A novel two-site immunoradiometric assay for beta-endorphin using nitrocellulose as solid phase.
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.
Voellmy
D R
DR
Institute of Forensic Medicine, University of Zurich, Switzerland.
Külling
P
P
Gramsch
C
C
Häni
M
M
Mehraein
P
P
Messiha
F S
FS
Pasi
A
A
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*
D001615:beta-Endorphin; D003101:Collodion