The reduction in animal usage by multiple antigen immunisation schedules

Antibodies are important biological molecules having widespread use within clinical diagnosis and biological research. Conventional protocols for their production involve immunising animals with single targets of interest in order to elicit an immune response and subsequently, either harvesting antibodies from serum (polyclonal antibodies) or immortalising hyperimmunised spleen cells in order to generate cell lines secreting antibody with singular specificity to the target antigen (monoclonal antibodies). Our project was undertaken with a view to reducing the number of animals on procedure and was achieved using a strategy of co-injecting multiple antigens into single animals. For polyclonal sera, subsequent steps involved affinity purifying samples to produce antibodies with similar yield and specificity to those obtained from animals immunised with single antigens, whilst for monoclonal antibodies, populations of immortalised spleen cells (hybridomas) were extensively screened in order to identify cell lines expressing antibody with defined recognition of each single component of the multiple antigen. Our studies showed that in schedules where single antigen immunisations elicited a strong response, the parallel multiple component immunisation induced similar responses. Subsequent affinity chromatography of serum from animals immunised with multiple antigens yielded up to 70% of pure antibody as that recovered from control animals immunised with single antigens, giving similar efficacy and specificity. Moreover from mice co-injected with 2 and 3 antigens we successfully selected hybridoma colonies secreting monoclonal antibodies with precise binding characteristics for the individual components. The study raised interesting logistical issues which have particular relevance to a Service Licence incorporating many disparate antibody production regimes; specifically the importance of introducing sufficient multi component antigen without compromising the parameters of the licence and timescale considerations in situations where one component induced a response more rapidly than others, necessitating further boosts with the latter. We are continuing to employ the technique whenever the research programme allows resulting in a reduction in the number of animals used for antibody production.