Typical recommended antigen concentration per immunisation are as follows:

Rabbit:   50 µg -200 µg/immunisation - 4 immunisations per animal
Sheep and Goat:    minimum 200 µg/immunisation - 4 immunisations per animal

Whatever antigen is used there will probably be multiple epitopes against which antibodies could be generated.

When choosing epitopes for your antibody production it is best to look for  those with exposed hydrophilic regions as these will be easily accessible for antibody binding. Hydrophobic regions are usually hidden within the protein.

• Peptides
• Proteins
• Bacteria/viruses
• Plant and yeast antigens
• Other antigens
• Antigen Preparation
• Peptides (see link page)

Proteins

Protein antigens can be either naturally occurring or recombinant when choosing your protein antigen you should consider the following:-

The size of the protein, the amount of aggregation and the relative nativity of the protein can all  affect the quality and quantity of antibody produced.  Generally, the larger the immunogenic protein the better.

Soluble, non-aggregated proteins may induce tolerance in the host rather than a satisfactory antibody response.

Antibodies to native proteins react best with native proteins and antibodies to denatured proteins react best with denatured proteins.

For example if antibodies are to be used on membrane blots, or other assays where the proteins are subjected to denaturing conditions, then antibodies should be made against denatured proteins. Alternatively, if antibodies are to be used to react with a native protein or to block a protein's active site, then antibodies should be made against the native protein.

Bacteria/Viruses

Live bacteria or viruses, should not be used except as a last resort. There are many fixatives and inactivation techniques and one can generally be found that does not damage the antigenic determinants.

Plant and Yeast antigens

Sometimes high backgrounds can be found in host animal sera for  plant or yeast antigens. We can supply  pre-immune serum for screening for selecting  candidate animals for immunisation.


Other Antigens

Small polypeptides, polysaccharides and other  non-protein antigens generally need to be conjugated or cross-linked to larger, immunogenic carrier protein such as Bovine Serum Albumin to increase immunogenicity.


Antigen Preparation

Antigens should always be prepared using techniques that ensure that they are free of microbial contamination. Most protein antigen preparations can be sterilized by passage through a 0.22u filter.

Where possible, antigens should be free of preparative byproducts and chemicals such as polyacrylamide gel, urea, endotoxin, particulate matter and extremes of pH.

The buffer used to re-suspend your protein is important as some buffers may have an adverse effects on the animal's welfare. The least toxic option available should be used.

The final immunogen used is an oil/water emulsion so therefore detergents should be avoided if possible as these may affect the stability.

SDS can be used provided that the concentration is less than 0.1%.

If you have any queries on your antigen or buffer please This e-mail address is being protected from spambots. You need JavaScript enabled to view it for advice.