J. Biol. Chem., Vol. 260, Issue 29, 15655-15661, Dec, 1985
A monoclonal antibody capable of modulating opioid binding to rat neural membranes
JM Bidlack and RR Denton
A monoclonal antibody capable of inhibiting opioid binding to rat neural
membranes has been produced. Spleen cells from a BALB/c mouse, immunized
with a partially purified opioid receptor complex, were fused with
P3-X63.Ag8.653.3 myeloma cells. The cell line OR-689.2.4 secreted an IgM
that was capable of partially inhibiting opioid binding to rat neural
membranes under equilibrium binding conditions, while not affecting the
binding of nonopioid ligands. Control mouse immunoglobulins and
heat-denatured OR-689.2.4 did not inhibit opioid binding to membranes. The
purified immunoglobulin inhibited the binding of [3H]dihydromorphine in a
titrable, saturable, and reversible manner, as well as the binding of the
delta-ligand [3H][D-Ala2,D- Leu5]enkephalin, the kappa-ligand [3H]
ethylketocyclazocine, and 3H- labeled antagonists. In addition to blocking
the binding of opioids to membranes, the immunoglobulin could also displace
bound [3H]dihydromorphine from neural membranes. The 125I-labeled
immunoglobulin specifically bound to neural membranes with a Kd of 1.3 nM
and a maximal number of binding sites of 41.8 fmol/0.25 mg of membrane
protein. In a titrable manner, the immunoglobulin precipitated opioid
binding sites from a solubilized preparation of neural membranes. When
OR-689.2.4 conjugated to Sepharose was incubated with the partially
purified opioid receptor complex, labeled with 125I, a 35,000-dalton
protein was specifically bound by the immunoglobulin. This antibody
provides a tool for probing the multiple opioid binding sites.
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