Subtractive Immunization Introduction for Hybridoma Generation

Subtractive immunization is a method that excludes or blunts the response of the humoral immune system to non-target antigens by altering the response mechanism of the immune system, thus allowing the immune system to directly target the target antigenic determinant cluster and produce antibodies that are difficult to obtain through conventional immunization. Subtractive immunization not only eliminates non-target monoclonal antibodies but also increases the chance of monoclonal antibody production against antigens such as sparse or weakly immunogenic antigenic determinants.

Types of Subtractive Immunization

In general, subtractive immunization is divided into three categories depending on how the host animal acquires tolerance to the tolerant agent—high zone tolerization, neonatal tolerization, and drug-induced tolerization. Varied tolerance strategies have varied impacts on the immune system of the host animal, as well as diverse pathways for the subsequent production of antibodies.

High zone tolerization method

The adult animal no longer produces an immune response to this specific antigenic stimulus under repeated stimulation with high doses of foreign soluble antigen (without adjuvant), resulting in high zonal tolerance, but the animal can still react with other antigenic determinants to produce a specific spectrum of antibodies against the target antigen.

Advantages of the high zone tolerance method
It provides a good way for the target antigen to react directly with the host animal for immunization.
Disadvantages of the high zone tolerance method

The antigen molecule composition of the specific antibody obtained is relatively homogeneous, and the tolerogen must be purified, soluble, and non-aggregated.
The dose of the tolerated antigen needs to be determined by a dose-response experiment.
This method doesn't work as well when the target antigen is a small part of the mixture or can't be separated in a way that can be dissolved in water.

Neonatal tolerization method

The normal mature immune system is specific to antigenic responses, recognizing self and non-self, and avoiding autoimmune reactions. The developing immune system, on the other hand, readily adapts to foreign substances. Thus, if foreign substances are appropriately introduced into the host when the immune system is developing at maturity (i.e., embryonic or neonatal stages), the host will clear these B cells that specifically target the foreign substance while activating specific T suppressor cells, thereby inducing lifelong tolerance to the foreign substance without eliciting an immune response.

The neonatal tolerance method has a wide range of applications in many fields. For example, it has a wide range of applications in the preparation of antibody species with weak immune epitope antigenic determinants, and the highly specific antibodies obtained by neonatal tolerance have high potential applications in differentiating normal and diseased tissues, etc. In addition, neonatal tolerance is an effective way to induce tolerance to foreign antigens in animals, but the amount of antigen that causes neonatal tolerance is mainly determined by the antigen type.

Drug-induced tolerization method

Cyclophosphamide makes host animals tolerant to foreign antigens. The hypothesis of cyclophosphamide-mediated subtractive immunization is based on the fact that in a given immune response, antigens trigger the proliferation of specific B cells and T cells, and alkylating agents such as cyclophosphamide preferentially kill these sorted cells, selectively removing proliferating B and T cell clones. Thus, cyclophosphamide can alter the immune response by selectively affecting immunodominance or immunosuppression, such that when the same component of the antigen re-enters the body, the immune response to it is suppressed.

There are many steps to producing monoclonal antibodies, and each step is very important and will have an impact on the final antibody. Creative Biolabs would like to discuss and solve production problems with you in order to complete your research project faster.

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