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

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? 

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.

What is the Adaptive Immune Response?

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.

What is Immune Memory? 

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 Vaccines

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 Research

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

 

 

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

Tetanus Toxin’s Use as a Protein Carrier and Antigen

Tetanus toxin (TT) is the major virulence factor of the Gram-positive bacterium Clostridium tetani. Infection with this bacterium in unvaccinated persons produces muscle spasms by binding to nerve endings and moving throughout the nervous system in a specific way. Eventually, almost total paralysis results. The deactivated toxin is the basis for a vaccine, which can even be administered to pregnant women, usually as part of a combination vaccine also aimed at preventing neonatal pertussis.¹ Worldwide, the mortality of infection among unvaccinated persons reaches 10%.² It is the binding specificity of this dangerous toxin that makes it valuable as a research material. List Biological Laboratories (List Labs) offers inactivated tetanus toxin and six related products, used in intricate and fascinating ways in research as a model antigen and protein carrier.

 

List Labs’ Tetanus Toxoid in Immunosupression Research

Existing antibody treatments for rheumatic arthritis (RA), for example doses of rituximab every six months, suppress autoreactive B cells by killing them. The effects of the treatment fade over the 6-month period; this increases the inherent risk of infection and progressive multifocal leukoencephalopathy. Chu et al studied a treatment that characterized B-cell immunosuppression by an engineered antibody; List Labs’ tetanus toxoid (TTd)

was used in an elegant series of explorations of the mechanism of action of an engineered antibody (XmAb581) that enhanced the action of a B-cell antigen receptor complex currently under clinical development for treatment of RA, and enhanced both its safety and efficacy.³

 

Tetanus Toxoid in B Cell-Driven Autoimmune Disease Research

In a study with a broader purpose, Klose et al, expanding a previous murine study, developed a protein engineering strategy to selectively target and eradicate human memory B cells. These authors built a fusion protein that combined a model antigen TTd fragment C with a truncated version of exotoxin A derived from Pseudomonas aeruginosa. A fluorescein isocyanate-labelled TT fragment C produced by List Laboratories, used as a control in the binding analysis, played an important supporting role. This research offers a promising approach for the specific depletion of autoreactive B-lymphocytes in B cell-driven autoimmune diseases.⁴

 Prompted by the lower prevalence of these diseases in children who lived near farm animals and in unhygienic environments, Iwasaki et al studied the key role of intestinal infection in development of allergic respiratory disease in children. Their study used List Labs’ TTd antigen

to compare total antibody binding between asthmatic and non-asthmatic children. The authors found an association between lower antibody titers in asthmatic children to echovirus, and in a previous study, a heightened response to rhinovirus. These findings support a key role for intestinal infection in the development of allergic respiratory disease.⁵

 

Further Innovative Applications of List Labs’ Tetanus Toxin and Tetanus Toxoid

List Labs’ tetanus toxin (TT)

was used in a study that challenged an accepted mechanism for cell death in injured human retinal ganglion cells; Li Y et al showed that dysregulation of mobile zinc is to blame. In anesthetized animals, they used TTd to cleave the synaptic vesicle protein and then injected the zinc formulation. Fluorescent images showed that there was a rapid accumulation of Zn²⁺ in amacrine cell processes after optic nerve injury.⁶ Investigating yet another problem that affects injured persons, i.e., the necessity to keep an injured limb immobile, which results in muscle atrophy, Matthews et al reported that inactivity can result in 20% to 30% atrophy despite the use of exercise-based or neuromuscular electronic stimulation. They injected either saline or a very dilute solution of List Labs’ TT in physiological saline in the tibialis anterior of rats and compared the health of muscle fibers after immobilization. They found that the TT prevented loss of size in all 3 myofiber types, and therefore was protective against muscle loss during immobility.⁷

Finally, animal studies are used to evaluate the efficacy of pharmaceutical and other products for human use; although there are stringent conditions that assure ethical treatment of animals, the process is often time-inefficient, inaccurate, and costly. Temann et al explored using precision-cut lung slices (PCLS) from lungs of donors that were not suitable for use in transplantation as an alternative to animal studies. They evaluated a culture system using PCLS stimulated by List Labs’ TTd

and found that these slices could be held in culture for up to 14 days to study cytotoxic, inflammatory, and immune responses.⁸

The studies we cite here are only a small sample of what can be accomplished using List Labs’ TT and related products. We invite you to visit our citations page to explore how TT and our other products can augment your experimental design.

