ABSTRACT
Binding of 3H-etorphine and 3H-D-Ala2-D-Leu5-enkephalin to opiate receptors in synaptosomal and microsomal fractions prepared from guinea pig ileum homogenates has been studied. It is found that the dissociation constants for etorphine from all fractions are the same. The binding capacity for etorphine for the purified synaptosomal fraction is greater than for other fractions by a factor of 5. For the enkephalin derivative binding to the microsomal fraction the dissociation constant is greater than for etorphine while the binding capacity is a factor of 3 lower. These results are in contrast to the case for binding to central nervous system subcellular fractions.
Subject(s)
Ileum/innervation , Receptors, Opioid/metabolism , Animals , Enkephalin, Leucine/analogs & derivatives , Enkephalin, Leucine/metabolism , Enkephalin, Leucine-2-Alanine , Etorphine/metabolism , Guinea Pigs , Ileum/metabolism , Ileum/ultrastructure , In Vitro Techniques , Subcellular Fractions/metabolismABSTRACT
Four lines of high affinity monoclonal antibodies directed against morphine have been isolated and affinity purified. Some of their properties, including cross-reactivities to a large set of selected opiate agonists and antagonists are described. Importantly, none of the immunoglobulins cross-react with D-Ala2-D-Leu5-enkephalin.
Subject(s)
Antibodies, Monoclonal/immunology , Morphine/immunology , Animals , Chromatography, Affinity , Cross Reactions , Enkephalin, Leucine/analogs & derivatives , Enkephalin, Leucine/immunology , Enkephalin, Leucine-2-Alanine , Female , Immunoglobulin G/immunology , Mice , Mice, Inbred BALB C , Narcotic Antagonists/immunologyABSTRACT
This paper describes the conditions under which a short column packed with Sephadex G-25 may be eluted centrifugally to separate labeled ligands from binding immunoglobulins in a manner which can yield quantitative binding information. This method may be used as an alternative to the classical binding assay involving ammonium sulfate precipitation. The advantage is a large saving in total assay time over that procedure. The column assay is applicable to antisera and purified polyclonal and monoclonal immunoglobulins. The restrictions are that the labeled ligands must be of molecular weight less than approximately 5000 Da.