Monoclonal Antibodies to Benzodiazepines

Blas, Angel L. De; Park, Dongeun; Vitórica, Javier; Sangameswaran, Lakshmi

doi:10.1111/j.1471-4159.1985.tb10530.x

Cited by 32 publications

(8 citation statements)

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“…In these studies we used an anti-benzodiazepine monoclonal antibody (mAb) (1,2). This finding was supported by immunocytochemistry and immunoblotting experiments as well as by the purification by immunoaffinity chromatography of a rat brain substance that inhibited [3H]flunitrazepam binding to the neuronal-type benzodiazepine receptor (3,4).…”

mentioning

confidence: 95%

“…Useful spectra appeared as the probe was heated to 100°C, about the same temperature as required by authentic N-desmethyldiazepam. (2). The blockage of immunoreactivity by Ro 5-4864 and diazepam is due to the high affinities of these benzodiazepines for mAb 21-7F9 and does not necessarily reflect the specificity of the endogenous substance for the "peripheral" or '"central" type of benzodiazepine receptor.…”

mentioning

confidence: 97%

“…Some of these results have been communicated in preliminary form (11)(12)(13) The anti-benzodiazepine mAb 21-7F9 was obtained after immunizing BALB/c mice with 3-hemisuccinyloxyclonazepam coupled to bovine serum albumin. The production, purification, and characterization of mAb 21-7F9 have been reported (2).…”

mentioning

confidence: 99%

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Purification of a benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain.

Sangameswaran¹,

Fales²,

Friedrich³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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An endogenous brain substance that binds to the central-type benzodiazepine receptors with agonist properties is present in both rat and bovine brains. This substance has been purified to homogeneity from bovine brain by immunoafflmity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reversed-phase HPLC steps. The purified substance was characterized as the benzodiazepine N-desmethyldiazepam (nordiazepam The anti-benzodiazepine mAb 21-7F9 was obtained after immunizing BALB/c mice with 3-hemisuccinyloxyclonazepam coupled to bovine serum albumin. The production, purification, and characterization of mAb 21-7F9 have been reported (2).Immunoaffinity Chromatography. Fifty grams of bovine cerebellum and cerebral cortex were homogenized (Waring blender, 6 min) in 60 ml of 5 mM Tris-HCl, pH 8.1/0.3 mM phenylmethylsulfonyl fluoride/1 mM EDTA containing soybean trypsin inhibitor (20 pug/ml) and pepstatin (1 pug/ml). The preparation of the soluble extract and the procedure for immunoaffinity chromatography have been described (1). The sample was loaded on a 10-ml Affi-Gel 10 (Bio-Rad) column containing 50 mg ofimmobilized anti-benzodiazepine mAb 21-7F9, and 100 ml of each washing solution was passed through the column at a flow rate of 10 ml/hr. The retained material was eluted with 100 ml of 0.2 M acetic acid at a flow rate of 50 ml/hr.Gel Filtration on Sephadex G-25. The immunoaffinity eluate was dried under reduced pressure, dissolved in 0.9 ml of 40% formic acid, and layered on a Sephadex G-25 column (51.5 cm x 1.2 cm i.d.) that had been equilibrated with 40% formic acid. Fractions (2 ml) were collected at a flow rate of 5 ml/hr. The active fractions were pooled, concentrated under reduced pressure, and resuspended in 600 Al of 0.1% trifluoroacetic acid. The benzodiazepine receptor binding activity of the fractions was assayed as indicated below.Reversed-Phase HPLC.Step I. A 200-Al sample of the Sephadex G-25-purified substance was subjected to reversed-phase HPLC on a C8 column [length, 25 cm; particle size, 10 /Lm; pore size, 300 A; Brownlee Laboratory (Santa Clara, CA)] and eluted with a linear gradient (5-60%) of acetonitrile in 0.1% trifluoroacetic acid.Step II. tTo whom all reprint requests should be addressed. 9236The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Purification of a benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain.

Sangameswaran¹,

Fales²,

Friedrich³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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“…The production of the hybridomas and the comparative study of the BZD binding properties of this and other antibodies will be reported elsewhere (38). The hybridomas secreting mAbs to BZD were identified by assaying the conditioned culture media in three ways: (i) binding of 3H-labeled flunitrazepam (FNZ) tp the mAbs, (ii) antibody competition with tie [3H]FNZ binding to the brain membrane BZDR, and (iii) antibody binding to the 3-hemisuccinyloxyclonazepam conjugate with keyhole limpet hemocyanin (BZD-KLH) in a standard ELISA assay.…”

Section: Methodsmentioning

confidence: 99%

Demonstration of benzodiazepine-like molecules in the mammalian brain with a monoclonal antibody to benzodiazepines.

