Optimize your assays using recombinant antibodies selected for desired affinity
Selection of the best antigen and/or antigen generation option to meet your project goals is an important part of the HuCAL® custom antibody generation process.
Whole protein antigens are usually the method of choice and the preferred target for antibody generation; protein can be provided on the part of our customers or protein antigens can be produced as part of an antibody generation project. Only 0.25 - 0.5 mg of protein at 80% purity by SDS PAGE is required. The sample buffer must not contain primary amines such as tris or glycine, additives such as bovine serum albumin (BSA), or detergents.
If the protein contains a linker, a purification tag or a fusion partner, then 0.25 - 0.5 mg of an unrelated protein with the same linker, tag or fusion partner should also be provided. This material will be used for counter-selection and counter-screening to make sure that the antibodies delivered will be specific for the protein of interest, and do not bind to the linker or tag.
Bio-Rad offers in house antigen generation options as well as access to trusted external partners for a full range of options:
Peptides have limitations as antigens, due both to the reduced number of epitopes available and to their flexible structure. Antibodies against peptides are often of lower affinity than antibodies against proteins, and there is no guarantee that the anti-peptide antibody will recognize the natively folded parent protein. In some cases, however, peptides are the only option and selection of certain epitope-specific antibodies (e.g. phospho-specific) often requires the use of a peptide antigen.
Bio-Rad routinely conjugates peptides to the carrier proteins BSA and human transferrin (Trf), with coupling via an N- or C-terminal cysteine. Alternative coupling strategies can be used where necessary. We can provide peptide synthesis and coupling services de novo or for existing peptides that have qualified as selection antigen.
When antigen material is unavailable, there are still other excellent alternatives to peptide antigens. Bio-Rad can arrange for cDNA clone purchase (where available), or gene synthesis of a selected antigen domain or of the full-length antigen in order to generate an appropriate antigen for the HuCAL antibody generation process.
AgX technology provides rapid, high-level bacterial expression and purification of protein antigens from gene optimized DNA fragments. These protein domains (100 - 300 aa) are usually a good choice for the generation of highly-specific monoclonal antibodies from the HuCAL PLATINUM® antibody library.
Bio-Rad will assist in determining which regions of the protein are the most suitable for antigen generation based on the requirements of the antibody generation project, and then use our advanced bioinformatics tools to find the best sequences for subsequent antigen expression. Once the ideal sequence has been identified, AbD will arrange for synthesis of the gene optimized DNA fragment for expression in bacteria.
N-terminal fusion of the AgX to the N1 domain of the pIII protein results in strong expression of protein fragments, while the C-terminal His-6 enables purification of protein samples under denaturing conditions. Therefore, the AgX antigen generation platform is especially useful as source for the generation of antibodies for Western Blots or other assays in which denatured (including fixed) antigens shall be detected.
Mammalian antigen expression systems are often preferred for the production of mammalian proteins. The period of time required to produce antigens in sufficient amounts using mammalian cell culture is a little longer than with E. coli; however, mammalian expression offers posttranslational modification e.g. glycosylation. Together with the mammalian protein folding system (i.e. chaperones), the resulting proteins often resemble the native conformation and show better solubility.
Bio-Rad offers the expression of proteins fused to the Fc domain of human IgG1, or fused to other domains. Additional fusion domains, like the Fc domain assist in protein folding and improve the solubility of the protein. The Fc-fusion protein is secreted into the medium and can be affinity purified via the Fc domain.
Mammalian antigen expression is therefore the system of choice for many proteins that are difficult to produce and for antibody generation projects where detection of native antigen is required.
Cell-free antigen expression systems take the cells’ protein production machinery out of the cell into a vial. DNA encoding the protein of interest is added to a reaction mix and in a coupled transcription-translation reaction only this protein is produced at the ribosomes. The result is a fast expression system that is independent of cell viability, making it ideal for expression of cytotoxic molecules. The system can be adjusted (e.g. temperature, addition of detergents, chaperones etc.) to the requirements of the protein to be produced in order to get the best possible yield and solubility. Furthermore, the open system is well suited for the production of modified proteins such as site-specific biotinylated proteins.
Bio-Rad offers the cell-free expression of proteins using E. coli and wheat germ based systems. Site-specific incorporation of a biotinylated amino acid allows reduction of the required antigen material down to as little as 5-10 µg for an entire antibody generation project. Therefore, cell-free expression of antigens in combination with site-specific biotinylation is a good choice if it is possible to produce only very small amounts of antigen.
If you have a custom monoclonal antibody generation project in mind, please complete our HuCAL Project Outline Form or contact our HuCAL specialists to begin the consultation process today, firstname.lastname@example.org