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Cytokines play an important role in the induction or inhibition of synthesis of the particular isotypes of immunoglobulin after antigenic stimulation. Studies of these effects in vivo require substantial amounts of reagents that are difficult and time-consuming to prepare. The present paper describes methods for immunization and in vitro culture that permit the investigation of effects of cytokines and anti-cytokines on a secondary, antigen-specific IgE response, using much lower quantities of materials than are required in vivo. The results are compared with those reported from in vivo studies.

Syngeneic monoclonal anti-IgE antibodies are of value in studies of the suppression of IgE synthesis. Procedures are described here for the production of high titers of murine anti-IgE antibodies by initiating immunization in the perinatal period, before mice develop tolerance to their autologous IgE. This in turn facilitates the production of monoclonal anti-IgE antibodies. Properties of some of these mAbs are reported, including affinity, fine specificity and ability to bind to IgE on B lymphoma cells or mast cells.

Conditions are described for the preparation of F(ab')2 and Fab fragments of mouse IgE. Papain, pepsin or trypsin each produced F(ab')2 fragments with Mr approximately equal to 130,000 which yielded Fab fragments on further digestion. The release of Fab fragments from F(ab')2 resulted from further cleavage of the H chain. Pepsin, and especially trypsin appear more suitable for the preparation of F(ab')2 because of the difficulty of separating a 93 kDa by-product from the F(ab')2 produced by papain. The best yields of purified Fab were obtained with papain. Rates of digestion were in the order, pepsin approximately equal to trypsin much greater than papain.

This paper describes the development of methods for quantitation of an intrastrain cross-reactive idiotype (CRIA) in IgE antibodies directed to the p-azophenylarsonate (Ar) group and their application to normally immunized and idiotypically suppressed mice. The data were validated by the use of monoclonal and serum anti-Ar antibodies known to possess or lack CRIA. The idiotype was absent in the IgE as well as the total anti-Ar population of idiotypically suppressed mice. A good correlation was noted between the CRIA content of total and IgE anti-Ar with a trend toward a somewhat lower content in the IgE population. Irradiated mice that received immune cells prior to further immunization expressed relatively low concentrations of the idiotype (per weight unit of anti-Ar). The method will be useful in studies of the regulation of the switch to IgE synthesis.

Radioimmunoassays for mouse IgE antibodies, based on adherence to an antigen-coated surface, are precise and sensitive, but errors can be introduced by the presence of relatively high concentrations of non-IgE antibodies of the same specificity. Such errors are caused by competition for the limited number of antigenic determinants on the antigen-coated surface. In this paper we explore further the quantitative aspects of the 'competition effect'. An easily applied method is described, based on preferential precipitation of non-IgE antibodies by ammonium sulfate, that permits analysis of IgE antibodies in the presence of large amounts of non-IgE antibodies (that are principally IgG). For IgE anti-Ar, the maximum permissible ratio is extended from approximately 1500:1 to at least 40,000:1. We have also determined the effect of IgG antibodies or whole mouse serum on PCA reactions of mouse IgE antibodies, carried out in rats.

Methods are described for the production of syngeneic mouse anti-IgE monoclonal antibodies (mAb). Hybridomas were prepared by using spleen cells from mice immunized with a conjugate of keyhole limpet hemocyanin with monoclonal IgE. Serum titers varied from approximately 40 to 1000 micrograms/ml. The anti-IgE mAb were isolated by affinity chromatography on columns containing immobilized monoclonal IgE. The mAb are specific for isotypic determinants of IgE and do not react with other immunoglobulin isotypes. One of the mAb, which has a high affinity for IgE (Ka = 4.7 X 10(8) M-1), should be useful for studies of regulation of IgE. The applicability of the mAb to quantitative assays for IgE was demonstrated.