RESUMO
A mouse monoclonal, anti-idiotypic, anti-opioid receptor antibody (Ab2-AOR) has been generated from monoclonal anti-morphine antibodies (Ab1). Hybridoma culture supernatants were screened by a solid phase radioimmunoassay (RIA), based on their competition with radiolabelled morphine for Ab1. One of the Ab2s that gave a positive RIA also competed at rat brain opioid receptors with tritiated opioid ligands dihydromorphine (DHM), naloxone, etorphine, Tyr-D-Ala-Gly-Phe-D-Leu (DADLE), Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE). SDS-PAGE revealed Ab2-AOR to be highly purified after successive affinity and protein A-Sepharose chromatography. Ab2-AOR at concentrations of 10-100 nM competed with both mu- and delta-selective specific ligands for brain opioid receptors. Less than 13 micrograms/ml Ab2-AOR completely inhibited specific opioid radioligand binding to both soluble and membrane-bound opioid receptors. To demonstrate its anti-delta receptor activity further, a double-antibody ELISA procedure was developed that is based on the binding of Ab2-AOR to immobilized NG 108-15 cells (which contain only delta opioid receptors). Dose-dependent, opioid peptide- and opiate alkaloid-competitive binding of Ab2-AOR-containing ascites fluid to NG 108-15 cells was observed. A mu opioid agonist effect was demonstrated for Ab2-AOR, in that it decreased by 70% [3H]thymidine incorporation into DNA of fetal brain cell aggregates. This agonist-like action of Ab2-AOR was blocked by naltrexone. The antibody bound specifically to brain tissue sections and the presence of diprenorphine blocked this interaction. Hence, an Ab2 with mu and delta specificity has been characterized.
Assuntos
Anticorpos Anti-Idiotípicos , Anticorpos Monoclonais , Receptores Opioides/metabolismo , Animais , Anticorpos Anti-Idiotípicos/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Ligação Competitiva , Encéfalo/metabolismo , Cromatografia de Afinidade , Replicação do DNA/efeitos dos fármacos , Hibridomas/imunologia , Técnicas In Vitro , Cinética , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Naltrexona/farmacologia , Entorpecentes/metabolismo , Radioimunoensaio , Receptores Opioides/imunologia , Receptores Opioides delta , Receptores Opioides mu , Timidina/metabolismoRESUMO
A case is presented of a massive, full-thickness, trunk-wall defect successfully reconstructed with a free-tissue transfer of the entire vastus lateralis muscle. Indications, technical details, and donor-site considerations are discussed.