines.  A paper published in 2013 by Xie et al describes a TNA developed for the evaluation of hyperimmune sera raised in animals against potential C. difficile toxin A (TcdA) and toxin B (TcdB) toxoid vaccine candidates.  The authors optimized the assay using Vero cells for detection of neutralizing antibodies and for the determination of toxin potency.

TNA Assays for Anthrax Vaccines

Similar toxin neutralization assays have been developed and optimized for anthrax vaccines.  These assays have been in use for over 10 years.  A good review of these assays using different types of cultured cells to measure antibody levels created in response to different vaccines was provided by Ngundi et al, 2010.  Anthrax toxin products available from List Labs are protective antigen (PA), lethal factor (LF), edema factor (EF), as well as the respective antibodies, all of which are used in these assays.

References:

Alfa et al (2013) Combination of culture, antigen and toxin detection, and cytotoxin neutralization assay for optimal Clostridium difficile diagnostic testing. Can J Infect Dis Med Microbiol 24(2) 89-92. PMCID 3720004

Ngundi et al (2010) Comparison of Three Anthrax Toxin Neutralization Assays. Clinical and Vaccine Immunology 17(6) 895–903. PMID: 20375243

Xie et al (2013) Development and Optimization of a Novel Assay to Measure Neutralizing Antibodies Against Clostridium difficile Toxins. Clinical and Vaccine Immunology 20(4) 517-525. PMID: 23389929