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By: Mary N. Wessling, Ph.D. ELS

Bacterial Toxins used for Vaccine Research

List Biological Laboratories’ (List Labs) catalog of products is related to furthering research in human health and preventing disease, most commonly as the starting materials for vaccine research & development or production around the world. Vaccines are mainly identified for their capacity to prevent diseases that the body’s innate defensive mechanisms (the skin and specialized cells in the blood, for example) can’t resist unaided. However, there are many other uses for these purified materials in medical research, and you will likely encounter wording on our website that is not part of everyday vocabulary for non-scientists. This article is intended to provide a basic understanding of some of the more frequently used terms and aid you in selecting the products most essential to your projects.

Toxin vs. Toxoid

For starters, what is the difference between “toxin” and “toxoid”. Broadly defined, anything that can cause harm to an organism is a toxin. However, for List Labs’ products and in biological usage, a toxin refers to a bacterial or viral product that has harmful effects when it enters the body (List Labs’ toxins are in a highly purified form). A toxoid is a chemically altered toxin that has reduced or no toxicity and is used for its remaining antigenic activity, which can stimulate an immune response.

Take, for example, cholera, a disease produced by Vibrio cholerae bacteria, possibly through contact with body fluids from a person ill with cholera or through contaminated water supply. Cholera causes severe diarrhea, and untreated, it can be fatal. However, the purified List Labs’ cholera toxin by chemical modification becomes a toxoid that lacks toxic activity but retains structures that make it useful for immunization of research animals or stimulation of immune cells in vitro.

How do Toxoids Impact the Immune Process? 

To understand how some List Labs products work, an overview of the immune process is helpful. During the course of a day, we frequently touch, ingest, or breathe in something that has potential to harm the body. Our cells react to this invader: is this a threat, or not, and if so, how serious is the threat?

What is the Innate Immune Response and Its Role in Disease Prevention? 

The innate immune response is the first order of defense in the immune process. There are many different cell types in our body. Some of these cells are equipped by their structural and biochemical components to destroy dangerous microbial invaders–pathogens–quickly. The inflammation that we experience from minor infections is often a sign of this process as cells from the blood destroy the pathogen. This happens quickly, within hours.

Adaptive Immune Response: How B-Cells and T-Cells Defend Against Invaders

Another cellular response system requires a longer time to react to the threat. These cells react by changing from an inactive form to one that will start a more complex defensive process: this is the second step, the adaptive immune response. There are two different classes of cells that comprise the adaptive immune response; they differ by the structures that give them their ability to bind antigens– the invading bacteria and viruses. Both these cells are called lymphocytes; individually, they are the B-lymphocytes (B-cells) and T-lymphocytes (T-cells). Both originate from stem cells in the bone marrow; B and T refer to the place in the body where they mature. T-cells mature in the thymus into several subclasses of T-cells that circulate in the blood and lymph. “Killer” T-cells recognize foreign antigens on cell surfaces (e.g. from viral infection or malignancy). “Helper” T-cells induce B-cells to produce antibodies. “Suppressor” T-cells dampen or regulate the immune response to prevent over-reaction. B-cells mature in the bone marrow and migrate to secondary lymphoid tissues (e.g. spleen and lymph nodes). When they encounter foreign antigens and/or helper T-cells, they are stimulated to divide and expand clonally to produce antibodies and differentiate into plasma cells.

The Importance of Immune Memory in Vaccine Development 

After the B and T-lymphocytes react to an antigen, two results are possible. The first, and desirable result, is that the invader is identified and defeated, leaving behind what might be called its criminal record: immune memory. When the antigen comes creeping back in the future, the adaptive immune system recognizes it and attacks. The second possibility is an over-reaction and lack of cessation of the adaptive immune process that is harmful to the body: an autoimmune condition.

Antigens, Epitopes and Their Role in Vaccines Design

Where do vaccines come into this process? An antigen is a substance that causes the body to mount an immune response against it. Antigens include toxins, bacteria, viruses, or other substances that the body recognizes as foreign or not “self”. Vaccines have structural features similar to structures of the toxin or invading pathogen that can elicit adaptive immunity.

