Immunoassays For Diphtheria Toxin

Immunoassays are based on the ability of specific polyclonal or monoclonal antitoxic antibodies to bind diphtheria toxin. Typically, immunoassays do not distinguish between biologically active forms of diphtheria toxin and biologically inactive forms that retain full or partial antigenicity (such as diphtheria toxoid or partially denatured diphtheria toxin). Immunoassays provide inexpensive and fairly rapid substitutes for bioassays in determining the toxigenicity of clinical isolates of

C. diphtheriae. Each reference laboratory must perform periodic controls to demonstrate that the results of any particular immunoassay correlate well with the results of standard bioassays for diphtheria toxin.

For many years, the Elek test has been used most commonly in diagnostic laboratories to determine whether isolates of C. diphtheriae are tox+. In Elek tests, a strip of filter paper impregnated with diphtheria antitoxin is applied to the surface of an agar plate containing medium that supports growth of C. diphtheriae and production of diphtheria toxin. Isolates of C. diphtheriae of unknown toxigenicity are streaked on the surface of the medium at right angles to the strip and parallel to known toxigenic and nontoxigenic control isolates. Toxin produced during bacterial growth and antitoxin from the strip diffuse into the medium, forming precipitin lines where toxin and antitoxin are present at equivalence. Precipitin lines formed by tox+ unknown and tox+ control isolates at adjacent positions fuse to give lines of identity. Elek tests are highly reliable when all media, antisera, growth conditions, and bacterial standards are rigidly controlled, but results are likely to be unreliable in laboratories that perform Elek tests infrequently and lack experienced personnel. Incorrect interpretation of nonspecific precip-itin lines can produce false-positive results, whereas false-negative results due to low sensitivity of the assay system may occur if test conditions are not carefully controlled. The large epidemic of diphtheria in Russia and the Newly Independent States during the 1990s stimulated interest in developing improved diagnostic tests and increasing the competence of laboratory personnel for identifying C. diphtheriae and performing toxigenicity tests. Modifications in procedures for the Elek test improved the reproducibility of results, decreased the amounts of reagents required, and decreased the time required for results from 48 hr to 16-24 hr.[13,14]

Subsequently, a quantitative antigen-capture enzyme immunoassay and a qualitative immunochromatographic strip (ICS) test for diphtheria toxin were developed, both of which offer rapid, sensitive, and specific alternatives to the Elek test for toxigenicity testing.[15,16] The enzyme immunoassay used an equine polyclonal antibody for capture and an alkaline phosphatase-conjugated monoclonal antifragment A antibody for detection of diphtheria toxin. The limit of sensitivity was 0.1 ng of diphtheria toxin/mL, and results available within 3 hr of colony selection agreed uniformly with Elek tests. The ICS test also used equine polyclonal antibody for capture but substituted colloidal gold-labeled monoclonal antifragment A antibody for detection of diphtheria toxin. The limit of sensitivity for the ICS test was 0.5 ng of diphtheria toxin/mL, and results were available within 10 min. Furthermore, when the ICS test was used to compare 850

throat swabs that were inoculated directly into broth for 16 hr or analyzed by conventional culture methods, the concordance for detecting diphtheria toxin by the two methods was 99%, and the sensitivity and specificity of the ICS test for detecting diphtheria toxin were 98% and 99%, respectively. The ICS test has significant advantages over the enzyme immunoassay with respect to ease of test performance, stability of reagents, and documented ability to detect diphtheria toxin production within 16 hr from initial collection of a throat swab from a patient with suspected diphtheria.

were infected by clonally related C. diphtheriae isolates that were not highly prevalent before the epidemic.[21] Use of single-stranded conformation polymorphism and direct DNA sequencing methods demonstrated allelic variations in tox among clinical isolates of C. diphtheriae, but all mutations in tox were silent and did not change the amino acid sequence of diphtheria toxin.[22] Therefore the epidemic was not caused by evolution of C. diphtheriae strains that produced a variant of diphtheria toxin resistant to neutralization by antitoxic antibodies, and intensive immunization with diphtheria toxoid succeeded in bringing the epidemic under control by the late 1990s.[23]

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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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