A monoclonal antibody recognizing κ- but not μ- and δ-opioid receptors

Katalin Maderspach, Klára Németh, József Simon, S. Benyhe, M. Szücs, Mária Wollemann

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

A monoclonal antibody (mAb), KA8, that interacts with the κ-opioid receptor binding site was generated. BALB/c female mice were immunized with a partially purified κ-opioid receptor preparation from frog brain. Spleen cells were hybridized with SP2/0AG8 myeloma cells. The antibody-producing hybridomas were screened for competition with opioid ligands in a modified enzyme-linked immunosorbent assay. The cell line KA8 secretes an IgG1 (κ-light chain) immunoglobulin. The mAb KA8 purified by affinity chromatography on protein A-Sepharose CL4B was able to precipitate the antigen from a solubilized and affinity-purified frog brain κ-opioid receptor preparation. In competition studies, the mAb KA8 decreased specific [3H]ethylketocyclazocine ([3H]EKC) binding to the frog brain membrane fraction in a concentration-dependent manner to a maximum to 72%. The degree of the inhibition was increased to 86% when μ-and δ-opioid binding was suppressed by 100 nM [D-Ala2,NMe-Phe4,Gly-ol]-enkephalin (DAGO) and 100 nM [D-Ala2,L-Leu5]-enkephalin (DADLE), respectively, and to 100% when μ-, δ-, and κ2-sites were blocked by 5 μM DADLE. However, the μ-specific [3H]DAGO and the δ-preferring [3H]DADLE binding to frog brain membranes cannot be inhibited by mAb KA8. These data suggest that this mAb is recognizing the κ- but not the μ- and δ-subtype of opioid receptors. The mAb KA8 also inhibits specific [3H]naloxone and [3H]EKC binding to chick brain cultured neurons and rat brain membranes, whereas it has only a slight effect on [3H]EKC binding to guinea pig cerebellar membranes. These findings suggest horriologies in the κ-opioid binding site of frog brain and rat brain as well as chick neurons, but the κ-opioid receptor subtype in the guinea pig cerebellum may be different.

Original languageEnglish
Pages (from-to)1897-1904
Number of pages8
JournalJournal of Neurochemistry
Volume56
Issue number6
Publication statusPublished - Jun 1991

Fingerprint

Opioid Receptors
Brain
Monoclonal Antibodies
Leucine-2-Alanine Enkephalin
Anura
Ala(2)-MePhe(4)-Gly(5)-enkephalin
Opioid Analgesics
Membranes
Neurons
Rats
Guinea Pigs
Ethylketocyclazocine
Binding Sites
Immunoglobulin Light Chains
Affinity chromatography
Immunosorbents
Enkephalins
Hybridomas
Naloxone
Affinity Chromatography

Keywords

  • κ-Opioid receptor
  • Brain (frog, chick, rat, guinea pig)
  • Monoclonal antibody

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Maderspach, K., Németh, K., Simon, J., Benyhe, S., Szücs, M., & Wollemann, M. (1991). A monoclonal antibody recognizing κ- but not μ- and δ-opioid receptors. Journal of Neurochemistry, 56(6), 1897-1904.

A monoclonal antibody recognizing κ- but not μ- and δ-opioid receptors. / Maderspach, Katalin; Németh, Klára; Simon, József; Benyhe, S.; Szücs, M.; Wollemann, Mária.

In: Journal of Neurochemistry, Vol. 56, No. 6, 06.1991, p. 1897-1904.

Research output: Contribution to journalArticle

Maderspach, K, Németh, K, Simon, J, Benyhe, S, Szücs, M & Wollemann, M 1991, 'A monoclonal antibody recognizing κ- but not μ- and δ-opioid receptors', Journal of Neurochemistry, vol. 56, no. 6, pp. 1897-1904.
Maderspach, Katalin ; Németh, Klára ; Simon, József ; Benyhe, S. ; Szücs, M. ; Wollemann, Mária. / A monoclonal antibody recognizing κ- but not μ- and δ-opioid receptors. In: Journal of Neurochemistry. 1991 ; Vol. 56, No. 6. pp. 1897-1904.
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abstract = "A monoclonal antibody (mAb), KA8, that interacts with the κ-opioid receptor binding site was generated. BALB/c female mice were immunized with a partially purified κ-opioid receptor preparation from frog brain. Spleen cells were hybridized with SP2/0AG8 myeloma cells. The antibody-producing hybridomas were screened for competition with opioid ligands in a modified enzyme-linked immunosorbent assay. The cell line KA8 secretes an IgG1 (κ-light chain) immunoglobulin. The mAb KA8 purified by affinity chromatography on protein A-Sepharose CL4B was able to precipitate the antigen from a solubilized and affinity-purified frog brain κ-opioid receptor preparation. In competition studies, the mAb KA8 decreased specific [3H]ethylketocyclazocine ([3H]EKC) binding to the frog brain membrane fraction in a concentration-dependent manner to a maximum to 72{\%}. The degree of the inhibition was increased to 86{\%} when μ-and δ-opioid binding was suppressed by 100 nM [D-Ala2,NMe-Phe4,Gly-ol]-enkephalin (DAGO) and 100 nM [D-Ala2,L-Leu5]-enkephalin (DADLE), respectively, and to 100{\%} when μ-, δ-, and κ2-sites were blocked by 5 μM DADLE. However, the μ-specific [3H]DAGO and the δ-preferring [3H]DADLE binding to frog brain membranes cannot be inhibited by mAb KA8. These data suggest that this mAb is recognizing the κ- but not the μ- and δ-subtype of opioid receptors. The mAb KA8 also inhibits specific [3H]naloxone and [3H]EKC binding to chick brain cultured neurons and rat brain membranes, whereas it has only a slight effect on [3H]EKC binding to guinea pig cerebellar membranes. These findings suggest horriologies in the κ-opioid binding site of frog brain and rat brain as well as chick neurons, but the κ-opioid receptor subtype in the guinea pig cerebellum may be different.",
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