A fast, sensitive, specific and accurate detection method to determine active infection
Dr. Nancy Shine
[Campbell, CA, 11/29/2018]
• Method to detect Anthrax before it’s deadly
• Anthrax is a problem for livestock
• Test method is both specific and sensitive for Anthrax
• List Labs is looking to partner on this newly discovered method
Since the intentional release of anthrax spores leading to lethal inhalational
anthrax in 2001, the need for rapid and sensitive detection of infection has been critical.
Unfortunately, early symptoms of infection are similar to those of common illnesses.
While the symptoms are not remarkable, the Bacillus anthracis bacteria enter the
patient’s blood stream and rapidly multiply. This expanding population of bacteria
produces deadly proteins which will eventually overcome the patient. Classical
techniques to detect and identify bacteria in blood take too long. We have devised a
rapid method for detecting one of the proteins produced in the infection. This protein,
anthrax lethal factor, is produced early in infection in a quantity sufficient for detection
making it possible to rapidly determine that a patient is infected and to initiate therapy. A
quick diagnosis is essential for successful treatment of the disease.
Anthrax is not only a bioterrorism threat. There are many areas in the world
where anthrax is endemic. Efforts have been focused on surveillance in countries where
livestock are infected. Contact between infected animals and humans leads to disease.
A quick diagnosis depends on the availability of a rapid, sensitive and simple test.
This paper reports the design of a sensitive and specific test for anthrax infection.
A reagent that specifically detects the presence of low amounts of lethal factor from
anthrax infection is described. This study sets a new standard for a sensitive, simple,
and specific method to detect anthrax infection.
List Labs is looking to partner with an organization that can take this biotechnology to the level of application in the field. Please contact Dr. Shine if you’re interested in partnering.
About List Biological Labs, Inc.
Established since 1978, List Biological Labs, Inc. specializes in native toxins, recombinant proteins, bacteria, Biotherapeutics and GMP products. We develop assays, perform contract manufacturing and produce our own GMP LPS product.
List Labs produces toxins for the research community, including: C. difficile toxin A and toxin B, shiga toxins, cholera toxin, anthrax toxins (PA, LF, and EF), pertussis toxin, diphtheria toxin, CRM197, tetanus toxin, staphylococcal enterotoxin B, botulinum toxins as well as several types of lipopolysaccharides (LPS) or endotoxin for purchase by the research community.
By: Nancy Shine, PhD, Director of R&D, List Labs
Nancy Shine presenting her poster at ASM Biothreats February, 2018
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!
Nancy Shine, PhD, Director R&D, List Labs and author of posters
Over 20 Scientific Posters Available on our website
Scientific posters are a great way to visually display complex scientific issues. List Labs has a large list of scientific posters published on our website under the specific products they pertain to. We have been getting a lot of requests lately to compile a list of all of our published posters into one place. Please see below for a comprehensive list of all List Labs’ scientific posters to date.
Click on the one you are interested in to check it out.
- Evaluation of Three Vamptide® Substrates For Botulinum Neurotoxin Type B
- FRET Peptide Substrates for Botulinum Neurotoxin Types A, B, C, E
- Sensitive and Specific Bifunctional Assay for Botulinum Neurotoxin Type A
- Sensitive Detection of Botulinum Neurotoxin Type A Using High Affinity Polyclonal Antibodies
- New FRET substrate for Botulinum Neurotoxin Type A
- Ultra Sensitive HPLC Detection Assay for Botulinum Neurotoxin Type A
- Evaluation of a Small Peptide Inhibitor and A Control Peptide for Botulinum Neurotoxin, Type A
- Sensitive, In Vitro, Bifunctional Potency Assay for Botulinum Neurotoxin Type A
- Ultra Sensitive HPLC Detection Assay for Botulinum Neurotoxin Type A and SYNTAXtide®, FRET Substrate for Botulinum Toxin C.
- Functional Assay for Botulinum Neurotoxin Type A Utilizing the Neuronal Receptor Protein SV2c
- Capture Assay for Botulinum Neurotoxin Type A Utilizing the Neuronal Receptor Protein SV2c
- New High Affinity Antibodies Against Botulinum Neurotoxin Type A
- FRET Peptide Substrates for the Botulinum Toxins Type A, B, and E and for Anthrax Lethal Factor
- SNAP Etide®, a FRET Substrate for Botulinum Toxin Type E
- Sensitive and Specific Assay to Measure the Endoprotease Activity of Botulinum Toxin Type A Holotoxin in Milk
- Comparison of Activity of Botulinum Neurotoxin Type A Holotoxin and Light Chain Using SNAPtide® FRET Substrates
- Rapid, Sensitive, and Specific Assay to Measure the Endoprotease Activity in Botulinum Toxin Type A
- New SV2c Construct for Use in Binding and Detecting Botulinum Neurotoxin Type A
By: Suzanne Canada, Ph.D.
