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A double determinant sandwich immunoassay for quantitation of serum monoclonal anti-I-A antibody
A double-determinant sandwich radioimmunoassay (RIA) is described for the specific detection of anti-I-Ak monoclonal antibody (mAb) in the sera of non-Ighb murine hosts undergoing anti-Ia immunotherapy. This RIA utilizes 2 previously undescribed mAb reagents generated against an Ia.17-specific mAb secreted by the 10-3.6 hybridoma. The first reagent, 7.34, is specific for Ighb-linked allotypic determinants on the Fc portion of IgG2a immunoglobulins as defined by the pattern of reactivity with normal sera from a panel of inbred and Igh recombinant inbred strains. The second reagent, 58.3, is an anti-idiotypic mAb recognizing unique determinants in the combining site of 10-3.6 immunoglobulins, as determined by the specificity of the 58.3 mAb in solid-phase RIA and the capacity of this reagent to inhibit the binding of labeled 10-3.6 mAb to I-Ak-expressing spleen cells. In an RIA procedure using purified 58.3 mAb as substrate and 125I-labeled 7.34 as the detection reagent, serum concentrations of 10-3.6 as low as 1-5 ng/ml can be measured reproducibly after mathematical linearization of the sigmoid standard curve. In the present studies, the serum half-life of 10-3.6 mAb was calculated from assay data to be 3-5 h in I-Ak homo- or heterozygotes and 72 h in non-I-Ak mice. The serum level of 10-3.6 as a function of the mAb treatment protocol was also examined and results are considered with respect to the efficacy of different therapeutic regimens in prolonging transplant survival. Sandwich immunoassays of this type (RIA or ELISA) should provide a highly sensitive and specific means for monitoring serum mAb levels in individuals subjected to antibody immunotherapy for treatment of autoimmune disease, transplant rejection or tumor progression.
A double-determinant sandwich radioimmunoassay (RIA) is described for the specific detection of anti-I-Ak monoclonal antibody (mAb) in the sera of non-Ighb murine hosts undergoing anti-Ia immunotherapy. This RIA utilizes 2 previously undescribed mAb reagents generated against an Ia.17-specific mAb secreted by the 10-3.6 hybridoma. The first reagent, 7.34, is specific for Ighb-linked allotypic determinants on the Fc portion of IgG2a immunoglobulins as defined by the pattern of reactivity with normal sera from a panel of inbred and Igh recombinant inbred strains. The second reagent, 58.3, is an anti-idiotypic mAb recognizing unique determinants in the combining site of 10-3.6 immunoglobulins, as determined by the specificity of the 58.3 mAb in solid-phase RIA and the capacity of this reagent to inhibit the binding of labeled 10-3.6 mAb to I-Ak-expressing spleen cells. In an RIA procedure using purified 58.3 mAb as substrate and 125I-labeled 7.34 as the detection reagent, serum concentrations of 10-3.6 as low as 1-5 ng/ml can be measured reproducibly after mathematical linearization of the sigmoid standard curve. In the present studies, the serum half-life of 10-3.6 mAb was calculated from assay data to be 3-5 h in I-Ak homo- or heterozygotes and 72 h in non-I-Ak mice. The serum level of 10-3.6 as a function of the mAb treatment protocol was also examined and results are considered with respect to the efficacy of different therapeutic regimens in prolonging transplant survival. Sandwich immunoassays of this type (RIA or ELISA) should provide a highly sensitive and specific means for monitoring serum mAb levels in individuals subjected to antibody immunotherapy for treatment of autoimmune disease, transplant rejection or tumor progression.