What is an antigen?

An antigen is a compound that will interact with a B cell, T cell or antibody but may or may not induce an immune response. An immunogen can induce an immune response. Therefore an antigen is not necessarily an immunogen.

What types of antigens can be used to make antibodies?

We can develop antibodies against a variety of antigen sources including: synthetic peptide, native protein, recombinant protein, whole mammalian cells, carbohydrates, whole bacteria, small molecules and DNA. We also make anti-idiotypic antibodies. Please contact us to discuss your antibody goals.

What are some factors that affect an antigen?

Antigen Size:
Haptens and peptides require coupling to carrier proteins to be immunogenic. Antigens greater than 10,000 Da are considered immunogenic.
Antigen Purity:
The immunizing antigen should be the highest purity available. Greater than 90% purity is suggested. For screening the antigen can be less pure.Rule of thumb: inject with purest available so as not to generate antibodies to any immuno-dominant contaminants.
Antigen Type:
Approach will depend on the target (specific epitope, cross reactivity, how conserved it is etc.), what the end use of the antibody is (FACS, ELISA, W. blot etc.) and specific antigen information (published results, current commercially available antibodies etc.). Please contact us to discuss your specific antibody goals so we can suggest the best approach possible.

How much antigen is needed to do a Project?

Mice and Rat
Antigen type Monoclonal
Peptide10 – 25 amino acids 2mg peptide coupled to KLH for immunizing
2mg peptide coupled to BSA for screening
Proteins (recombinant, native, fusion proteins etc.) 2mg protein in sterile buffer (does not need to be soluble)
1 – 2mg soluble protein (does not need to be sterile)
Small molecules 2mg hapten coupled to carrier for immunizing
1 – 2mg hapten coupled to carrier for screening
Whole bacterium or whole mammalian cells Contact us for exact amount required
Amount is dependent on target
Rabbit
Antigen type Monoclonal Polyclonal
Peptide10 – 25 amino acids 2 – 3mg peptide coupled to KLH per rabbit for immunizing
2mg peptide coupled to BSA for screening
2 – 3mg peptide coupled to KLH per rabbit for immunizing
0.2mg peptide coupled to BSA for testing serum
Proteins (recombinant, native, fusion proteins etc.) 2mg protein in sterile buffer per rabbit
2mg soluble protein (does not need to be sterile) for screening
2mg protein in sterile buffer per rabbit
0.2mg soluble protein (does not need to be sterile) for testing serum
Small molecules 2mg hapten coupled to carrier for immunizing
1 – 2mg hapten coupled to carrier for screening
Whole bacterium or whole mammalian cells Contact us for exact amounts required
Amount is dependent on target
Contact us for exact amounts required
Amount is dependent on target

Does ImmunoPrecise make peptides?

Yes, we can design your peptide (free of charge) when you develop your antibody with us. We can select the most immunogenic sequence from your full length protein. We can then generate your peptide for you. Peptide synthesis and carrier conjugation takes about 3 weeks. Please contact us to discuss your project or to get a quote on peptide synthesis.

How long should my peptide be?

The length of the peptide should be between 12 and 25 amino acids. This is the standard size as based on a B cell epitope, which is roughly 10 – 12 amino acids. Longer peptides may adopt non-native structures that give rise to antibodies that do not recognize the native protein. Shorter peptides do not provide sufficient epitopic diversity, thereby producing a constricted range of antibodies, limiting the opportunity to generate antibodies that recognize the protein. In the case of extremely short peptides selected from the interior of the protein sequence, for example, the overwhelming majority of antibodies may be against the charged N or C terminal residue carrying the respective amide or carboxyl functional group, which would impair recognition by antibodies of the sequence in a native protein where the residue is linked to its flanking neighbors by a peptide bond. The exception to this length requirement occurs in the case of antibodies to phosphor- or other modification-specific sites. Phosphorylation of a single residue produces minor conformational changes in an epitope. It is desirable to “focus” antibodies on this site, so peptides of 8-12 amino acids with the modification centrally located are selected to enhance the production of phospho-specific antibodies.

What form should my antigen be in?

If antigen is solubilised we prefer a minimum concentration of 1mg/mL in a sterile neutral buffer (with no additives) for immunization purposes. Please ensure that your immunizing antigen contains nothing that may have harmful/toxic effects in animals. For example: urea, formalin, sodium azide, glycerol, extreme pH, high salt, high concentrations of imidazole or guanidine. If you are unsure, contact us for more information. If antigen is lyophilized please let us know how best to rehydrate your antigen.

My antigen is a fusion protein. Is this a problem?

Fusion proteins are added for ease of purification or to increase the immunogenicity of the target. For example, GST, SOD and His tags. There is a good chance that antibodies will be made against the tag. Providing a screening antigen that has had the tag removed or contains a different tag avoids detection of these unwanted antibodies.

What information does ImmunoPrecise require about my antigen?

The more information you can tell us the better (stability, storage conditions, concentration etc.). This will help us design and tweak the process to give you the best antibodies possible. Please fill out a Project Information Sheet and include it when submitting your antigen.

How do I ship my antigen to ImmunoPrecise?

Shipping address
ImmunoPrecise Antibodies, Ltd.
Vancouver Island Technology Park,
#3204-4464 Markham Street,
Victoria, B.C., V8Z 7X8, Canada

Please let us know when you are shipping antigen to us so we can track the shipment and ensure its safe arrival. Please download and complete our Project Information Sheet, and send it along with your antigens. Antigens that require -20°C or colder storage temperatures should be shipped on dry ice. Antigens that are stable at 4 – 8°C can be shipped on ice packs. Contact us if you have any questions regarding packaging, how to do a dry ice shipment, import permits etc. We are shipping experts!