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Lack of pokeweed mitogen induced immuno globulin e formation in vitro by human peripheral blood mononuclear cells detection of cross reacting idiotypic determinants on poly clonal immuno globulin by antibodies to a single immuno globulin e myeloma protein



Lack of pokeweed mitogen induced immuno globulin e formation in vitro by human peripheral blood mononuclear cells detection of cross reacting idiotypic determinants on poly clonal immuno globulin by antibodies to a single immuno globulin e myeloma protein



Journal of Immunology 131(6): 3001-3005



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 inhertent qualitative differences in the anti-IgE antibodies used to measure low concentrations of IgE in culture supernatants, the specificities of anti-IgE reagents prepared by various methods were compared. 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 3 ways: 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; antibodies specific for the .epsilon.-chain (anti-.epsilon.) were purified by affinity chromatography with IgE myeloma proteins that were not used for immunization; and 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 3 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. Anti-.epsilon. antibodies did not detect a significant PWM-induced IgE formation. An increase in the concentration of idiotypic antigen was detected in 9 of 10 culture supernatants by anti-id PS and in 5 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 .apprx. 100 IU IgE/ml more than anti-.epsilon. antibodies isolated by affinity 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.

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Accession: 005790175

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