Role of sodium azide in reducing nonspecific color development in enzyme immunoassays

Serology Branch, U.S. Department of Agriculture, Beltsville, MD 20705, USA.

Improved enzyme immunoassay (EIA) procedures achieved by incorporating sodium azide during predilution of serum samples in a solid-phase EIA for the detection of anti-Toxoplasma antibody in swine using a peroxidase conjugate and in all washes of a bovine brucellosis rapid card test EIA using alkaline phosphatase conjugate are reported. Without this modification, substantial background interference was encountered that showed direct correlation with the degree of hemolysis of the serum samples. Anti-Toxoplasma gondii antibody-negative samples, separated by subjective groupings based on degree of hemolysis, into "clear", "slight", and "gross/total" samples, had a mean +/- standard deviation of 0.150 +/- 0.072, 0.187 +/- 0.105, and 0.232 +/- 0.108, respectively. The incorporation of sodium azide during the initial step of serum dilution dramatically eliminated the background, giving a mean +/- standard deviation of 0.079 +/- 0.029, 0.076 +/- 0.022, and 0.081 +/- 0.029, respectively. The level of endogenous peroxidase activity, a possible factor for this nonspecific interference, was considerably elevated in some of the swine sera. The clear, slight, and gross/total categories had relative levels of 1%, 2%, and 51% peroxidase activity compared to the conjugate peroxidase activity of 100%. Whereas sodium azide could be used only in sample predilution in the swine toxoplasmosis peroxidase-conjugate test, in the bovine brucellosis alkaline phosphatase-conjugate card test it could be used in all wash cycles. Many brucellosis card test results were visually uninterpretable because of significant background color when the manufacturer's wash reagent was used. The substitution of a wash reagent containing sodium azide eliminated background color, giving a visually unambiguous test.

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