An Approach to the Quantitation of Immunogenic Antigen

Using passively administered isotope-labeled anti-KLH to suppress the antibody response of rabbits to KLH, we have attempted to estimate the amount of antigen actually involved in stimulating antibody formation. Single and paired label tracer studies of passively administered anti-KLH IgG indicated that from 0.7 to 2.9 microg were utilized or involved by the antigen in the course of a 90% suppression of the response to 2 mg KLH. Tracer studies of labeled anti-KLH F(ab')(2) fragments revealed the retention of from 2 to 3 microg of these fragments in the entire rabbit during a 60% suppression of the response to 1 mg KLH. Based on previously determined ratios of mixtures of KLH and suppressive amounts of anti-KLH in adjuvant, the antibody utilization data were converted to the probable amount of antigen or immunogen involved. It appears that after an injection of 2 mg of KLH approximately 0.2-0.5 microg of antigen persisted and reacted with antibody given 24 hr later. Since all of this persisting, reactive antigen may not be immunogenic, the above estimate of immunogen is probably high, but may serve to establish upper limits for the amounts of immunogen involved in stimulating antibody formation and provide a meaningful frame of reference for antigen tracer studies.