An epitope is a specific molecular region on the surface of an antigen, typically one of many on the antigen, that elicits an immune response and is capable of binding with the specific antibody produced by the response. A toxin has many epitopes that can be recognized by the immune response. The epitopes that are required for toxicity have been altered chemically in toxoids or by specific genetic mutations in inactive mutants; however, many epitopes are retained and can stimulate an adaptive or memory immune response that will be effective against the toxin.

Toxins and Toxoid Products for Vaccine Research at List Labs

Below is a list of toxin and toxoid or inactive mutant pairs of products available to support your research.

Toxin and product numbers Toxoid Inactive Mutant
Botulinum Neurotoxin Type A from Clostridium botulinum – 130A, 130B, 9130A 133L
Botulinum Neurotoxin Type B, Nicked, from Clostridium botulinum – 136A, 136B 139
Toxin A from Clostridium difficile – 152C 153
Toxin B from Clostridium difficile – 155A, 155B, 155L 154A
Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae – 150 151 149
Enterotoxin Type B from Staphylococcus aureus – 122 123
Tetanus Toxin from Clostridium tetani – 190A, 190B 191A, 191B

 

 

List Labs Citations PageBy: Rachel Berlin, Marketing Manager

The List Labs website hosts a library of scientific article abstracts related to the research performed using our products called the Citations Page. Visitors can search this library to learn how others have used List Labs’ reagents in their research. This valuable resource is updated monthly with new articles from a wide variety of publications. Check out a few recent articles below:

Botulinum Neurotoxin

Carrier Proteins

Clostridium difficile toxin

Lipopolysaccharide (LPS)

Diphtheria toxin

Don’t see the reagent you’re interested in? You can search the citations by product, year, publication, or by the type of cell, animal, assay, protein or research. Check it out today!

By: Mary N. Wessling, Ph.D. ELS

List Biological Laboratories, Inc.’s products are being used to confront one of the most pressing problems in health care today: stopping the worldwide spread of illness caused by Clostridium difficile (CD, C Difficile Toxin), a gram-positive, spore-forming anaerobic bacillus. The spores are highly resistant to adverse environmental conditions and are frequently among the contaminants in food products, where they germinate [1]. The CD pathogen causes severe diarrhea and pseudomembranous colitis, among other dangerous gastrointestinal ailments. At present, the estimate is 500,000 cases in hospitals and long-term care facilities, with an annual mortality rate of 15,000 to 30,000 in the US and worldwide [2]. The estimated annual cost of treating CD infections ranges from $436 million to $3.2 billion per year in the US alone [3].

C Difficile Toxin in Hospitals

Historically, CD was mainly a problem for hospitals and long-term care facilities, but infections may spread rapidly into the community, especially among persons who have required antibiotic treatments that kill off competing strains in the intestines and allow CD to multiply. CD is a rapidly evolving bacterium, with hypervirulent strains contributing to the increase in mortality. At present, 234 unique genomes have been identified that cause most of the hospital outbreaks in the US and Europe. The current testing of stool samples to confirm diagnosis requires up to 3 days to differentiate between dangerous CD infection (CDI) and less harmful causes of diarrhea. A delay in diagnosis of CD presents a difficulty in treatment, making the development of more rapid diagnostic techniques a high priority.

C Difficile Toxin Types A & B

Clostridium difficile produces two major toxins, C Difficile Toxin A aka TcdA (Product #152) and C Difficile Toxin B aka TcdB (Product #155), the latter being the more virulent. These toxins inactivate the Rho-GTPase through glycosylation, and the structural bases for their activities have been clarified by X-ray diffraction, biochemical assays, and molecular dynamics [4].  The List Labs Toxin A and B products are playing an important role in the search for a more rapid and accurate methods to diagnose CDI. To meet the demands of those more sensitive and exacting methods, List’s difficile toxins are of higher purity than previously available products.  The purity of List Labs’ toxins enables diverse creative and fascinating scientific approaches that adapt and modify known analytical techniques to CDI testing. What follows (in chronological order of publication) are recent studies that relied on List Labs’ products for their results.

