Antigen Expression Services

Selecting the best antigen or antigen generation option to meet your project goals is an important part of the HuCAL® antibody generation process.

Protein Antigens

Whole protein antigens are usually the method of choice; the protein can be provided by you or they can be produced as part of the project. All that is required is 0.25-0.5 mg of protein at 80% purity. 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 or purification tag, then 0.25 mg to 0.5 mg of an unrelated protein with the same linker or tag should also be provided. This is used for counter-selection and screening to make sure that the antibodies delivered will be against the protein of interest, and are not binding to the linker or tag.

We offer in house antigen generation options as well as access to trusted external partners for a full range of options:

• Gene Synthesis and Acquisition Services, obtaining antigen DNA
• Peptide synthesis services, including design and conjugation
• Bacterial expression of recombinant protein fragments using our AgX® system
• Mammalian expression of epitope-tagged or Fc-fusion proteins
• Baculovirus expression
• Cell-free expression

Gene Synthesis and Acquisition Services

When DNA fragments are unavailable, there are still excellent alternatives to peptide antigens. We can arrange for clone purchase (where available), gene synthesis of the selected domain, or cloning of the gene from an existing library.

Peptide Synthesis Services

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. However, in some cases they are the only option. Furthermore selection of certain epitope-specific antibodies (e.g. phospho-specific) often requires a peptide antigen.

AbD Serotec 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 for de novo or existing peptides.

AgX® Antigen Expression Service

AgX provides rapid, high-level bacterial expression and purification of protein antigens from cDNA or PCR fragments. These protein domains (100 aa to 300 aa) are ideal for the generation of highly-specific monoclonal antibodies from the HuCAL PLATINUM® antibody library, and will add just 3 weeks to the HuCAL antibody generation process.

We can assist in determining which regions of the protein are of the greatest interest, and then use advanced bioinformatics tools to find the best sequences for expression and antigenicity. We start with just 1 ug of purified DNA, and if that is unavailable we can also include gene synthesis or acquistion services.

When purified protein antigens are unavailable, researchers often substitute peptide antigens. Unfortunately, this is not always the best solution for producing antibodies capable of detecting native proteins. Instead, we recommend using AgX expressed domains because they are typically longer than peptides, providing more epitopes for antibody generation and allowing a greater chance of representing 3-dimensional epitopes from the native parental protein.

In addition, 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.

In our own HuCAL antibody generation projects, we have found that antibodies generated using protein domains as antigens generally show higher affinity than antibodies generated using peptides.

If you have a custom monoclonal antibody project in mind, please complete our HuCAL Inquiry Form or contact our HuCAL specialists at sales.muc@abdserotec.com to begin the process today.

Further Reading about AgX Technology:

1. Frisch, C. et al. (2003) From EST to IHC: human antibody pipeline for target research.
   J. Immunol. Methods 275:203-212.
2. Sun, C. et al. (2003) Kirrel2, a novel immunoglobulin superfamily gene expressed primarily in beta cells of the pancreatic islets.
   Genomics 82:130-142.