Development of a monoclonal antibody sandwich ELISA for the determination of antigen content and quality in diphtheria vaccines.

At present, quality control of diphtheria vaccines by both manufacturers and national control laboratories relies heavily on in vivo assays to confirm potency. As part of the VAC2VAC project we have developed a monoclonal antibody (mAb) enzyme-linked immunosorbent assay (ELISA) to measure the relative amount and quality of diphtheria toxoid (DTxd) in diphtheria-tetanus based vaccines and believe this test has the potential to play a key role in a control strategy no longer including an in vivo potency test. The mAb ELISA is highly specific, has good dilutional linearity, and is suitable for detecting DTxd in a range of different human vaccine products. We demonstrate the ability of the assay to discriminate between batches of different content and quality using vaccine batches that were prepared to contain differing amounts of DTxd or were altered by exposure to heat or oxidative stress. We also demonstrate successful transfer of the method to other laboratories and show that different diphtheria antigen materials may be able to serve as a reference antigen for local standardization of the method. The assay is ideally suited for incorporation into a consistency approach for routine diphtheria vaccine quality control testing and may be suitable to serve as the stability indicating test in replacement of the current in vivo potency test.

Diphtheria vaccines help to protect against diphtheria infection. Currently, animal tests are used to ensure the potency of such vaccines. Since these tests were first introduced, there have been improvements in non-animal technologies that can be used to ensure consistent production of potent vaccine batches. To demonstrate that a new batch of diphtheria vaccine is consistent with a previous batch of known potency, the quality and amount of the component that stimulates the immune response upon vaccination must be assessed in comparison. We have developed an assay that can measure the quality of a range of different diphtheria vaccine product types. The assay is very specific and reliable, and different laboratories obtained comparable results, showing that the assay is suited for routine use. Once validated by manufacturers and recognized by regulators, this assay will greatly reduce the number of animals needed for batch release of diphtheria vaccines.

Characterisation of tetanus monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay.

Batch release testing for human and veterinary tetanus vaccines still relies heavily on methods that involve animals, particularly for potency testing. The quantity and quality of tetanus antigen present in these products is of utmost importance for product safety and clinical effect. Immunochemical methods that measure consistency of antigen content and quality, potentially as an indicator of potency, could be a better choice and negate the need for an in vivo potency test. These immunochemical methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against tetanus with a view to select antibodies that can be used for development of an in vitro potency immunoassay. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined using an in-house cell-based neutralisation assay to support prior in vivo potency data that was available for some, but not all, of the antibodies. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of "superiority" of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development.

Characterisation of diphtheria monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay.

Immunoassays are used for routine potency assessment of several vaccines, in some cases having been specifically developed as alternatives to in vivo potency tests. These methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against diphtheria with a view to select antibodies that can be used for development of an in vitro potency immunoassay for diphtheria vaccines. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined by a cell-based toxin neutralisation test and diphtheria toxin-domain recognition was determined by Western blotting. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of "superiority" of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development.

Evaluation of a capture antigen ELISA for the characterisation of tetanus vaccines for veterinary use.

We previously developed an ELISA assay for detection of tetanus toxoid antigen in tetanus vaccines for human use. Tetanus vaccines for veterinary use are qualitatively different to those used in humans, often containing a larger number and variety of non-tetanus antigens in the multi-valent products, and adjuvants that are not found in human vaccines. We assessed performance of the capture ELISA with a range of veterinary tetanus vaccines as a first step towards development of an immunoassay as a potential in vivo potency substitute. Nine tetanus vaccines were tested and all produced a good dose response in the ELISA. The shape of the dose response curve for the whole vaccine compared to a matched non-adjuvanted tetanus toxoid antigen was more comparable for vaccines containing a non-aluminium adjuvant than products containing aluminium adjuvants. Elution of the antigen from aluminium adjuvant did not improve the comparability of the dose response curve but did increase the total amount of tetanus antigen available for detection. The ELISA was highly specific for tetanus with no signal obtained for a large number of non-tetanus antigens. These results suggest that a capture ELISA assay can be applied to a control strategy for veterinary tetanus vaccines.