C Difficile Infection Studies

Molecular diagnostic techniques are increasingly being used to quantify the seriousness of infection and to distinguish CDI from other causes. The study by Moura et al [2] used List Labs’ products (purified TcdA and TcdB) for a proteomic analysis that identified and quantified the protein factors involved in CD toxin production through an enhanced mass spectrometric (MS) method. This method provided a basis for development of improved MS methods that demand only small samples and contribute to a better understanding of toxin-mediated illnesses, their prevention and therapy.

In the same year, Lei and Bochner [5] used List Labs’ Toxins A and B in phenotype microarrays (PMs) under different culture conditions. Noting that with the evolution of the CD genome, multiple lineages evolved independently; they examined how the Toxin B cytopathic effect caused cell rounding and used that to measure the virulence of CD under different conditions. Using the PMs, they developed a 1-day test that compared the pure List Labs’ toxins and unpurified toxins to see parallels, which could be measured by colorimetry. This provided a more rapid test than the 3-day diagnostic tests currently in use.

In 2014, Huang et al [6] used CD toxin B to approach the problem of distinguishing between infection, colonization, and live and dead CD organisms. Their real-time microelectronic sensor-based analysis had high sensitivity and relied on reading the impedance of cells applied to microelectrodes to detect specific cellular processes through quantification over time. The method is rapid: the authors reported that 80% positive results were obtained within 24 hours. Concentration of the CD in stool measured by their method correlated with clinical severity, providing a method to monitor the progress of CDI in patients.

In 2014, an innovative study by Leslie et al [7] used human intestinal organoids (HOIs) derived from stem cells to model the disruption of barrier functions in the human intestine by CD. The HOIs were generated by directed differentiation of human pluripotent stem cells, which were then further differentiated into intestinal tissue. These HOI’s were subjected to the C. difficile strain, TcdA or TcdB. Increased toxicity under conditions favorable to production of toxins by CD was measured by presence of cell rounding using fluorescent dextran. Injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable CD.

In 2015, Hong et al [3] used an extension of a method they had developed previously combined with ELISA, eventually applying single-stranded DNA molecular recognition elements (MRE) to microchips. They used List Labs’ lyophilized toxin B, reconstituted and immobilized on magnetic beads; after incubation with an ssDNA library, 80 randomly selected clones were sequenced and analyzed. The goal was to identify ssDNA MREs that bind to toxin B; eventually, fluorescence was used to examine the structure of one selected MRE bound to toxin B. This very complex process yielded an MRE bound with high specificity with toxin B in human fecal matter; it demonstrated a proof-of-concept diagnostic application.

In quite another approach to preventing the morbidity and mortality attributed to CD, a study by Zilbermintz et al [8] showed that the antimalarial drug amodiaquine has a protective effect against CD. The drug was one of a group of existing FDA-approved compounds screened to extend their use to as broad-spectrum, host-oriented therapies. Amodiaquine interferes with the functioning of the host protein cathepsin B targeted by CD and other pathogens as well. The aim of their approach is to find therapies that circumvent the effect of pathogen mutations that lead to drug resistance. All toxins used in the study were purchased from List Biological Laboratories. Though not a new diagnostic technique, this discovery promised an approach to CDI using existing pharmacology, which then could reduce the immense cost of treating CD patients [8].

 

References:

