Abstract
BackgroundMonoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas.ResultsIncreased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos.ConclusionsThis method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.
Highlights
Monoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process
A method for the rapid selection of recombinant mouse monoclonal antibodies Our aim was to rapidly select monoclonal antibodies of high affinity and specificity that could be used in wholemount immunohistochemistry protocols and could be distributed
Throughput was increased by immunizing mice with pools of antigens from our library of zebrafish cell surface and secreted proteins, so that antibodies to several proteins could be selected simultaneously from a single animal
Summary
Monoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. The ability of antibodies to bind, with high selectivity and affinity, to diverse chemical structures has made them an extremely important tool for biomedical research [1]. Their value is underlined by the numerous ongoing international efforts, both academic and commercial, to produce large antibody resources for the scientific community [2]. By providing essentially limitless amounts of a defined reagent, monoclonal antibodies selected from an immunized animal remain the reagent of choice They are, generally regarded as unsuitable for building large resources because of the requirement for long immunization schedules and a large tissue culture demand. Antibodies raised against peptides, often recognize only denatured proteins (for example on western blots) and do not stain native proteins found in wholemount fixed tissue, limiting their usefulness
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