T.Tuthill - A simple universal test to quantitate 146s antigen during production of FMD vaccines

Session: Improving conventional vaccines

Open Session of the EuFMD – 2018 – Increasing Global Security in the supply of effective vaccines – 29-31 October 2018 -Borgo Egnazia, Italy

Introduction
Conventional foot-and-mouth disease (FMD) vaccines are produced by the chemical inactivation of virus preparations grown in cell culture. The efficacy of inactivated vaccines is dependent on the presence of intact virus particles, distinguished by their sedimentation of 146S in sucrose gradient centrifugation. Such intact 146S antigen is unstable and can dissociate into capsid subunits (pentamers, 12S) during preparation or storage, resulting in a marked reduction in immunogenicity. Yield and stability of 146S antigen is therefore a crucial parameter for vaccine development and must be optimised for each new vaccine strain. The ‘gold standard’ in vitro test for assessing 146S particles involves analysis of particle sedimentation in sucrose density gradients. This is a laborious and low throughput method. Immunological reagents that specifically recognise 146S antigen have previously been reported but such reagents are specific for a single serotype of FMDV. Here, we describe the characterization of a 146S-specific monoclonal antibody (5B6) that recognizes all FMDV serotypes and its use in a simple, universal test to quantitate 146S antigen.

Materials and Methods
The reactivity of monoclonal antibody 5B6 was characterised using ELISA, confocal microscopy and western blot. The 146S test was developed as a sandwich ELISA using recombinant bovine integrin to capture FMDV particles and 5B6 as a 146S specific detector. Preparations of FMDV antigens of various serotypes were used to evaluate the test, including parallel testing of samples before and after heating to induce complete dissociation of antigen.

Results
The monoclonal antibody reacted with virus of all serotypes. The sandwich ELISA has specificity for 146S of all serotypes tested.

Discussion
In summary, we have developed a pan serotypic detection system specific for 146S particles. This has potential to be further validated as a high throughput test for quantitation of FMDV 146S particles during vaccine manufacture and quality control.

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