Antibody Generation Method
Anti-HCP antibodies can be, and it is thought should be, generated using a variety of immunization protocols. The use of different immunization strategies offers the advantage of overcoming the limitations found in any of the individual methods.
In the classical approach, the HCP immunogen is immunized as a whole complex mixture in adjuvant. This can be successful, particularly if using bacterial host immunogen. One downside to the classical strategy is that often times the larger immunogens (greater than 50 kDa) tend to dominate and the low molecular weight proteins do not generate a robust immune response leading to insufficient HCP antibody reagent. Using an HCP sample that is fractionated and immunizing distinct animal cohorts allows for the generation of antibodies specific for the high molecular weight and the low molecular weight samples.
Another pitfall is that some immunogens are not immunogenic in all hosts. To overcome this limitation, anti-HCP antibodies should be generated in 2 or 3 different hosts, which could include one or more of the following species: rabbits, goat/sheep/donkey, and chickens. The large animals have the added benefit of a large long term supply of HCP antibody reagent.
The third commonly used method recommended at Rockland Immunochemicals is the cascade immunization. This method uses the antibodies generated after HCP immunization to immunodeplete the immunodominant HCP species. The antibodies are first purified, immobilized, and the starting HCP is reacted with the anti-HCP. The unbound HCP fraction is used to boost the immunization to develop a more comprehensive immune response.