 

1. Chu HY, Englund JA. Maternal immunization. Birth Defects Research. 2017;109(5):379-386. PMID: 28398678
2. da Silva Antunes R, Paul S, Sidney J, et al. Definition of Human Epitopes Recognized in Tetanus Toxoid and Development of an Assay Strategy to Detect Ex Vivo Tetanus CD4+ T Cell Responses. PloS One. 2017;12(1):e0169086. PMID: 28081174
3. Chu SY, Yeter K, Kotha R, et al. Suppression of rheumatoid arthritis B cells by XmAb5871, an anti-CD19 antibody that coengages B cell antigen receptor complex and Fcgamma receptor IIb inhibitory receptor. Arthritis & Rheumatology (Hoboken, NJ). 2014;66(5):1153-1164. PMID: 24782179
4. Klose D, Saunders U, Barth S, Fischer R, Jacobi AM, Nachreiner T. Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen. BMC Biotechnology. 2016;16:18. PMCID: PMC4756516
5. Iwasaki J, Chai LY, Khoo SK, et al. Lower anti-echovirus antibody responses in children presenting to hospital with asthma exacerbations. Clinical and Experimental Allergy : Journal of the British Society for Allergy and Clinical Immunology. 2015;45(10):1523-1530. PMID: 25640320
6. Li Y, Andereggen L, Yuki K, et al. Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration. Proceedings of the National Academy of Sciences of the United States of America. 2017;114(2):E209-e218. PMCID: PMC5240690
7. Matthews CC, Lovering RM, Bowen TG, Fishman PS. Tetanus toxin preserves skeletal muscle contractile force and size during limb immobilization. Muscle & Nerve. 2014;50(5):759-766. PMID: 24590678
8. Temann A, Golovina T, Neuhaus V, et al. Evaluation of inflammatory and immune responses in long-term cultured human precision-cut lung slices. Human Vaccines & Immunotherapeutics. 2017;13(2):351-358. PMID: 27929748

By:
Debra Booth, VP of Operations
Linda Eaton, Ph.D., VP of Research & Development
Stacy Burns-Guydish, Ph.D., Senior Director of Microbiology
PJ Nehil, Sales & Distribution Coordinator

Dear Researchers Everywhere,

List Labs recently exhibited at the 2016 BIO International Convention. As a producer of bacterial products for research, as well as a provider of custom laboratory services, we were excited to meet with current and potential customers. We were eager to gain some insight into the current and future state of biotechnology and the up and coming field of microbiome research. But you never know exactly how you’ll feel until you spend the time in the conference exhibit hall. We were very pleased to see that we are a part of an industry that is moving forward at the pace of a start up, fueled by the novel ideas and intellect of many scientists.

Last week, thousands of people filled the Moscone Convention Center in San Francisco. The event was brilliantly organized and the venue was strategically arranged. The various pavilions were organized by state or country and some by specialty. It was a great opportunity to network and identify new sources for projects and services. Attendees were CEO and business development folks interested in learning more about what exhibitors have to provide. At our booth, we talked about immunotherapy, live biotherapeutics, contract manufacturing, GMP production, and more. It was a pleasure to shake hands with many customers, distributors, and colleagues and discuss ways we can partner with them to move their research forward.