Tanager Medical Writing
An exciting report was released in October about a new class of targeted anti-tumor drugs, in which genetically engineered stem cells were used to deliver cytotoxins to brain tumors.1 Brain cancers known as glioblastomas (GBM) are notoriously difficult to treat because the tumors often re-grow after surgery and because most standard cancer therapies cannot pass the blood-brain barrier. Those cancer therapies that can reach the tumors must be delivered at high doses which can be toxic to the entire body, without specifically targeting the GBM tumor. In this case, a research team at Massachusetts General Hospital (MGH) in Boston used stem cells, added to mouse brain tumors after surgery, to deliver Pseudomonas exotoxin directly at the site of the tumor itself. 2
Although this research is cutting-edge and an exciting development for GBM patients, the idea of using toxins attached to targeting molecules such as antibodies or specific ligands has long been explored as a way of fighting diseases, especially cancer. One popular approach has been to use antibodies linked to toxins to aid in targeting the therapy. (See Chari 2008 and Goldmacher 2011 for reviews).3, 4 An example is the approach taken by group of researchers looking for ways to increase the effectiveness of Herceptin®, a monoclonal antibody that is best known for targeting HER-overexpressing malignant breast cancer tumors. Antibody was coupled to both diphtheria toxin and multi-walled carbon nanotubes. They found that both conjugates were more effective in specifically killing HER-2 expressing cells than Herceptin® alone.5
An elegant approach to targeting toxins is to activate the toxin by cleavage at the site of therapy. This is precisely the approach used by Schafer and colleagues.6 Their model system exploited the fact that metalloproteinases are commonly overexpressed on the surface of squamous cell cancers. Anthrax toxin was engineered to be activated by cleavage by urokinase plasminogen activator (uPA) on the cell surface and metalloproteinases. This approach seemed to work on xenografted human head and neck squamous cell carcinoma (HNSCC) cell lines by inducing apoptotic and necrotic tumor cell death. However, cultured cancer cell lines were found to be insensitive to the engineered toxin, so the researchers concluded that the regulation of two-fold activation was not straightforward as anticipated.
Shiga toxin– produced by an organism responsible for bacterial dysentery – has properties that could be harnessed for cancer research7. A group of researchers took advantage of the binding of the Shiga toxin B pentamer to the glycosphingolipid globotriaosylceramide (Gb3) on the cell surface. After binding, the Shiga toxin complex is internalized by eukaryotic cells where the Shiga toxin A moiety can exert its toxic effect. Gb3 is reportedly over-expressed in throat, gastric, and ovarian cancers—and researchers hope that this overexpression pattern could be used to attain more targeted therapy. Specific binding of GB3 by the Shiga toxin B pentamer could also be exploited for imaging of these tumors and for delivering a genetically engineer Shiga toxin A chimera that would only be activated in cancer cells.
In their quest for new and more effective therapies, researchers have noted that bacterial toxins are examples of highly toxic, but also targeted and regulated systems that have co-evolved with the eukaryotic hosts (humans).8, 9 In the words of Fabbri et al., “Knowledge of their properties could be used for medical purposes.” List Biological Laboratories, Inc. provides purified bacterial toxins for research purposes, including Anthrax toxins (Product # 169, 172, & 176), Shiga toxins (Product # 161 & 162), Diphtheria toxins (Product # 149, 150, & 151), and others.
- Paddock C., (2014) Stem cells that release cancer-killing toxins offer new brain tumor treatment. Last accessed: 06 January 2015.
- Stuckey DW, Hingtgen SD, Karakas N, Rich BE, Shah K (2015) Engineering toxin-resistant therapeutic stem cells to treat brain tumors Stem Cells 33(2):589-600. doi: 10.1002/stem.1874. PMID: 25346520
- Chari RV (2008) Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc Chem Res 41(1):98-107. PMID: 17705444
- Goldmacher VS, Kovtun YV (2011) Antibody-drug conjugates: using monoclonal antibodies for delivery of cytotoxic payloads to cancer cells Ther Deliv 2(3):397-416. PMID: 22834009
- Oraki KM, Mirzaie S, Zeinali M, Amin M, Said HM, Jalaili A, Mosaveri N, Jamalan M (2014) Ablation of breast cancer cells using trastuzumab-functionalized multi-walled carbon nanotubes and trastuzumab-diphtheria toxin conjugate Chem Biol Drug Des 83(3):259-65. PMID: 24118702
- Schafer JM, Peters DE, Morley T, Liu S, Molinolo AA, Leppla SH, Bugge TH (2011) Efficient Targeting of Head and Neck Squamous Cell Carcinoma by Systemic Administration of a Dual uPA and MMP-Activated Engineered Anthrax Toxin. PLoS ONE 6(5): e20532. PMID: 21655226
- Engedal N, Skotland T, Torgersen ML, Sandvig K (2011) Shiga toxin and its use in targeted cancer therapy and imaging Microb Biotechnol 4(1):32-46. PMID: 21255370
- Barth H, Aktories K, Popoff MR, Stiles BG (2004) Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins Microbiol Mol Biol Rev 68(3):373-402.
