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A method which allows an accurate determination of very low titers of antibodies able to neutralize diphtheria toxin has been developed. The assay is based on the incubation of a reference amount of diphtheria toxin with different dilution of the serum and the evaluation of the residual toxicity of diphtheria toxin on Vero cells. The cells are seeded in 96-well plastic plates with the toxin-serum mixtures and the residual toxin activity is measured as the block of cell protein synthesis after incubation with [14C]leucine. Antitoxin titers as low as 0.002 IU can be reproducibly and accurately measured and the method gives a linear response in the range 0.002 to 8.0 IU/ml.
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