Evaluation of alternative methodologies for the generation of murine monoclonal anti-idiotypic antibodies to human B cell leukemias and lymphomas.

The standard immunization procedure for the development of monoclonal antibodies to human malignant B cell idiotype immunoglobulin in our laboratory consists of intraperitoneal immunization with 50-100 micrograms of purified immunoglobulin followed 7 days later by intravenous immunization with an equal quantity of protein. In order to shorten the process and decrease the amount of idiotypic immunoglobulin necessary for successful immunization, we have evaluated 2 alternative immunization procedures. When immunized by the standard method, the percentage of hybridomas demonstrating anti-idiotype activity was 0.3-1.0%. In order to increase the proportion of anti-idiotype hybrids tolerance to the constant regions of human IgM was established by intraperitoneal administration of disaggregated human gamma globulin 14 days prior to immunization of both male and female mice. The percentage of wells with anti-idiotype activity rose to 16-52% in tolerant male mice. The percentage of anti-idiotype hybrids generated was significantly lower in tolerant female mice. To accelerate the process of anti-idiotype development, a single intrasplenic immunization with soluble idiotype IgM was also evaluated. No anti-idiotype or anti-IgM secreting heterohybrids were formed out of nearly 1400 wells seeded in 3 separate fusions using soluble IgM. When the idiotype IgM was immobilized onto protein A-Sepharose, and then injected intrasplenically, approximately 1% of the wells seeded showed anti-idiotype activity. Thus, a single intrasplenic immunization with immobilized immunoglobulin resulted in significant time saving, while prior tolerization may greatly increase the percentage of anti-idiotype hybrids.