  1. Xiao Y et al. Clostridial spore germination versus bacilli: genome mining and current Food Microbiol. 2011 Apr;28(2):266-74. doi: 10.1016/j.fm.2010.03.016. Epub 2010 Apr 1. PMID: 21315983 
  2. Moura H et al. Proteomic analysis and label-free quantification of the large Clostridium difficile toxins. Int J Proteomics. 2013;2013:293782. doi: 10.1155/2013/293782. Epub 2013 Aug 27. PMID: 24066231 
  3. Hong KL et al. In vitro selection of a single-stranded DNA molecular recognition element against Clostridium difficile toxin B and sensitive detection in human fecal matter. J Nucleic Acids. 2015;2015:808495. doi: 10.1155/2015/808495. Epub 2015 Feb 5. PMID: 25734010
  4. Yin JC et al. Structural insights into substrate recognition by Clostridium difficile sortase. Front Cell Infect Microbiol. 2016 Nov 2a2;6:160. eCollection 2016. PMID: 27921010
  5. Lei XH , Bochner BR. Using phenotype microarrays to determine culture conditions that induce or repress toxin production by Clostridium difficile and other microorganisms. PLoS One. 2013;8(2):e56545. doi: 10.1371/journal.pone.0056545. Epub 2013 Feb 20. PMID: 23437164
  6. Huang B et al. Real-time cellular analysis coupled with a specimen enrichment accurately detects and quantifies Clostridium difficile toxins in stool. J Clin Microbiol. 2014 Apr;52(4):1105-11. doi: 10.1128/JCM.02601-13. Epub 2014 Jan 22. PMID: 24452160
  7. Leslie JL et al. Persistence and toxin production by Clostridium difficile within human intestinal organoids result in disruption of epithelial paracellular barrier function. Infect Immun. 2015 Jan;83(1):138-45. doi: 10.1128/IAI.02561-14. Epub 2014 Oct 13. PMID: 25312952
  8. Zilbermintz L et al. Identification of agents effective against multiple toxins and viruses by host-oriented cell targeting. Sci Rep. 2015 Aug 27;5:13476. doi: 10.1038/srep13476. PMID: 26310922 

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

While you go about your day, you are surrounded by micro-organisms.  Although most of us spend a lot of time washing up and trying not to think about the propensity of creatures that share our personal space, scientists have been studying them.  Due to their great progress, we are reaching an understanding of how these bacteria and fungi affect our bodies’ functions [1]. The evidence indicates that this inner-ecosystem can not only cause disease if perturbed, but also influence our overall health!  Organisms including Escherichia coli, Helicobacter pylori, Streptococcus thermophilus, and species of Clostridia, Lactobacillus, and Bacterioides inhabit our gut. Corynebacterium jeikeium as well as Staphylococcus species live on our skin, and other Streptococci as well as Neisseria and Candida albicans inhabit our mouth and upper respiratory system [2].  The makeup and diversity of organisms has been found to be strongly influenced, not only by what you eat [3], but also by who you live with [4, 5].  With greater understanding of this rich soup of life that we carry with us, the microbiome has become the new frontier in cutting-edge drug development [6].

In the last three years, research into the molecular basis of microbial influence has blossomed.  The first and most obvious application for this information was in treating C. difficile infections; which result from overgrowth of the opportunistic pathogen after an antibiotic regimen or hospital stay.  Researchers found that fecal transplants from a healthy individual were an effective way to treat this potentially fatal infection [7, 8]. The role of intestinal microbes in Inflammatory Bowel Disease (IBD) has been established [9] in the last year or two.  Based on this knowledge, possible treatments for IBD, such as ulcerative colitis and Crohn’s disease, are in development. Other publications point to microbes’ role in inflammation of the skin and respiratory tract, including acne [10]and asthma[11].  More excitement has been generated as investigators have found links to other chronic diseases including diabetes [12, 13], hypertension [14, 15], and chronic liver disease [16].  Preliminary investigations suggest a connection between overall gut microbial composition and obesity [17].  Some studies in mouse models have even linked the microbiome to the neurological conditions of Alzheimer’s [18] and autism [19, 20].

With all this research going on, you need a great resource like LIST Biological Laboratories, with experience and expertise with microbial products spanning over 25 years. LIST has several products available that can serve as positive controls for your microbial research.  Potent toxins from C. difficile are available (LIST products #157, #158), as well as antibodies that aid in their detection (LIST products #753, #754). Lipopolysaccharides are also available, which cause inflammation and activation of immune signaling cascades, and are extracted from bacterial cell walls of E. coli O111:B4, O55:B5, O157:H7, J5 and K12; Salmonella typhimurium, Salmonella minnesota and Bordetella pertussis. Other acute immune system activators such as Staphylococcal toxins (LIST products #120, #122) and Shiga toxins (LIST products #161 & #162) are also available.

