Antigens

The ideal antigen: large, pure, and removed in evolution

Although a wide range of substances are antigenic, not all antigens are equal in their ability to elicit an immune response. We have experience in producing antibodies in a variety of species. In short, ideal antigens are large, pure, removed in evolution from the host species, and available in milligram amounts.

With the recent explosion in gene sequences, many scientists and researchers are requesting antibodies to protein antigens that have never been isolated. Two methods have evolved to prepare antigens from gene sequence data: 1) chemical peptide synthesis, and 2) expression of recombinant proteins. Because what we are learning in charting this new territory can help you, please contact us before you prepare your antigen.

Chemical peptide synthesis
Making monoclonal antibodies to synthetic peptides which bind native antigen can be challenging. If this is your chosen method, call us before you synthesize a peptide so we can recommend ways to optimize the process. For example, one approach is to immunize with a peptide conjugate and screen hybridoma supernatants using native antigen.

Peptides and other small molecules are weakly immunogenic and must be coupled to a carrier, such as keyhole limpet hemocyanin or ovalbumin. We recommend that you quantify the amount of peptide conjugated to your carrier (not all conjugation reactions occur with high efficiency) and consider preparing a second conjugate using a different carrier for use in screening.

Expression of recombinant proteins
Recombinant proteins should be 20,000 daltons or larger and may contain their expression tags (GST, FLAG, His) during the immunization stage. Ideally, purified antigen with the tag removed should be available for screening. A screening strategy using recombinant protein with and without the tag is one reasonable approach.

Highly conserved antigens
Highly conserved antigens are a challenge to prepare antibodies against since most species are tolerant to self. The greater degree of sequence differences between your antigen and the host sequence, the greater the likelihood of producing antibodies.

Other antigen properties
The more you know about your antigen the more straightforward it is to make antibodies. Important properties include: purity, biological activity, solubility, and sequence homology to the host animal. All of these factors come into play when there is a low or marginal immune response.