Bioassays For Diphtheria Toxin

Bioassays are based on the ability of diphtheria toxin to cause death, dermonecrosis, inhibition of protein synthesis, or other toxic effects in highly susceptible animals (such as rabbits or guinea pigs) or in cultured cells derived from them. The molecular basis for the toxicity of diphtheria toxin has been well studied (reviewed in Ref. [10]). Diphtheria toxin is secreted by C. diphtheriae as a single mature polypeptide of 28,350 Da, containing 535 amino acid residues. Proteolytic nicking of diphtheria toxin and reduction of its two internal disulfide bonds generate fragments A and B. The receptor-binding domain of fragment B binds to the precursor form of a heparin-binding epidermal growth factor-like protein, the receptor for toxin on the surface of susceptible eukaryotic cells. The toxin-receptor complex is endocytosed, and upon acidification of the endosome the translocation domain of fragment B undergoes a conformational change, causing it to insert into the endosomal membrane and form a pore through which fragment A is translocated into the cyto-sol. Fragment A catalyzes the transfer of the adenosine diphosphate ribose moiety from nicotinamide adenine dinucleotide to a posttranslationally modified histidine residue (called diphthamide) on elongation factor 2 (EF-2), thereby inactivating EF-2 and inhibiting protein synthesis. A single molecule of fragment A within the cytoplasm is sufficient to kill a eukaryotic cell.

Because bioassays provide direct measures of toxicity, they have long been accepted as the ''gold standard'' for detecting diphtheria toxin. Because the observed biological endpoints (such as death or dermonecrosis) are not necessarily unique for diphtheria toxin, controls are needed to demonstrate specificity. Typically, duplicate assays are performed in the presence and absence of diphtheria antitoxin, and neutralization of toxicity by antitoxin proves that observed toxic effects are caused by diphtheria toxin. Assays for dermonecrosis following intradermal injections of diphtheria toxin into susceptible animals are more sensitive than tests for lethality following subcutaneous injections, and intradermal tests can detect as little as 50 pg of diphtheria toxin.[11] Several different endpoints have been used to measure toxicity for cultured cells, including cell death, cytotoxicity, inhibition of protein synthesis, etc. Tests on Vero cells in microtiter cultures using a tetrazolium dye to assess cell viability could detect 3 pg of diphtheria toxin per assay, and the results of cell culture assays were in complete concordance with toxigenicity tests performed in animals for a collection of 55 isolates of potentially tox+ corynebac-teria.[12] The advantages of bioassays for diphtheria toxin include high sensitivity and specificity. The disadvantages are that bioassays are labor intensive, costly (especially if rabbits or guinea pigs are required), slow (several days to obtain results), and require facilities for experimental animals or tissue cultures that are not always available in diagnostic laboratories.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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