To better understand the interaction between f3-adrenergic ligands and their specific membrane receptors, we have undertaken to isolate and purify the various components intervening in the catecholamine-mediated activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. For this purpose we have developed an affinity chromatography method that allows physical separation of the functional cyclase and f3-adrenergic receptors from turkey erythrocyte membranes (1).Various biochemical and pharmacological properties of the receptors were described (2). The minute amounts of protein prepared by this method and the risk of losing essential components of the receptor cyclase complex during the membrane purification and solubilization steps prompted us to develop a second line of investigation based on immunological methods.Anti-receptor antibodies can be obtained by immunization of rabbits and mice with purified ,B-adrenergic receptor, but most of the antibodies react with parts of the molecule other than the binding site (ref. 3; unpublished data). This is in agreement with observations made in other systems: animals do not make anti-receptor binding site antibodies upon immunization with insulin, acetylcholine, or thyrotropin receptors despite the fact that in pathological conditions such as autoimmune diabetes (4), myasthenia gravis (5), Graves disease (6), and 02-adrenergic hyporesponsiveness in allergic rhinitis (7) anti-binding site antibodies probably constitute an important basis for the etiopathogeny.

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A fluorometric assay is described for the detection of red blood cell antibodies. The assay reveals as little as 600 molecules of bound, fluoresceinated rabbit anti-human IgG antibodies per erythrocyte. Eleven patients with possible autoimmune erythrocyte disorder and negative direct antiglobulin test were studied by the fluorometric assay. The outcome of the fluorometric assay was compared with that of the human allogeneic rosette test. Results obtained by the two methods were in complete agreement. Five of the patients were shown to possess unexpectedly high levels of erythrocyte-bound IgG in spite of a negative, direct antiglobulin test. These findings and the validity of the fluorometric assay are discussed.

The Simian virus 40-transformed rabbit spleen cell TRSC-1 synthesizes intracellular whole IgG molecules of the alb4 allotype. Two hypoxanthine-guanine phosphoryl transferase-deficient mutants were derived from this line. One of these, TRSC-1-8, was used in somatic cell fusion experiments together with gangliocytes from a rabbit immunized against beta-galactosidase. Out of nineteen hybrid clones surviving in selective medium, only one, L17, was shown to produce free gamma chains which express the a2 allotype of the donor rabbit rather than the al marker of the parents TRSC-1-8 line. The inability to restore IgG secretion in hybrids suggests that dominant regulatory controls are exerted by the TRSC-1 genome on Ig reduction. This supports the notion that the TRSC-1 line originated from a splenocyte that had not reached the final plasmocyte differentiation stage at the time of viral transformation.