Specificity and detection limit of a dermal temperature histamine sensitization test for absence of residual pertussis toxin in vaccines.

Currently, an assay based on fatal sensitization of mice to histamine challenge is widely used for testing absence of residual pertussis toxin in acellular pertussis containing vaccines. For replacement of this lethal end-point assay, an alternative method based on body temperature measurement in mice has been presented, and in this study the specificity and detection limit of a dermal temperature-based assay were assessed. Test preparations containing pertussis toxin were prepared in aluminum-adjuvanted pertussis toxoid vaccine and injected intraperitoneally in histamine sensitive mice. Later the mice were challenged with histamine and the pertussis toxin-induced decrease in dermal temperature recorded. By comparison of mice treated with pertussis toxoid vaccine spiked with pertussis toxin with mice treated with pertussis toxoid vaccine alone, the assay gave a response that specifically could detect presence of pertussis toxin. The acellular pertussis containing vaccine did not interfere with the pertussis toxin-induced temperature response recorded. In tests for presence of pertussis toxin in the pertussis vaccine preparation, the detection limit of the assay was estimated to approximately 5 ng pertussis toxin per human dose of pertussis toxoid. The dermal temperature-based assay was found to be a valid method to be applied in routine quality control of vaccines.

Development and validation of an ATP method for rapid estimation of viable units in lyophilised BCG Danish 1331 vaccine.

An assay for quantifying viability in BCG vaccine by determining intracellular ATP content was developed and validated. ATP content was determined by measuring bioluminescence in the presence of luciferin/luciferase. During development and validation the ATP method was compared to the conventional viable count method. A key step to obtain correlation between ATP content and CFU was found to be a period of pre-incubation in a growth medium before ATP determination. During the validation, the robustness, linearity, accuracy, precision, and range were studied. The method validation study showed that the method applied was robust and applicable to determine ATP content in lyophilised BCG for estimating viability in the BCG samples. By comparison with a conventional viable count method, a high correlation between ATP content and the viable count was found; this relationship can be applied in routine quality control to estimate viable count from the ATP content determined in a sample.

Report of an international collaborative study to establish the suitability of using modified ATP assay for viable count of BCG vaccine.

As part of the World Health Organisation (WHO) initiative to update the current requirements for BCG vaccine a collaborative study was carried out to establish the robustness, reproducibility and the suitability of the modified ATP assay. This assay was developed by Statens Serum Institut, Denmark, as a potential replacement of the method for detection of viable counts of BCG vaccine which is routinely used as a quality control test for lot release. Two BCG preparations, of same strain but different production methods, were tested. For each preparation, two different storage conditions of -20 or 37 degrees C were used in order to establish the suitability of this assay for testing heat-treated BCG vaccine as in the temperature stability test. The lyophilised BCG samples were tested using the ATP reagents from the same source and same principle of testing but some procedural modifications were allowed to accommodate different equipment and resource availability in different laboratories. Data from four laboratories showed that the heat-treated BCG samples contained significantly lower ATP content per sample than the untreated control stored at -20 degrees C. Three laboratories gave consistent mean ATP contents, especially for control samples, even with variations in testing protocol. The present study showed that this modified ATP assay is very robust and can be reproducible. Once the correlation of cultural viable count and ATP content of a BCG vaccine product has been established, this rapid alternative assay may be used to monitor BCG viable count. Due to the fact that this study was small, further investigation is planned. A collaborative study will be carried out using this modified ATP assay in parallel with the cultural viable count method in the establishment of the replacement of the WHO International Reference Preparation of BCG vaccine.