Lack of pokeweed mitogen-induced IgE formation in vitro by human peripheral blood mononuclear cells: detection of cross-reacting idiotypic determinants on polyclonal Ig by antibodies to a single IgE myeloma protein.

The ability of human peripheral blood mononuclear cells to synthesize IgE in vitro in response to pokeweed mitogen (PWM) is controversial. To determine whether the conflicting results obtained by different laboratories could be due to inherent qualitative differences in the anti-IgE antibodies used to measure low concentrations of IgE in culture supernatants, we compared the specificities of anti-IgE reagents prepared by various methods. Immunoadsorbent-purified antibodies were isolated from a goat antiserum to the lambda, IgE myeloma protein PS and a rabbit antiserum to the kappa, IgE protein Bed in three ways: 1) antibodies to IgE PS (anti-PS) were isolated from the goat antiserum by affinity chromatography with PS coupled to Sepharose 4B; these antibodies consisted of anti-epsilon chain-specific and anti-idiotypic antibodies to protein PS; 2) antibodies specific for the epsilon-chain (anti-epsilon) were purified by affinity chromatography with IgE myeloma proteins that were not used for immunization; and 3) antibodies to idiotypic determinants of proteins PS (anti-id PS) and Bed (anti-id Bed) were isolated on affinity columns with the respective myeloma proteins after absorption of the epsilon-chain-specific antibodies. These three types of antibodies were then used in a solid phase radioimmunosorbent test to quantitate the amount of "IgE" synthesized by peripheral blood mononuclear cells from nonatopic and atopic donors cultured for 7 days in the presence and absence of PWM. ANTI-PS antibodies detected a PWM-induced "IgE formation" in cell culture supernatants of both non-atopic and atopic donors. In contrast, anti-∈ antibodies did not detect a significant PWM-induced IgE formation. An increase in the concentration of idiotypic antigen however, was detected in nine of 10 culture supernatants by anti-id PS and in five of 10 culture supernatants by anti-id Bed. The anti-id PS and anti-id Bed antibodies also detected antigens in F(ab')2 (but not Fc) fragments of polyclonal IgG. Anti-PS antibodies measured approximately 100 IU IgE/ml more than anti-∈ antibodies in sera having low IgE levels. Thus, anti-IgE antibodies isolated bya ffinity chromatography with the same myeloma protein that was used for immunization cannot be used for measuring IgE concentrations in cell culture supernatants or sera from non-atopic donors because anti-idiotypic antibodies in these preparations react with Fab determinants of polyclonal Ig. Because the concentration of these cross-reactive idiotypic determinants is at least an order of magnitude higher than the concentration of IgE in culture supernatants, or sera from nonatopic donors, the use of such antibodies leads to an overestimation of the IgE concentration.