The objective of this study is to develop a suitable in vitro potency assay to measure the diphtheria component in biological products using sera from guinea pigs immunized with these products. A microtiter plate assay has been developed using VERO cells as the target for the diphtheria toxin. The antitoxin product is diluted serially on the plate and a constant volume of toxin is added to each well. This antitoxin-toxin mixture is incubated for one hour and then the VERO cells are added and the plate is incubated for 5 days. The neutralization endpoint, or the last well exhibiting growth, is determined by microscopic examination of the cell sheet. This endpoint is used to calculate the potency of the diphtheria antibody. The Standard Diphtheria toxin #35119 has been chosen as the toxin for both the in t""""""""r assay and the rabbit skin test. A minimum cytopathic dose (MCD) value for the toxin has been established and is reproducible. This MCD value has been used to calculate the appropriate concentration of toxin for the Toxin Neutralization test described above. Additionally, the minimal reactive dose (MRD) of the toxin has been measured using the rabbit skin test and the two values are comparable. The correct toxin and antitoxin concentrations have been determined. Currently, the validity of the assay is being tested in a blind study measuring the antibody level of the Standard Antitoxin #49 against itself. In addition the assay is being used to measure the diphtheria component in various ISG and IGIV products as well as guinea pig sera. These samples are also being studied in the rabbit skin test and the results of the two assays are being compared.
