April 28, 2014

By: dpinkston@listlabs.com

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