In case the assortment of purified bacterial products on hand are insufficient for your research needs, LIST also provides contract manufacturing for biotherapeutics, as well as microbial purification services.

References

  1. Human Microbiome Project, C., A framework for human microbiome research. Nature, 2012. 486(7402): p. 215-21. PMID: 22699610
  2. Human Microbiome Project, C., Structure, function and diversity of the healthy human microbiome. Nature, 2012. 486(7402): p. 207-14. PMID: 22699609
  3. David, L.A., et al., Diet rapidly and reproducibly alters the human gut microbiome. Nature, 2014. 505(7484): p. 559-63. PMID: 24336217
  4. Yatsunenko, T., et al., Human gut microbiome viewed across age and geography. Nature, 2012. 486(7402): p. 222-7. PMID: 22699611
  5. La Rosa, P.S., et al., Patterned progression of bacterial populations in the premature infant gut. Proc Natl Acad Sci U S A, 2014. 111(34): p. 12522-7. PMID: 25114261
  6. Donia, M.S., et al., A Systematic Analysis of Biosynthetic Gene Clusters in the Human Microbiome Reveals a Common Family of Antibiotics. Cell, 2014. 158(6) p1402 – 1414. PMID: 25215495
  7. Seekatz, A.M., et al., Recovery of the gut microbiome following fecal microbiota transplantation. MBio, 2014. 5(3): p. e00893-14. PMID: 24939885
  8. Scott, K.P., et al., Manipulating the gut microbiota to maintain health and treat disease. Microb Ecol Health Dis, 2015. 26: p. 25877. PMID: 25651995
  9. Huttenhower, C., A.D. Kostic, and R.J. Xavier, Inflammatory bowel disease as a model for translating the microbiome. Immunity, 2014. 40(6): p. 843-54. PMID: 24950204
  10. Christensen, G.J. and H. Bruggemann, Bacterial skin commensals and their role as host guardians. Benef Microbes, 2014. 5(2): p. 201-15. PMID: 24322878
  11. Martin, C., et al., Host-microbe interactions in distal airways: relevance to chronic airway diseases. Eur Respir Rev, 2015. 24(135): p. 78-91. PMID: 25726559
  12. Tang, D., et al., Comparative investigation of in vitro biotransformation of 14 components in Ginkgo biloba extract in normal, diabetes and diabetic nephropathy rat intestinal bacteria matrix. J Pharm Biomed Anal, 2014. 100: p. 1-10. PMID: 25117949
  13. Sato, J., et al., Gut dysbiosis and detection of “live gut bacteria” in blood of Japanese patients with type 2 diabetes. Diabetes Care, 2014. 37(8): p. 2343-50. PMID: 24824547
  14. Pluznick, J., A novel SCFA receptor, the microbiota, and blood pressure regulation. Gut Microbes, 2014. 5(2): p. 202-7. PMID: 24429443
  15. Pluznick, J.L., Renal and cardiovascular sensory receptors and blood pressure regulation. Am J Physiol Renal Physiol, 2013. 305(4): p. F439-44. PMID: 23761671
  16. Minemura, M. and Y. Shimizu, Gut microbiota and liver diseases. World J Gastroenterol, 2015. 21(6): p. 1691-702. PMID: 25684933
  17. Al-Ghalith, G.A., P. Vangay, and D. Knights, The guts of obesity: progress and challenges in linking gut microbes to obesity. Discov Med, 2015. 19(103): p. 81-8. PMID: 25725222
  18. Bibi, F., et al., Link between chronic bacterial inflammation and Alzheimer disease. CNS Neurol Disord Drug Targets, 2014. 13(7): p. 1140-7. PMID: 25230225
  19. De Angelis, M., et al., Fecal microbiota and metabolome of children with autism and pervasive developmental disorder not otherwise specified. PLoS One, 2013. 8(10): p. e76993. PMID: 24130822
  20. Pequegnat, B., et al., A vaccine and diagnostic target for Clostridium bolteae, an autism-associated bacterium. Vaccine, 2013. 31(26): p. 2787-90. PMID: 23602537

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).

