Antigen Design
Antigen Design and Production
The design of your antigen is one of the most important steps in the whole process of immunoassay development. The choices made in antigen design depend on the nature of the target antigen, the performance requirements, and in the desired immunoassay applications. SDIX has developed a sophisticated antigen design process to ensure the highest probability of success in your project (see below). We accomplish this by considering the impact of the antigen design on all steps of the process from producing the antigen through to success in the final immunoassay application.
The type of target protein influences the choices in antigen design and production. Amongst mammalian proteins there are a number of differences that can produce specific challenges for antibody production. Critical differences are; whether they are produced intracellularly or passage the secretory pathway and get modified in a variety of ways such as glycosylation, whether they are soluble or imbedded in the membrane, and whether they have a single or multiple membrane spanning domains. These differences can be used to group mammalian proteins into four general groups each of which requires different considerations for antigen design and production.
Traditional antigens can be grouped into five general categories:
synthetic peptides (<20aa),
recombinant protein fragments or full length (expressed in a variety of hosts such as E. coli, insect cells etc),
native proteins purified from the natural source, and
whole cells.
Each of these categories has different strengths and weaknesses.
Synthetic peptides can be made quickly and cheaply but can fail to produce useful antibodies especially for immunoassays that display folded protein.
Recombinant protein overcomes the limitations of short peptides, but requires more effort and cost to produce. Fragments of proteins are generally easier to produce than full length protein but larger proteins contain more epitopes.
Native protein is most likely to produce useful antibodies but is very difficult to produce in sufficient amounts.
Whole cells can present membrane proteins in their native state, but can be difficult to produce and requires significant screening to isolate specific antibodies.
Genomic Antibody (DNA) antigen combines the advantages of peptides (cheap and fast to produce) with recombinant protein (multiple folded epitopes).
The table below summarizes the different means or expression systems for producing antigens and notes advantages and weaknesses of a given system.
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Chemical Synthesis
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E. coli
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Insect
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Mammalian Cells
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Yeast
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Peptides
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Best for post translational modifications like phospho groups or small unstructured regions of proteins.
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Not typically used for peptide production.
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Not typically used for peptide production.
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Not typically used for peptide production.
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Not typically used for peptide production.
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Recombinant intracellular proteins
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Not typically used unless a small (<30AA) specific sequences needs to be targeted.
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A simple and scalable way to make a recombinant protein but problems with correct folding can occur. Endotoxin should also be removed before immunization.
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A good way to produce modest yields of folded intracellular proteins. Development of baculoviruses can be time consuming
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A good way to produce folded intracellular proteins though yields can be low and production of stable cell lines can be time consuming.
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A simple and scalable way to make an intracellular recombinant protein.
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Recombinant extracellular proteins
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Not typically used unless a small (<30AA) specific sequences needs to be targeted.
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Not recommended for non prokaryotic extracellular proteins with post translational modifications.
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Not recommended for non insect based proteins as some specific post translational modifications may not occur.
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The best way to produce correctly folded and posttranslational modified mammalian proteins. Cell growth can be slow and yields can be low however.
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Not recommended for producing secreted mammalian proteins due to differences in post translational processing.
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Transmembrane proteins
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Not typically used unless a small (<30AA) specific sequences needs to be targeted.
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Not recommended for non prokaryotic proteins since post translational processing and folding can be a problem.
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Not recommended for non insect proteins since post translational processing can vary.
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The best way to produce correctly folded and posttranslational modified mammalian Transmembrane proteins. Cell growth can be slow and yields can be low however.
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Not recommended for non yeast proteins since post translational processing can vary.
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Whole Cells
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NA
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Applicable if target organism is prokaryotic.
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Useful if the cell surface expressed protein is correctly modified. Background to other insect proteins can be a problem.
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The best way obtain correctly folded an modified cell surface antigen. High background (immune response) to other cell surface proteins can be a problem however.
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Useful if the cell surface expressed protein is correctly modified. Background to other yeast proteins can be a problem however.
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Synthetic peptides are a popular antigen for antibody development. They are easy and cheap to produce and enable the antibody to be targeted with fine specificity to small regions of the protein to either detect subtle differences in the amino acid sequence or post-translational modifications such as phosphorylation.
Limitations of peptide antigens. Antibodies raised against peptides can fail to work in immunoassays especially where the target protein is folded. Reasons for the failure include the peptide sequence is buried in the folded protein, the binding site is blocked by glycosylation or other post-translational modifications, or the antibodies are unable to bind the folded structure. There are also concerns about the specificity of the resulting antibodies that may derive from the conformational and rotational freedom of the peptide antigen. [Reference: Martin C. Michel & Thomas Wieland & Gozoh Tsujimoto Naunyn-Schmied Arch Pharmacol (2009) 379:385–388 How reliable are G-protein-coupled receptor antibodies?].
Antigens that are composed of large regions (ie. a protein) of the target are in general more successful at producing antibodies that work in immunoassays, especially assays such as sandwich ELISA and flow cytometry. This is because a protein is able to fold and thus generate antibodies to folded structure. Proteins also have multiple epitopes giving it a built in redundancy and generates a diversity of epitopes to increase the chance of it working in the immunoassay.
The SDIX antigen design process utilizes a broad range of biological data. The different criteria are weighed and risks assessed to arrive at a design that enhances the probability of success in the final immunoassay. SDIX has developed a proprietary software to rapidly identify suitable designs.
SDIX offers 3 options for our antigen services:
Genomic Antibody Technology antibodies,
Peptide Antibodies and
Antigen from Protein Antibodies.
Consideration of which
area of the protein to bind to is highly important for a successful
antibody. The following need to be considered:
Highly conserved regions,
Paralogous regions,
Interior/transmembrane region and more.