The ideal antigen: large, pure, and removed in evolution.
Although a wide range of substances are antigenic (cells, proteins, organelles, small molecules, conjugated peptides, carbohydrates, lipids), not all antigens elicit an immune response. In addition, individual animals in a given species have different humoral immune responses. We find the best approach is to use large (greater than 20kD) proteins or peptides conjugated to carrier proteins. For proteins with high homology to rat or mouse, we suggest varying the immunization strategy, typically by varying the adjuvant, route of administration, or length of the protocol. It may also be necessary to increase the number of animals to increase the likelihood of getting a rare unique idiotype.
With the recent explosion in gene sequences, many scientists and researchers are requesting antibodies to protein antigens that have never been isolated. Two methods are useful in preparing antigens from gene sequence data: 1) chemical peptide synthesis of the expressed protein, and 2) expression of recombinant proteins. Because there are pros and cons to both approaches, please contact us to discuss your options in the early stages of your project.
Chemical peptide synthesis
Making monoclonal antibodies to synthetic peptides that also bind the protein can be challenging. If this is your chosen method, call us before you synthesize a peptide so we can recommend ways to increase our chances of success. For example, one approach is to immunize with a peptide conjugate and screen hybridoma supernatants using native protein. Since some anti-peptide antibodies fail to bind native protein, this approach can be used to obtain antibodies that blot or bind antigen using immunofluorescence following methanol or glutaraldehyde fixation.
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, 6 x His, etc.) 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 since the tag itself is often immunogenic.
Highly conserved antigens
Highly conserved antigens are a challenge to prepare antibodies against since most species are tolerant to self. The greater the degree of sequence differences between your antigen and the host sequence, the greater the likelihood of producing antibodies. In general, the more homologous the antigen, the greater the need to try alternative immunization strategies including using higher numbers of animals of different strains.
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. One common suggestion is to prepare a rabbit antisera before attempting the production of a monoclonal antibody. This oftentimes identifies difficulties in making antibodies to a particular antigen. The serum is useful as a positive control in other immunoassays.