 

  1. Hammond B., Sipics M., Youngdhal K., (2013). From the History of Vaccines, a project of the college of physician of Philadelphia. ISBN: 9780988623101
  2. Svennerholm AM., (2011) From Cholera to Enterotoxigenic Escherichia coli (ETEC) vaccine development.  Indian J Med Res. 133(2): 188–194. PMID: 21415493
  3. 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

List Biological Laboratories, Inc. is pleased to announce a key addition to our Clostridium difficile Toxin A and Toxin B product line. Our new product, 155L is a liquid formulation of Toxin B. This product allows for the highest activity level of any of our C.difficile offerings and the most stability.  It already shows stability for several months when stored at 2-8°C and we will continue to update.

This product is offered in a 50 µg size for $450.

We are very excited about the liquid formulation and the flexibility it will allow in the lab. It is now available from stock, and ships on Blue Ice throughout the U.S. and Canada and worldwide with special arrangements.

Clostridium difficile Toxin A
We have enjoyed tremendous success with our Toxin A. We have determined that the best activity and stability post-reconstitution (up to 2 months) is in the 100 µg size, #152C. The price for this product has been lowered from $535 to $300 based on the dramatic improvements in our yield and the substantial growth in overall sales. We are happy to pass this savings on, making it a terrific value for our C.difficile customers.

Clostridium difficile Toxin B
Products 155A and 155B have a newly formulated buffer which allows for highly active Toxin B. After reconstitution, this product is stabile for days when stored at 2-8°C and is now available from stock.

Related Products
We offer a variety of Binary Toxins and Antibodies to complement your C.difficile research. We have Binary Toxins, both A and B subunit. Check our website for more information.

Bulk Product Orders
Should you have need of a substantial quantity of C.difficile in bulk, please let us know what quantity and formulation you desire. With sufficient quantity, we can often tailor to your specific needs. We generally have bulk available for all C.difficile offerings.  We also offer other types of bulk reagents for purchase.

Coming Soon-Pipeline
Clostridium difficile produces and secretes a glutamate dehydrogenase (cdGDH). This enzyme is highly conserved among different ribotypes, and antibodies to cdGDH are often included in detection kits for C. difficile infection (CDI), increasing sensitivity. While cdGDH is available recombinantly, we have isolated and purified the cdGDH from a native strain of C. difficile. In addition to the enzyme, chicken antibodies will be generated and made available soon.

We welcome your feedback on these changes and would love to hear how the new formulations and new products work for you. Please contact us with your comments and feedback.

What is Clostridium Difficile?

Clostridium difficile is the causative agent of antibiotic-associated diarrhea and pseudomembranous colitis.  It produces two major exotoxins, Toxin A and Toxin B; however, about 10% of strains isolated from patients with colitis also contain genes, coding for a unique ADP-ribosylating toxin, CDT Binary Toxin. Additionally, Clostridium difficile produces and secretes a glutamate dehydrogenase (cdGDH).

List Labs offers four new products in the C. difficile family

New product offerings from List Labs cover other proteins produced concurrently with the exotoxins.  These proteins are valuable as alternate markers allowing more sensitive or more accurate determination of C. difficile infections (CDI).

These new products, all related to C. difficile, will be of interest to diagnostic developers, vaccine manufacturers, as well as, to those doing research in infectious diseases.  List Labs is notably offering antigens and antibodies for two C. difficile proteins which are present in C. difficile infections.  These products add to our C. difficile reagents which also include the main virulence factors Toxin A and Toxin B, and the antibodies: Goat Anti-Toxin A, Chicken Anti-Toxin B and Chicken Anti-Toxin B.

