Failure of PFC inhibition assays to distinguish idiotypically between clonotypes that are readily distinguishable by RIA analysis.
Two different hemolytic plaque assay protocols that are commonly used to quantitate idiotype-positive antibody-secreting cells have been compared to a standard radioimmunoassay (RIA) to test for their ability to discriminate between related, but idiotypically distinct, clonotypes. The idiotype proband used in this analysis is the individual specific idiotype associated with the dextran-binding myeloma protein M104E (M104E IdI). Antibodies specific for this private idiotype (anti-M104E IdI) were purified by a combination of adsorption and affinity chromatography of the immunoglobulin fraction isolated from the sera of rabbits repeatedly immunized with M104E. The same affinity-purified anti-M104E IdI antibodies were used in the hemolytic plaque assays and in the RIA. In one of the plaque assays, the putative idiotype-positive antibody-forming cells were scored by lysis of target erythrocytes to which the anti-idiotype had been covalently coupled. In the other plaque assay, the idiotype-positive cells were determined indirectly by anti-idiotype inhibition of PFC produced on dextran-coupled target erythrocytes. The fidelity of these two assays to quantitate the M104E private idiotype expression in individual BALB/c mice after a single immunization with dextran B-1355S was determined by comparing the plaque assay data to the data generated by a double-antibody, post-precipitation RIA of either the antibodies in the serum or of monoclonal antibodies produced by hybridomas. Our data indicate that both plaque assay protocols reflect an overestimate of the actual expression of M104E private idiotype. By using a library of dextran-specific hybridomas (that have been characterized in an RIA with respect to their M104E IdI cross-reactivity), we have shown that the PFC overestimate of the M104E expression observed in dextran-immune mice is due to the inability of both plaque assay protocols to distinguish between dextran-specific clonotypes that express idiotypes cross-reactive with, but not identical to, the 104E IdI. We conclude that the plaque assay should be used only in conjunction with an RIA to estimate the idiotype expression. This is especially true in situations where closely related cross-reactive idiotype families exist.
B S
BS
Schepart
G L
GL
Mayers
R B
RB
Bankert
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000918:Antibody Specificity; D003911:Dextrans / Q000276:immunology; D005260:Female; D006462:Hemolytic Plaque Technique; D007130:Immunoglobulin Idiotypes / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D011863:Radioimmunoassay; D013154:Spleen / Q000276:immunology
D000888:Antibodies, Anti-Idiotypic; D003911:Dextrans; D007130:Immunoglobulin Idiotypes