The Full-EpitopeTM service will produce polyclonal antibodies with the best quality for your downstream applications.
Polyclonal antibody production is to acquire antibodies that react with the antigen in qualitative or quantitative immuno-detection assays. The performance of the antibody in the downstream applications is the foremost consideration when selecting antibody production options.
For using a peptide as the antigen, even with careful epitope selection, there remains a considerable risk that peptide-based antibodies may not recognize the native protein (Greenbaum et al, 2007). Another possible issue is the titer and specificity. Peptide-based antibodies normally have a relatively low titer. Compounding with that the antibody usually just recognizes a small epitope (typically 5-8 aa), low dilution applications of the antibody may cross-react with other non-specific proteins.
Using recombinant proteins as antigens, we have produced numerous antibodies (through affinity purification) that essentially all react strongly with the original proteins. From our experiences, it is more productive to use recombinant proteins (even with semi-soluble proteins, or sub-domains) instead of short peptides.
|Full-EpitopeTM Polyclonal Antibody Service|
|Protein antigen||Soluble protein 10-15mg, >90% purity|
|Antibody||3~8 mg (antigen affinity purified)|
|Price||Inquiry (30% below industry average)|
|Protein Preparation||Codon optimization||4-6 weeks||For in vivo soluble protein (SupernateIN technology), please inquire.|
|Recombinant protein production||0.1mg purified protein to customer, 10-15mg for antibody production.|
|Immunization & bleeding||Two rabbits, 5-8 injections per rabbit||6 weeks||For additional rabbits or other hosts such as chicken, goat or mouse, please inquire.|
|Purification||Antigen affinity purification||1 week||Best specificity (better than protein A/G)|
With our SupernateINTMprotein expression platform, there is a high probability for the protein antigen to be expressed as a soluble form in E. coli. The soluble protein antigen most likely presents the native structure, which in turn translating into presenting most of the native epitopes. This will be superior to peptides and denature proteins, which may only show limited epitopes on their primary sequences. It will be also superior to in vitro refolded proteins, which may present misfolded regions and non-native epitopes.
In additional to the normal immunological adjuvants, special innovative immune enhancers will be applied. The immune enhancers can boost and accelerate the response of immune system and continuously generate high-titer antibodies.
Special immune site will be applied. The recombinant protein antigen will be injected in special tissue/organ of the animal. This is different from the commonly applied multiple sites during subcutaneous injection. This technique will induce stronger immune response: the antigen can be absorbed with better efficiency and trigger accelerated antibody generation.
The immune enhancer plus special immune method has been proven to improve the antibody quality quite effectively. It also helps to shorten the turnaround time of antibody production.
In order to get high-titer and specific antibodies, weapply antigen affinity purification after serum harvest. To do so, the recombinant protein antigen will be conjugated on resin. The antibodies will be purified through the antigen affinity resin. The resulting antibodies will have a much higher titer and better specificity than the antibodies purified by traditional protein A/G method.
If preferred in case of tagged protein antigen, we may provide free custom anti-tagelimination purification. Preparation of tagged protein antigens (e.g., His6, GST, and etc.) is more economic than that of the tag-free versions. If desired, the potential anti-tag antibodies can be eliminated from the final materials. We conjugate the tag peptide/protein to resin and collect the flow-through of the column as the final materialsm in order to eliminate the anti-tag antibodies. The purified antibodies can be widely used in many applications such as IP, IHC, WB, ELISA, FACs, and etc.
Are Protein or Peptide Antigens Better for Antibody Production?
|Protein antigen||Peptide antigen|
|– Present all epitopes on the target
– Present epitopes on native structure
– broadest possible affinity and utility
– Medium cost
|– limited epitope on primary sequence
– antibodies most likely against denatured targets
– Narrow assay utility
– Low cost
For the purpose of antibody production, polypeptides are considered proteins if they are larger than nine kilodaltons (9kDa) and do not need to be conjugated to a carrier protein to be made immunogenic. Nearly any purified protein (>90% pure) can be used as an antigen for antibody production.
Protein antigens are generally best when the goal is to elicit production of as many different antibody clones to detect as many different possible epitopes on the target protein as possible. The result is production by the animal host of a broad range of antibodies (a) that can be screened to select particular clones for different epitopes during monoclonal antibody development or (b) can be used as a polyclonal population to provide the broadest possible affinity and utility for multiple applications. Given that whole proteins are more likely than peptides to present normal secondary and tertiary structure, they are more likely to elicit production of antibodies that bind certain epitopes that are present only in the native protein target, as is usually the case in ELISA and immunoprecipitation.
When they can be designed based on knowledge of the target protein structure and function, peptides allow focused production of antibodies against point mutations and post-translational modifications (such as phospho-specific site).
Peptide antigens are too small (usually 4 to 20 amino acids) by themselves to elicit an immune response, so they must be conjugated to an immunogenic carrier protein (e.g., KLH or BSA) for immunization.
Because peptides represent fewer epitopes based on primary sequence structure rather than whole protein secondary or tertiary structure, they elicit production of antibodies whose specificity is less likely to be dependent upon the target protein being in its native, biologically active form. As such, anti-peptide antibodies are more likely to bind denatured targets for use in some applications.