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5-hydroxytryptophol (5-HTOL) derivatives, antibodies, immunoassays and detection of recent alcohol consumption

a technology of 5hydroxytryptophol and derivatives, applied in the field of antibodies, can solve the problems of expensive instruments and drawbacks of methods

Inactive Publication Date: 2007-11-29
AXIS BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about a new antibody that can be used to measure the levels of a compound called 5-HTOL in various body fluids. This compound is a marker for alcohol consumption and is also associated with other metabolites of serotonin. The invention provides a simple and reliable method for measuring 5-HTOL without the need for expensive instruments. The invention also includes a method for synthesizing the immunogen used to raise the antibody. The technical effect of the invention is to provide a new tool for the diagnosis and treatment of alcohol dependence and forensic medicine.

Problems solved by technology

The presently used method for measuring 5-HTOL is gas chromatography in combination with mass spectrometry (GC / MS), a method requiring expensive instruments.
A high pressure liquid chromatography (HPLC) method has been developed but it suffers from the drawback that of “false peaks”, i.e. overlapping Rf for 5-HTOL and other compounds.

Method used

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  • 5-hydroxytryptophol (5-HTOL) derivatives, antibodies, immunoassays and detection of recent alcohol consumption
  • 5-hydroxytryptophol (5-HTOL) derivatives, antibodies, immunoassays and detection of recent alcohol consumption
  • 5-hydroxytryptophol (5-HTOL) derivatives, antibodies, immunoassays and detection of recent alcohol consumption

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0035] Synthesis and purification of 3-(2-hydroxyethyl)indole-5-yl-O-β-D-glucopyranosiduronic acid (I) (Scheme 1 as shown in FIG. 1): 2-(5-Hydroxy-3-indolyl)ethyl alcohol (151 mg), MgCl2 (50 mg), uridine 5′-diphosphoglucuronic acid triammonium salt (490 mg), uridine 5′-diphosphoglucuronyl transferase (182 mg) (Type III from bovine liver, Sigma) and 40 ml of 0.1 M pH 7.4 Tris-buffer were added to a 200 ml E-flask. The flask was sealed with parafilm and incubated in a shaker-incubator at 37° C. and 150 rpm. Small samples were extracted from the incubation at different timepoints, and analysed after cooling, centrifugation, filtration and acidification by analytical PepRPC on a FPLC system, in order to follow the reaction. The concentration of 3-(2-hydroxyethyl)indole-5-yl-O-β-D-glucopyranosiduronic acid (I) reached plateau-levels after 20 h and remained at that level for more than 7 h.

[0036] The incubation was stopped on ice for 15 min followed by centrifugation at 10000 rpm at 5° C....

example 2

[0037] Synthesis of N-(2-pyridyldithioethyl)-(2-hydroxyethyl)indole-5-yl-O-β-D-glucopyranosiduronic acid (II). Scheme 2 (as shown in FIG. 2). 3-(2-Hydroxyethyl)indole-5-yl-O-β-D-glucopyranosiduronic acid (I) (52 mg, 0,153 mmole) was dissolved in 1 ml DMF (dried with molecular sieves) in 5 ml reacti vial. Thereafter O-(N-succinimidyl)-N,N, N′,N′-tetramethyluronium tetrafluoroborate (TSTU) (73 mg, 0.243 mmole) and diisopropylethylamine (84 μl, 0.456 mmole) were added. The reaction was run for 35 min in room temperature.

[0038] Cysteamine-2-pyridyldisulfide hydrochloride (72 mg, 0.324 mmole) was added to another reacti vial followed by DMF (0.5 ml) and diisopropylethylamine (56 μ1, 0.324 mmole). To this solution the hydroxysuccinimidyl activated acid (I) was added. The reaction was run for 1 h and then evaporated in vacuum at low pressure giving an oil. TLC (n-butanol:ethyl acetate:acetic acid:water=1:1:1:1) showed that almost all (I) had reacted. The oil was dissolved in 3 ml of 27% m...

example 3

[0039] Synthesis of the KLH conjugate (III) (Scheme 2). Keyhole limpet hemocyanine (KLH) (73.5 mg) was dissolved in 6.5 ml of 0.1 M borate buffer keeping pH 9.5. Undissolved substance was removed by filtration and the protein content of the solution was analysed by amino acid analysis to be 37 mg / ml protein. To this ice-cold solution of KLH 0.8 ml of a solution of a mixed anhydride of 3-(2-hydroxyethyl)indole-5-yl-O-β-D-glucopyranosiduronic acid (0.025 mmole) was added. The latter compound was prepared by dissolving 3-(2-hydroxyethyl)indole-5-yl-O-β-D-glucopyranosiduronic acid (I) (10.6 mg, 0.0312 mmole) in 1 ml of tetrahydrofuran in a 5 ml reacti vial. Nitrogen gas was let into the vial and the latter was placed in an ice-bath. After 15 min isobutylchloroformiate (4.1 μl, 0.0312 mmole) and triethylamine (4.3 μl, 0.0312) were added and the reaction the acid (I) and isobutyl chloroformiate was completed in 30 min at 0° C. The reaction between KLH and the anhydride was run at 20 min a...

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Abstract

Antibody specific for a 5-HTOL compound. A glucuronide with 5-hydroxytryptophol (5-HTOL) characterized in that it comprises the structure of the β-glucuronide between the 5-hydroxy group of 5-HTOL and D-glucopyranosiduronic acid. An immunoassay and a diagnostic method utilizing the immunoassay wherein a 5-hydroxytryptophol (5-HTOL) compound is determined by the use of an antibody specific for a 5-HTOL compound is used.

Description

TECHNICAL FIELD [0001] The present invention concerns a novel antibody that may be used in immunoassays of components reflecting body fluid levels of 5-hydroxytryptophol [5-HTOL=2-(5-hydroxy-3-indolyl)ethyl alcohol]. Free 5-HTOL and glucuronide and sulphate conjugates thereof are known markers for recent alcohol intake in humans. Other aspects of the invention are as indicated in the title. BACKGROUND ART [0002] 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA) are human metabolites of serotonin (5-hydroxytryptamine, 5-HT) via the common intermediate 5-hydroxyindole-3-acetaldehyde (5-HIAL). 5-HTOL is excreted in the urine where it mainly occurs conjugated with a glucuronic acid (T-5-G (or GHTOL), 75-95%) and, to a lesser extent, in free form or conjugated as a sulphate. 5-HIAA is also excreted in the urine. After alcohol consumption, the 5-HTOL level in various body fluids will raise from normal values that for urine are in the range of 40-650 nmol / L. The 5-HTO...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07H17/02C07K16/44G01N33/53C12N5/10C12N15/02C12P21/08
CPCC07H17/02C07K16/44Y10T436/203332Y10T436/145555Y10T436/25Y10T436/17Y10T436/173845Y10T436/14
Inventor BRANDT, RAGNHILDBECK, OLOFHELANDER, ANDERSJONSSON, ROSE-MARIEUNGER, ERICBORG, STEFANAKERBLOM, EVA
Owner AXIS BIOCHEM
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