We also had the opportunity to meet with vendors, in shipping, supplies and services. These vendors are critical to delivery of our biological and therapeutic products, and we benefited from learning about their new offerings as strategic partners. The enthusiasm was palpable from both exhibitors and attendees. At this event, we didn’t just meet industry veterans. We met many young scientists and job seekers looking for their first break. Some even came directly to us to hand-deliver their resumes. The event had a job expo, fueling another layer of energy and opportunity for exhibitors. We were resident in the California Pavilion where we learned that the State of California has a Biosciences Training Program, which will help companies pay for new employment training. Community colleges around the country are encouraging students to contribute to the future of biotechnology through clinical and regulatory apprenticeships. It’s great to see that science is providing opportunity for students in so many ways.

In closing, we found the 2016 BIO International Convention to be highly productive for our company. For those of you who didn’t get a chance to meet us at the convention, it’s very easy to reach us online and on social media. We would love to connect with you on LinkedIn, tweet with you on Twitter, like each other on Facebook and Google+. You can also check these accounts if you’re curious about your next opportunity to meet us at a conference. We even have a YouTube channel and a blog where you can learn more about us. We’d love to see your YouTube videos and read your blog if you have them as well. The future of biotechnology looks bright and we’re more excited than ever to be a part of it. We will definitely attend more events like this and we hope to see you at BIO 2017 in San Diego!

Regards,
Debra, Linda, Stacy, & PJ

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

Concerns over outbreaks of potentially serious childhood diseases are in the news again in California with an increase in whooping cough infections in 2014, caused by the pathogen Bordetella pertussis, and a measles outbreak in December 2014 to February 2015.  Since outbreaks and epidemics may be infrequent, many of us do not realize that vaccine development is an ongoing process. Public health experts monitor changes in the predominant strains and pathogen virulence continuously.  Although the sudden upswing in cases is alarming for those with young children, fuller understanding of the cyclic nature of these outbreaks fosters refinements to public health practices as well as the vaccines.

Recent increases in the number of cases of pertussis infections are due to eroding immunity in the population of immunized individuals, as well as changes in the prevalent virulent strains.  Outbreaks of new strains or new agents occur every 6 months or so [1]. For example, every 4 or 5 years, a new increase in pertussis infections is observed [2]. However, development of safe and effective vaccines is a process that takes up to 15 years [1].  Therefore, health agency efforts must be ongoing and constant funding must be in place to have these vaccines ready when the public needs them.

Although we are observing outbreaks of whooping cough and measles in the past year, the rates of infection are still much lower than what was observed in the pre-vaccine era.  In the pre-vaccine era (prior to the 1940s in the USA), 157 serious pertussis infections occurred yearly per 100,000 cases, including ~15 infant deaths/100,000 cases [2].  The highest incidence recorded was 260,000 cases in 1934 [3]. Since the broad adoption of vaccinations, whooping cough is much rarer during most years; upswings were observed in California in 2010 and again in 2014.  In 2010, the most serious cases were seen among infants under one year old.  The experts then realized that they could best control the rate of serious infections in infants by immunizing mothers at G27-36 weeks or immediately after the birth [4], and by making sure everyone in the expectant family was also immunized.  Furthermore, the 2014 outbreak of whooping cough was highest among 15 year olds (137.8 cases/100,000) [2].  By gathering immunization records and analysis of blood samples for anti-pertussis antibodies among those who had an infection, the doctors deduced that eroding resistance to pertussis corresponded with the use of the acellular pertussis vaccine.

The acellular pertussis vaccine was developed in the 1990’s in response to a high rate of side effects (fever) in those receiving the whole cell vaccine.  The acellular vaccine uses a few especially prominent antigens (pertussis toxoid, filamentous heamagglutinin, and pertactin, among others) that are specific to all pertussis bacteria. In the late 1990s, the FDA recommended replacement of the whole cell vaccine with the acellular vaccine which doesn’t cause as much fever and discomfort following vaccination boosters.