Sangameswaran¹,

Blas²

1985

Proc. Natl. Acad. Sci. U.S.A.

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An anti-benzodiazepine monoclonal antibody has been used to demonstrate the existence of benzodiazepine-like molecules in the brain. Immunocytochemical experiments show that these molecules are neuronal and not glial and that they are ubiquitously distributed throughout the brain. Immunoblots indicate the presence of benzodiazepinelike epitopes in several brain peptides. Small benzodiazepinelike molecules were isolated from the brain soluble fraction by immunoaffinity chromatography. They block the binding of agonists, inverse agonists, and antagonists to the neuronal-type benzodiazepine receptor. The neurotransmitter ry-aminobutyric acid increases the affinity of the benzodiazepine receptor for the purified endogenous molecules. The results indicate that the immunoafIinity-purified molecules behave like the neuronal-type benzodiazepine receptor agonists. The purified molecules, however, do not inhibit the binding of tritiated Ro 5-4864 to the "peripheral-type" benzodiazepine receptor. The results demonstrate the existence of benzodiazepine-like molecules in the brain that bind to the benzodiazepine receptor.These molecules are different from the endogenous benzodiazepine receptor ligands reported by others.The mammalian brain has been shown to have receptors for benzodiazepines (BZDs) (1, 2). One could ask, why has evolution provided the brain with receptors for these synthetic drugs (e.g., Valium, Librium, etc.)? Possibly the brain could produce endogenous BZD-like molecules that bind to the BZD receptors (BZDRs), modulating y-aminobutyric acid (GABA) neurotransmission. This notion is supported by the existence in the brain of endogenous opiate peptides, which were discovered after the discovery of the brain opiate receptors. A number of substances extracted from brain and other tissues bind to the BZDR-e.g., purines and purine nucleosides (3, 4), nicotinamide (5), ,3-carbolines (6), and a number of peptides and proteins of Mr 1500-70,000 (7-11). However, there is no real evidence that these compounds play any physiological role as ligands for BZDR (12, 13). Thus, purines, purine nucleosides, and nicotinamide displace diazepam binding to the receptor with an IC50 of -1 mM, whereas diazepam has a Kd in the nanomolar range. Although the ,B-carbolines have high affinity (Kd in the nanomolar range) and high specificity for BZDR, they are not natural brain constituents but are rather artifacts formed during tissue extraction (12, 13). The best-characterized brain peptide that binds to BZDR is the diazepam-binding inhibitor (DBI), Mr 11,000. DBI has been purified and the amino acid sequence, including a 18 amino acid active fragment, is known (11,14,15 In this paper we describe the use of an anti-benzodiazepine monoclonal antibody (mAb) for the identification and preliminary characterization of BZD-like molecules that bind to the brain BZDR. Some of these results have been communicated in preliminary form (16). MATERIALS AND METHODSThe mAb to BZD, 21-7F9, was obtained after immunizing BALB/c mice with 3-hemi...

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“…This raises the possibility that such compounds could act as endogenous agonists (De Blas et al, 1985). However, there is no evidence that such compounds are synthesized in vivo (Izquierdo and Medina, 1993).…”

Section: Are There Natural Ligands For the Benzodiazepine Receptor Inmentioning

confidence: 97%

Therapeutic applications of benzodiazepine receptor ligands in anxiety

Leonard

1999

Hum. Psychopharmacol. Clin. Exp.

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In this short review, evidence is presented to show that the benzodiazepines produce their variety of pharmacological eects by activating speci®c receptors that form part of the main inhibitory neurotransmitter receptor system, the g-amino-butyric acid (GABA) system, in the mammalian brain. Dierent classes of benzodiazepine receptor ligands have been developed which can alleviate anxiety or produce anxiety according to the ®ne structural changes that occur when the ligands interact with the benzodiazepine receptor. There is some evidence that endogenous substances in the brain can cause either an increase or a reduction in the anxiety state by acting on the benzodiazepine receptor. The unique nature of the benzodiazepine receptor, and the disparate properties of the drugs that act on this receptor, should allow plenty of scope for the development of novel compounds with selective anxiolytic and other properties in the future. Lastly, despite the evidence from animal studies that benzodiazepine receptor function changes in response to drug treatment, there is little evidence from human brain studies that such changes are relevant to the phenomena of tolerance, dependence and withdrawal eects that have been the recent cause for public concern.

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Monoclonal Antibodies to Benzodiazepines

Cited by 32 publications

References 20 publications

Purification of a benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain.

Purification of a benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain.

Demonstration of benzodiazepine-like molecules in the mammalian brain with a monoclonal antibody to benzodiazepines.

Therapeutic applications of benzodiazepine receptor ligands in anxiety

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