Measurement of diphteria antitoxin level in human serum by using quantitative bioassay method

Diphtheria toxin is secreted as a single peptide and then cleaved to an A chain and B chain. The A chain contains the enzymatic site and the 6 chain contains the translocation and binding sites of the toxin. Diphtheria toxin expresses its toxicity by inhibiting eukaryotic cellular protein synthesis. The inhibition results from inactivation of a cytoplasmic enzyme required for protein synthesis, elongation factor 2. The A chain of diphtheria toxin is a transferase which links the ADP-ribose portion of NAD+ to elongation factor 2, ADP-ribosylated elongation factor 2 is inactivate at protein synthesis.Nowadays, diphtheria toxin is used in vaccine production and standardisation. In this event, one of the method that is used isin vitro neutralization test. This test depends on neutralizing the toxin's effect on Vero cells by using serum that contains antitoxin. In our investigation, we used MTT in Vero cell assay which is based on the conversion of the yellow tetrazolium salt to the coloured formazan product by mitochondrial enzymes in viable cells. The concentration of the coloured product can be measured spectrophotometrically and is proportional to the number of viable cells according to the amount of the antitoxin amounts in serum samples.In the first step of our investigation we tried to fix the minimum cytotoxic dose of diphtheria toxin. In this step, we found 100& lethal dose effect of toxin as 4 x 10"4 Lf/ml.. In the second step, we found the amount of antitoxin that Is neutralized the 100£ lethal dose effect of toxin. So, the amount of antitoxin which we used is 0.00976525 lU/rnl. In the third step, we found the amount of antitoxin at the serum samples as 1.28IU/ml., 2.56IU/rnl., 0.32IU/ml., 2.56IU/ml., 0.16IU/ml., 0.16IU/rnl. in order.