Investigations of why the acellular pertussis vaccine is not conferring resistance as durable as others, such as the tetanus or diphtheria vaccines, are ongoing.  Some vaccine experts point to evidence that resistance to whooping cough isn’t as durable with the acellular vaccine [5, 6]; however, other analyses conclude that the genes encoding antigens targeted by acellular pertussis vaccines are changing at higher rates than other surface-protein encoding genes of the pathogen [7, 8].  A meta-analysis of different immunization schedules found that resistance depended on time since last immunization or exposure [9], with resistance dipping by 5 years after the last booster shot.  Some researchers have found that having more anti-pertussis antigens in the vaccine conferred a higher level of protection [10], and new antigens for the vaccine are under evaluation (e.g., LpxL 1 [11]).

Several virulence factors of B. pertussis are available for research purposes from List Labs including pertussis toxin (Products #179, #180 and #181), pertactin (Product #187), fimbriae 2/3 (Product #186), adenylate cyclase toxin (Product #188 and 188L),  filamentous heamagglutinin (#170) and lipopolysaccharide (Product #400). Inactivated toxins, known as toxoids, are frequently used in the vaccines.  List Labs offers toxoids of C. difficile toxins (Products #153 and #154), diphtheria toxin (Product #151), Staphylococcus aureus enterotoxin B (Product #123), tetanus toxin (Product #191) and botulinum neurotoxin types A and B (Product #133 and #139, respectively).  List Labs’ vaccine carrier proteins are provided for research use only; however, GMP material may be produced on a contract basis.  More information is available on our website: www.listlabs.com.

 

References

1. Rappuoli, R., Vaccines, Emerging Viruses, and How to Avoid Disaster. BMC Biology, 2014. 12: p. 100. PMID: 25432510
2. Winter, K., et al., Pertussis epidemic–California, 2014. MMWR Morb Mortal Wkly Rep, 2014. 63(48): p. 1129-32. PMID: 25474033
3. CDC, Pertussis Vaccination: Use of Acellular Pertussis Vaccines Among Infants and Young Children Recommendations of the Advisory Committee on Immunization Practices (ACIP) Morbidity and Mortality Weekly Review, 1997. 46: p. 1-25. PMID: 9091780
4. Raya, B.A., et al., Immunization of Pregnant Women Against Pertussis: The Effect of Timing on Antibody Avidity. Vaccine, 2015. 33(16):1948-52 PMID: 25744227
5. Silfverdal, S.A., et al., Immunological Persistence in 5 y olds Previously Vaccinated with Hexavalent DTPa-HBV-IPV/Hib at 3, 5, and 11 Months of Age. Hum Vaccin Immunother, 2014. 10(10): p. 2795-8.
   PMID: 25483640
6. Hara, M., et al., Pertussis outbreak in university students and evaluation of acellular pertussis vaccine effectiveness in Japan. BMC Infect Dis, 2015. 15(1): p. 45. PMID: 25656486
7. Sealey, K.L., et al., Genomic Analysis of Isolates From the United Kingdom 2012 Pertussis Outbreak Reveals That Vaccine Antigen Genes Are Unusually Fast Evolving. J Infect Dis, 2014. 212(2): p. 294-301. PMID: 25489002
8. Torjesen, I., Proteins Targeted by Pertussis Vaccine Are Mutating Unusually Quickly, Study Finds. BMJ, 2014. 349: p. g7850. PMID: 25552634
9. McGirr, A. and D.N. Fisman, Duration of Pertussis Immunity After DTaP Immunization: A Meta-analysis. Pediatrics, 2015. 135(2): p. 331-343. PMID: 25560446
10. Tefon, B.E., E. Ozcengiz, and G. Ozcengiz, Pertussis Vaccines: State-Of-The-Art and Future Trends. Curr Top Med Chem, 2013. 13(20): p. 2581-96. PMID: 24066885
11. Brummelman, J., et al., Modulation of the CD4(+) T cell response after acellular pertussis vaccination in the presence of TLR4 ligation. Vaccine, 2015. 33(12): p. 1483-91. PMID: 25659267

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

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