Production of auto-anti-idiotypic antibody during the normal immune response. VI. Hapten augmentation of plaque formation and hapten-reversible inhibition of plaque formation as assays for anti-idiotype antibody

Abstract (1) Evidence has been presented that the detection of hapten-augmentable plaques indicates cells whose secretion of antibody had been blocked by the binding of auto-anti-id to cell surface idiotypes. Because of the dependence of the assay on the affinities of the various species for one another, the number of hapten-augmentable plaques detected should be regarded as a minimal estimate of the number of cells whose secretion of antibody is inhibited by auto-anti-id. For confirmation that hapten-augmentable PFC are due to auto-anti-id 2 principal controls are important: (a) incubation of the spleen cell population with hapten prior to plaquing should remove the hapten-augmentable PFC; (b) the dialyzed supernate from hapten incubated cells should inhibit plaque formation in a hapten-reversible manner. (2) Evidence has been presented that hapten-reversible inhibition of plaque formation can serve as an assay for anti-id. Apparent false positive assays can result from the presence of anti-hapten antibody or antigen-antibody complexes; however, these apparent false positives are rarely reversed by hapten. Removal of anti-hapten antibody, by passage over an antigen immunoadsorbent, will eliminate this source of false positives and the procedure is recommended. False negative results can arise from mismatching of the anti-ids in the sample to be assayed and the idiotypes of the target cells used in the assay. This can result from shifts in idiotype expression related to age and time after antigen injection. False negatives can also result from the presence of idiotype-anti-id complexes in the sample to be assayed. This source of false negatives can sometimes be eliminated by passage of the sample through an antigen immunoadsorbent. (3) The existence of a high degree of hapten-augmentable PFC introduces complexities into the interpretation of hapten inhibition of plaque formation in terms of the affinity of the antibody secreted by the PFC. In such cases more accurate data might be obtained by examining cells after a brief incubation with hapten followed by washing. (4) It should be recognized that while hapten augmentable plaques may indicate the presence of auto-anti-id they cannot determine the number or complexity of the idiotype-anti-idiotype sets. However, hapten augmentation of plaque formation may prove valuable because it permits detection of auto-anti-id in complex systems where anti-id may not be detectable by more conventional techniques.