Collaborative study for the calibration of the replacement International Standard for pertussis toxin for use in histamine sensitisation and CHO cell clustering assays.

Pertussis toxin (PT) in its detoxified form is one of the major protective antigens in vaccines against Bordetella pertussis (whooping cough). Reference preparations of native PT are required for the quality control of pertussis vaccines. Stocks of the first WHO International Standard (IS) for PT (JNIH-5) were low and a replacement was required. One candidate material was donated by a vaccine manufacturer to NIBSC. It was formulated, lyophilised into sealed glass ampoules and coded 15/126. An international collaborative study assessed the suitability of this material to replace JNIH-5. Fourteen laboratories from 12 countries took part in the study. Eleven laboratories performed lethal murine histamine sensitisation assay (HIST), 14 performed Chinese Hamster Ovary (CHO) cell clustering assay. International Units (IU) were assigned to the material using these assays as they were used to assign units to JNIH-5. It was found that, unlike JNIH-5, the activities of 15/126 in HIST and CHO cell assays did not agree and therefore different unitage for each assay was assigned. The preparation 15/126 was established as the Second WHO IS for PT for HIST and CHO cell assays. It was assigned a unitage of 1,881 IU/ampoule in HIST and 680 IU/ampoule in the CHO cell clustering assay.

An ELISA method to estimate the mono ADP-ribosyltransferase activities: e.g in pertussis toxin and vaccines.

ADP-ribosyltransferase activities have been observed in many prokaryotic and eukaryotic species and viruses and are involved in many cellular processes, including cell signalling, DNA repair, gene regulation and apoptosis. In a number of bacterial toxins, mono ADP-ribosyltransferase is the main cause of host cell cytotoxicity. Several approaches have been used to analyse this biological system from measuring its enzyme products to its functions. By using a mono ADP-ribose binding protein we have now developed an ELISA method to estimate native pertussis toxin mono ADP-ribosyltransferase activity and its residual activities in pertussis vaccines as an example. This new approach is easy to perform and adaptable in most laboratories. In theory, this assay system is also very versatile and could measure the enzyme activity in other bacteria such as Cholera, Clostridium, E. coli, Diphtheria, Pertussis, Pseudomonas, Salmonella and Staphylococcus by just switching to their respective peptide substrates. Furthermore, this mono ADP-ribose binding protein could also be used for staining mono ADP-ribosyl products resolved on gels or membranes.