Circulating and Mitogen-Induced Immunoglobulin-Secreting Cells in Human Peripheral Blood: Evaluation by a Modified Reverse Hemolytic Plaque Assay

Abstract The number of immunoglobulin-secreting cells (ISC) found circulating in normal human peripheral blood and the number generated after in vitro stimulation with pokeweed mitogen (PWM) were assessed by using a modified reverse hemolytic plaque assay (RHPA). This technique involves incubation of the lymphocyte population to be tested in agarose with sheep erythrocytes coated with antibody to human immunoglobulin (Ig). The subsequent addition of anti-Ig-developing antiserum and C results in the formation of hemolytic plaques around active Ig-secreting cells. The RHPA allows for quantitation of total ISC as well as cells individually secreting IgM, IgG, or IgA. Freshly prepared human peripheral blood mononuclear cells (PBM) from 15 individuals were assayed for ISC and found to contain a mean of 747 ± 364 ISC/106 PBM, of which a mean of 445 secreted IgG, 164 IgM, and 239 IgA. In vitro culture of PBM with PWM resulted in the generation of 10-fold more ISC than were found in unstimulated cultures (9353 vs 925). Increased numbers of ISC were evident after 3 to 4 days of incubation and reached a peak after 5 to 7 days. There was great variation in the number of ISC generated in response to PWM in cultures of PBM obtained from different individuals. Moreover, PWM induced a predominance of IgM-secreting cells in cultures of PBM from some individuals, whereas stimulating predominantly IgG-secreting cells in others. The predominant Ig isotype of the ISC generated in response to PWM in cultures of PBM from different individuals was found to vary with the race and sex of the donor.