Antigenic features of protein carriers commonly used in immunisation trials.

An aluminium hydroxide adjuvant induced a more elevated and rapid immune responses against short peptides conjugated to the Keyhole Lympet Hemocyanin carrier than immuneasy adjuvant. Furthermore, since carrier proteins may compete with the fused or chemically linked polypeptides in eliciting antigen-specific immune response, we classified the immunogenicity of the most common carrier proteins used in molecular biology for antigen expression and mouse immunisation. The disulfide isomerase protein A gave a carrier with the lowest immunogenicity whilst disulfide isomerase protein C gave the highest immunogenicity and therefore should be avoided as a fusion partner. Using this protein as a model, we identified and located the immunodominant epitopes along its sequence. These results now enable the combination of carrier and immunisation conditions to be optimized.

Comparison and critical analysis of robotized technology for monoclonal antibody high-throughput production.

We have previously demonstrated how to transform the conventional method of hybridoma production and screening into a fast, high-throughput technology. Nevertheless, there were still open questions related to automated procedures and immunization protocols that we address now by comparing the hybridoma production work-flow in automated and manually executed processes. In addition, since the animals' antibody responses to single or multiple antigen challenge affect monoclonal antibody throughput, different immunization and fusion strategies were tested. Specifically, the results obtained with multiplexing (multiple target antigens injected into a single animal) and single antigen immunization followed by splenocyte pooling immediately before fusion were compared with conventional methods. The results presented here demonstrate that the optimal protocol consists of automated somatic-cell fusion and hybridoma dilution followed by manual plating of hybridoma cells. Additionally, more specific and productive hybridoma clones were obtained with multiplexed immunization in a single animal with respect to the splenocyte pooling from single antigen immunized animals. However, in terms of overall antibody yield, the conventional method consisting of single immunization for each single animal assured ten times more specific hybridoma cell lines than the strategy based on the multiple antigen immunization followed by separate fusion step. In conclusion, the most productive approach for recovering a large number of suitable antibodies relies on single antigen immunization followed by automated fusion and dilution steps and manual plating.