The first two products are components of CDT Binary Toxin, an ADP-ribosyltransferase.  This toxin is composed of two independently produced components, the enzymatic subunit A, CdtA, and the binding and translocation subunit B, CdtB, which mediates cell entry of CdtA.  CDT Binary Toxin causes depolymerization of the actin cytoskeleton and formation of microtubule-based membrane protrusions,resulting in cell rounding and cell death, and it is suggested to be involved in enhanced bacterial adhesion and colonization of hypervirulent C. difficile strains.  The cell surface receptor has been identified as lipolysis stimulated lipoprotein receptor (LSR).

CDT Binary Toxin, A Subunit (CDTa), Product # 157, is recombinantly expressed in E. coli and purified using affinity chromatography.  The affinity tag has subsequently been cleaved from the protein prior to packaging.  Binary toxin A subunit has been tested in an in vitro ADP-ribosylation assay.  It is non-toxic and unable to penetrate cells in the absence of the B subunit binding and translocation domain.  Expression and purification of the A subunit from a recombinant setting ensures that there is no possible contamination with the B subunit.

1)      #157A, Binary Toxin from Clostridium difficile A Subunit 20 ug, price $350

CDT Binary Toxin, B Subunit (CDTb), Product # 158, is recombinantly expressed in E. coli, purified using affinity chromatography and the affinity tag cleaved.  Prior to packaging, the B subunit is nicked with trypsin for activation.  The B subunit of the Binary Toxin is non-toxic, and does not contain any enzymatic activity.

2)      #158A, Binary Toxin from Clostridium difficile B Subunit 40 ug, price $350

 The next products are chicken antibodies: Chicken Anti-CDT Binary B subunit antibodies, with and without biotin.  Antibodies have been raised against C. difficile Binary Toxin B Subunit and affinity purified on an antigen column, Product # 758.  These antibodies are suitable for use in Western Blot assays and ELISAs as an effective probe for C. difficile Binary Toxin B Subunit.  Additionally, purified antibody has been labeled with biotin, Product # 759, providing antibodies for both capture and detection.

3)      #758A, Anti – C. difficile Binary Toxin B Subunit (Chicken IgY) 0.1 mg, price $290

4)      #759A, Biotinylated Anti – C. difficile Binary Toxin B Subunit (Chicken IgY) 0.1 mg, price $315

Usage

Use of CDT on subconfluent Caco-2 cells is described by Schwan et al, 2009.  Toxin –induced cellular processes were observed on these cells after one hour treatment with CDT Binary Toxin, a mixture of 20 ng/ml of CDTa and 40 ng/ml of CDTb.

Use our new Citations Finder to see additional citations of how C. Difficile from List Labs has been used in research.

Reference

  1. Schwan C., Stecher B., Tzivelekidis T., van Ham M., Rohde M., Hardt WD., Wehland J., Aktories K., (2009) Clostridium difficile Toxin CDT Induces Formation of Microtubule-Based Protrusions and Increases Adherence of Bacteria. PLoS Pathog 5(10): e1000626. PMID: 19834554

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

By: Karen Crawford, Ph.D.
President, List Biological Laboratories, Inc.

 

You hear about Clostridium difficile at your doctor’s office and in news articles, but what does it mean and how does it affect the world around us?

C. Difficile Statisitcs

How C. Difficile Affects the Intestine

C. difficile enterotoxins A and B are the key to pathogenesis of CDI.  C. difficile toxin A (TcdA) and toxin B (TcdB) are both cytotoxic and cause inflammation in intestine, but they have slightly different activities (Theriot, 2013).  Toxin B is an extremely potent cytotoxin, that glycosylates small GTPase of the Rho family (Cdc42 and Rac) which control the actin cytoskeleton in eukaryotic host cells; this glycosylation disrupts signaling pathways of the cell cycle and lead to apoptosis.  TcdA has an activity like TcdB, but it is much less potent as a cytotoxin, but more commonly noted for its enterotoxic activity and large size (308 kDa vs 270 kDa for TcdB).  These toxins are the major virulence factors for C. difficile and cause inflammation and damage to cells in the intestine when the normal gut microflora are disrupted, such as after a round of treatment with antibiotics (Theriot, 2013; Carter, 2010).

