High-throughput antibody production.

Proteome-wide sets of antibodies would be an invaluable research resource for use in highly parallel assays such as microarrays. Such assays could provide deeper insights into biology and a wealth of information for clinical diagnostics. However, the rate of discovery of new proteins far exceeds the antibody supply currently produced from traditional animal-based systems. To address this problem, a variety of improvements in antibody production have been developed, including improved animal-based technologies, new antibody structures with superior performances, faster and more discriminating screening techniques, and rapid validation methods. Many of these technologies are amenable to automation, allowing antibody production throughput to significantly increase.

High-level generation of polyclonal antibodies by genetic immunization.

Antibodies are important tools for investigating the proteome, but current methods for producing them have become a rate-limiting step. A primary obstacle in most methods for generating antibodies or antibody-like molecules is the requirement for at least microgram quantities of purified protein. We have developed a technology for producing antibodies using genetic immunization. Genetic immunization-based antibody production offers several advantages, including high throughput and high specificity. Moreover, antibodies produced from genetically immunized animals are more likely to recognize the native protein. Here we show that a genetic immunization-based system can be used to efficiently raise useful antibodies to a wide range of antigens. We accomplished this by linking the antigen gene to various elements that enhance antigenicity and by codelivering plasmids encoding genetic adjuvants. Our system, which was tested by immunizing mice with >130 antigens, has shown a final success rate of 84%.

Antibody production using genetic immunization.

Extract: Antibodies are key tools in proteomics. Their high specificity and affinity allow them to be used to measure individual proteins in complex mixtures, and most importantly to provide quantitative information. There is now great interest in generating proteome-wide sets of antibodies in order to discover biomarkers of disease and to enhance drug development. Antibodies are traditionally produced by injecting a pure form of the protein mixed with an adjuvant into an animal. The pure protein is usually produced either by overexpression in a host such as E. coli and purifying it using a fused tag (e.g., GST, 6-histidines), or by synthesizing small peptide sequences from the whole protein. The breakthrough effort in the last decade of determining the sequences of entire genomes has discovered tens of thousands of new proteins. Antibodies are now the major limiting factor in quantitative proteomics. The protein-based technology has proven too slow and expensive to make it feasible to produce antibodies on the scale now needed by the pharmaceutical and biotechnology industries.