M O Dailey

Isolation of molecules recognized by monoclonal antibodies and antisera: the solid phase immunoisolation technique

A simple technique for the isolation of antigens recognized by antisera and monoclonal antibodies has been developed. This method, the solid-phase immunoisolation technique, employs the protein-binding properties of polyvinylchloride microtiter plates. Antibodies are adsorbed to the plates either directly or via an anti-immunoglobulin reagent. Antigen is then placed in the wells, and allowed to adsorb to the antibody. The well is washed, and the antigen is then eluted with a denaturing electrophoresis sample buffer for one- or two-dimensional analysis. The solid-phase immunoisolation technique has been used to isolate a variety of cell membrane antigens with high signals and low backgrounds. The ease of the procedure and the high signal-to-noise ratio make this method preferable to the use of a staphylococcal adsorbent for many applications.

Pharmacological characterization of histamine H2 receptors on clonal cytolytic T lymphocytes: Evidence for histamine-induced desensitization

Cultured cytolytic T lymphocytes of clonal origin were screened for histamine-stimulated cyclic AMP production. Histamine caused a 2- to 8-fold elevation of cyclic AMP levels in five independent clones. The EC50 for histamine of 1.7 X 10(-5) M and the rank order of potencies of H1 and H2 agonists [impromidine greater than histamine greater than dimaprit greater than 4-methylhistamine greater than 2-methylhistamine greater than 2-(2-aminoethyl)-thiazole] were characteristic of the conventional histamine H2 receptor. H1 and H2 antagonists inhibited histamine-stimulated cyclic AMP elevation with inhibition constants typical for those found on other H2 receptor systems. Prior incubation of cells with histamine resulted in a marked loss in responsiveness to subsequent histamine challenge. We demonstrate that this desensitization is dose and time dependent and results in a change in the efficacy and not the potency of histamine. Although cyclic AMP increases could also be elicited with isoproterenol, prostaglandin E1 or forskolin, desensitization of histamine had no effect on the ability of these agents to stimulate cyclic AMP production. In contrast to the rapid rate of histamine-induced desensitization, recovery of histamine responsiveness could not be detected for several hours.

T cell clones: a model of a non-recirculating phase of T cell differentiation.

Cloned lines of T lymphocytes have been valuable in investigating many of the in vitro properties of thymus-derived lymphocytes. When such clones have been tested in vivo, however, they have generally shown much less activity than they have in vitro. Thus, cytolytic T cell (Tc) clones that are highly active in lysing tumor target cells in vitro are poorly effective in causing rejection of the same cells in vivo, unless they are injected into the same site as the tumor cells1,2. Similarly, in vitro T cell clones efficiently mediate delayed hypersensitivity only when injected at the same site as the relevant antigen3.