Earlier studies using animal models of CDI had suggested that the toxins act synergistically because purified TcdA alone was able to induce C. difficile disease pathology and TcdB was not effective unless it was co-administered with TcdA.  However, the isolation of some new, clinically relevant toxin A-negative, toxin B-positive (A−B+) strains of Clostridium difficile from humans (Drudy 2010), indicated that toxin B may be the key to its virulence as a pathogen (Lyras 2009, Carter 2010).  The emergence of these new strains has prompted researchers to evaluate current C. difficile diagnostic methods (Alder 2014, Brown 2011, Garamella 2012, Grein 2014) and recommend ensuring that medical laboratories can detect both TcdA and TcdB in specimens.

List Labs Offers TcdA and TcdB for Purchase

List Biological Laboratories has been producing TcdA and TcdB since 2000. These toxins are purified proteins that are tested to ensure that the activity is preserved. Along with chicken antibodies to each toxin, TcdA and TcdB can be used in disease modeling as well as the development of diagnostic tools for CDI detection and diagnosis.

 

References

Adler A, Schwartzberg Y, Samra Z, Schwartz O, Carmeli Y, et al. (2014) Trends and Changes in Clostridium difficile Diagnostic Policies and Their Impact on the Proportion of Positive Samples: a National Survey. Clin Microbiol Infect Mar 27. doi: 10.1111/1469-0691.12634. [Epub ahead of print]. PMID: 24674056

 

Akerlund T, Persson I, Unemo M, Noren T, Svenungsson B, Wullt M, Burman LG (2008) Increased sporulation rate of epidemic Clostridium difficile type 027/nap1. J Clin Microbiol 46: 1530–1533. PMID: 18287318

Brown NA, Lebar WD, Young CL, Hankerd RE, Newton DW (2011) Diagnosis of Clostridium difficile infection: comparison of four methods on specimens collected in Cary-Blair transport medium and tcdB PCR on fresh versus frozen samples.  Infect Dis Rep 3(1):e5. PMID: 24470904

Carter GP, Rood JI, Lyras, D (2010) The role of toxin A and toxin B in Clostridium difficile-associated disease:  Past and present perspectives. Gut Microbes 1(1):58-64. PMCID: PMC2906822

 

Drudy D, Fanning S, Kyne L (2010) Toxin A-negative, toxin B-positive Clostridium difficile.  Int J Infect Dis 11(1):5-10. PMID: 16857405

Garimella PS, Agarwal R, Katz A (2012) The utility of repeat enzyme immunoassay testing for the diagnosis of Clostridium difficile infection: a systematic review of the literature.  J Postgrad Med 58(3):194-8. PMID: 23023352

Grein JD, Ochner M, Hoang H, Jin A, Morgan MA, Murthy AR (2014) Comparison of testing approaches for Clostridium difficile infection at a large community hospital. Clin Microbiol Infect 20(1):65-9. PMID: 23521523

 

Lanis JM, Barua S, Ballard JD (2010) Variations in TcdB activity and the hypervirulence of emerging strains of Clostridium difficile . PLoS Pathog 6:e1001061. PMID: 20808849 

 

Lyras D, O’Connor JR, Howarth PM, Sambol SP, Carter GP, et al. (2009) Toxin B is essential for virulence of Clostridium difficile. Nature 458(7242): 1176–1179. PMID: 19252482

 

McDonald LC, Killgore GE, Thompson A, Owens RC Jr, Kazakova SV, Sambol SP, Johnson S, Gerding DN (2005) An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med 353: 2433–2441. PMID: 16322603

 

Schwan C, Stecher B, Tzivelekidis T, van Ham M, Rohde M, et al. (2009) Clostridium difficile Toxin CDT Induces Formation of Microtubule-Based Protrusions and Increases Adherence of Bacteria. PLoS Pathog 5: e1000626. PMID: 19834554

 

Theriot CM, Young VB (2013) Microbial and metabolic interactions between the gastrointestinal tract and Clostridium difficile infection Gut Microbes 5(1). PMID: 24335555 

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