- Fabbri A, Travaglione S, Falzano L, Fiorentini C (2008) Bacterial protein toxins: current and potential clinical use Curr Med Chem 15(11):1116-25. PMID: 18473807
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.
- NIAID website: http://www.niaid.nih.gov/topics/anthrax/pages/default.aspx
- CDC website: http://www.cdc.gov/anthrax/
- 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
- 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
- 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
- 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
By: Suzanne Canada, Ph.D.
Tanager Medical Writing
Vaccines have been used to help control diseases for more than 200 years and are the common practice for children and adults. Childhood vaccination has substantially reduced the morbidity and mortality from infectious diseases in much of the developed world, and influenza vaccinations have reduced the impact of seasonal influenza infections.1 However, medical researchers are constantly looking for ways to improve the vaccines that are already used, and develop new ones.
Opportunities for improvement of vaccines abound. For example, although much attention is given to child vaccinations, a reservoir of infection could be eliminated through promotion of adult booster shots such as pertussis booster shots for expectant mothers and close family members, to help protect susceptible newborns. In addition, some diseases that have vaccines currently available still flourish in areas of the world where infrastructures for vaccination are poor and are too costly or cannot be delivered in their current forms.1 Researchers are still trying to develop vaccines for other important diseases, such as HIV/AIDS, malaria and leishmaniasis. Vaccines are also being developed for bacterial pathogens, such as Vibrio cholerae O1 and enterotoxigenic Escherichia coli (ETEC) that are responsible for a high proportion of diarrheal disease and death in adults and children in many countries in Africa and Asia.2
By modifying the factors included in the vaccine, researchers balance the effectiveness of the immune response with the side effects. Previously, whole cell vaccines containing whole organisms that had been chemically inactivated were the norm, but the side effects of fever and discomfort following injections were much more common. Many of the vaccines used today, including those for measles and some influenza vaccines, use live, attenuated viruses. Others use killed forms of viruses, pieces of bacteria (lipopolysaccharides), or inactivated forms of bacterial toxins, known as “toxoids.” Killed viruses, lipopolysaccharides and toxoids can evoke an immune response that protects against future infection.3 Acellular vaccines were introduced in the late 1990’s that contain either three or five key bacterial proteins and have been quite effective in protecting infants and children under four with a much lower rate of side effects.
List Labs offers several virulence factors which are used in vaccine testing. For testing C. difficile vaccines; available reagents are C. difficile Toxin A (Product #152), C. difficile Toxin B (Product #155), C. difficile Toxoid A (Product #153), C. difficile Toxoid B (Product #154), and both subunits of the Binary Toxin (Products #157 and #158). Numerous Bordetella pertussis virulence factors are available for use in testing including: Products #179, #180 or #181 Pertussis Toxin, Product #170 FHA, Product #186 Fimbriae, Product #187 Pertactin, Product #188 and #189 Adenylate Cyclase and Product #400 Highly Purified B. pertussis LPS. Anthrax vaccine testing maybe carried out using Protective Antigen (Product #171) with Lethal Factor (Product #172) in a toxin neutralization assay. Although these factors are not suitable for testing on humans, they are excellent research tools.
Inactive toxins are quite useful in making antibodies or in capturing antibodies from a vaccinated population on ELISA plates. Three of our inactivated toxins, which carry mutations in the toxin active site, are B. pertussis Adenylate Cyclase Toxoid, Pertussis Toxin Mutant, Product #184 and CRM197, a non-toxic Mutant Diphtheria Toxin, Product #149. Toxoids made by formaldehyde treatment of toxins include versions of C difficile Toxins (Products #153 and #154), Diphtheria Toxoid (Product #151), Staphylococcus aureus Enterotoxoid B (Product #123), Tetanus Toxoid (Product #191) and Toxoids of Botulinum Neurotoxins A and B (Product #133 and #139, respectively).
- Hammond B., Sipics M., Youngdhal K., (2013). From the History of Vaccines, a project of the college of physician of Philadelphia. ISBN: 9780988623101
- Svennerholm AM., (2011) From Cholera to Enterotoxigenic Escherichia coli (ETEC) vaccine development. Indian J Med Res. 133(2): 188–194. PMID: 21415493
- Leitner DR., Feichter S., Schild-Prüfert K., Rechberger GN., Reidl J., Schild S., (2013) Lipopolysaccharide modifications of a cholera vaccine candidate based on outer membrane vesicles reduce endotoxicity and reveal the major protective antigen. Infect Immun 81(7):2379-93. PMID: 23630951