title: A novel two-site immunoradiometric assay for beta-endorphin using nitrocellulose as solid phase.
abstract: A two-site immunoradiometric assay for the highly specific direct quantitation of nonacetylated beta h-EP in crude brain tissue samples has been developed with a detection limit of 10 fmol per well. The assay used two different antibodies with distinct specificities: a polyclonal rabbit anti-beta h-EP antibody binding between the middle portion and the C-terminal end of beta h-EP was bound to nitrocellulose membrane discs, a solid phase with a high protein binding capacity. In the following two incubation steps, the beta h-EP containing crude tissue extract--or the beta h-EP-standard--and, subsequently, the 125I-labeled monoclonal 3-E7 mouse antibody directed against the N-terminus of beta h-EP were added. Binding of beta h-EP to the solid phase antibody in the first incubation step was not affected by the addition of cross reacting opioid peptides derived from beta h-LPH up to 10 pmol per disc. Nonspecific binding of the labeled antibody to the solid phase could be lowered to 3% of total counts by the use of PBS containing nonfat dry milk as blocking solution and incubation buffer, a procedure that did not reduce maximum specific binding. Dilution studies performed with extracts sampled from the anterior hypothalamus excluded the interference of tissue factors in the assay.
authors.affiliation: Institute of Forensic Medicine, University of Zurich, Switzerland.
authors.forename: D R
authors.initials: DR
authors.lastname: Voellmy
authors.forename: P
authors.initials: P
authors.lastname: Külling
authors.forename: C
authors.initials: C
authors.lastname: Gramsch
authors.forename: M
authors.initials: M
authors.lastname: Häni
authors.forename: P
authors.initials: P
authors.lastname: Mehraein
authors.forename: F S
authors.initials: FS
authors.lastname: Messiha
authors.forename: A
authors.initials: A
authors.lastname: Pasi
mesh_terms: D001921:Brain / Q000378:metabolism*; D003101:Collodion; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D010902:Pituitary Gland / Q000378:metabolism*; D011863:Radioimmunoassay / Q000379:methods*; D001615:beta-Endorphin / Q000378:metabolism*
chemical_list: D001615:beta-Endorphin; D003101:Collodion