Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-06-07
2003-08-19
Chin, Christopher L. (Department: 1641)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C435S004000, C435S007800, C435S007100, C435S007210, C424S178100, C424S130100, C436S063000, C436S089000, C436S091000, C436S096000, C436S106000, C436S111000, C436S131000, C436S161000, C436S164000, C436S166000, C436S172000, C436S174000, C436S500000, C422S052000, C422S069000, C422S070000, C422S081000, C422S082050, C530S387200
Reexamination Certificate
active
06608178
ABSTRACT:
TECHNICAL FIELD
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
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-HTOL/5-HIAA ratio will simultaneously become elevated from normal values that are <0,01 (or <10 if expressed as picomolar
anomolar). The increase of both the 5-HTOL level and the 5-HTOL/5-HIAA ratio persist for several hours after alcohol has disappeared from the body. These findings have made 5-HTOL and the 5-HTOL/5-HIAA ratio markers of recent alcohol consumption in the treatment of alcohol dependence and in forensic medicine, e.g. during post-mortem body examination. The preference has been for measuring the ratio because 5-HTOL levels, but not the ratio, are increased also after ingestion of food rich in serotonin, and because the range for normal and abnormal 5-HTOL levels, but not the ratios, are overlapping. As an alternative also the ratio between urinary 5-HTOL and other urinary excretion products, such as creatinine, have been used.
The term “15-HTOL” includes 5-HTOL compounds, such as free 5-HTOL, its glucuronide (tryptophol-5-gucuronide, T-5-G) and its sulphate, if not otherwise specified.
Increased levels of 5-HTOL compounds after alcohol consumption have so far been detected in urine, plasma, cerebrospinal fluid (CSF). It is most likely that one can detect 5-HTOL compounds also in serum, tear fluid, sweat, saliva etc. The normal level of 5-HTOL compounds and the balance between free 5-HTOL and its conjugated forms may be different between various types of sample samples.
Compared to 5-HIAA, 5-HTOL is complicated to measure. It occurs in lower concentrations than 5-HIAA and the conjugated forms are in excess with a strong predominance for the glucuronide. 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 R
f
for 5-HTOL and other compounds. Both methods require that the glucuronide (T-5-G) has to be converted enzymatically to free 5-HTOL before the assay. Thus there is a need for a cheap, simple and reliable method for measuring 5-HTOL compounds in various body fluids.
For earlier published articles within the field of the invention see separate list at the end of the descriptive part.
Reference may also be made to Dabadie et al, Synapse 14: 178-183 (1993), Iijima et al, Acta histochem 89: 141-156 (1990), U.S. Pat. No. 4,762,781 (Geffard) and EP-A-216162 (A/S De Danske Sukkerfabrikker)
OBJECTIVES OF THE PRESENT INVENTION
The primary objective of the invention is to provide an immunoassay method for the measurement of a 5-HTOL compound in samples containing free 5-HTOL together with its conjugates without prior hydrolysis of endogenous 5-HTOL conjugates.
An second objective is to provide an antibody allowing for an immunoassay of a 5-HTOL compound.
A third objective is to provide derivatives of T-5-G that may be used as immunogens or as 5-HTOL analogues mimicking the structure of corresponding native 5-HTOL compound in various assays, e.g. competitive immunoassays for 5-HTOL compounds.
The Discovery Behind the Invention
The invention is based on our discovery that one can obtain antibodies that bind to a 5-HTOL compound by raising a humoral immune response in the appropriate animals using an immunogenic form of the &bgr;-glucuronide of 5-HTOL and D-glucopyranosiduronic acid (i.e. an immunogenic form of 3-(2-hydroxyethyl)indole-5-O-&bgr;-D-glucopyranosiduronic acid) as the immunogen.
The Inventive Antibody Preparation
The first aspect is thus an antibody specific against a 5-HTOL compound. By the term “specific” is meant that the antibody during immunoassay conditions preferentially will bind to a 5-HTOL compound with no disturbing binding activity against other endogenous substances in human samples, for instance serotonin (5-HT), 5-HIAA or other structurally related substances, such as other indoles or glucuronides. In the presently preferred mode the inventive antibody preparation preferentially binds to T-5-G compared to other endogenous 5-HTOL compounds. The antibody preparation has an acceptable low binding activity to potential disturbing substances, such as other indoles and glucuronides, that may be present in body fluids. See the experimental part. Since the levels of free 5-HTOL, T-5-G and sulphated 5-HTOL all are elevated as a consequence of recent alcohol intake, useful antibodies may react with one, two or all of these 5-HTOL compounds.
The term antibody encompasses various antibody preparations having the above-mentioned specificity, such as the intact antibody and various active fragments (antigen/hapten binding fragments), such as Fab, Fab′, Fv, F(ab′)
2
etc. It also covers derivatized antibodies, such as antibodies to which labels have been attached covalently, such as biotin, hapten, enzymes, enzyme substrates, cofactors, fluorophors, chemiluminescers, chromophors, radioactive isotopes, metals, particles etc, and carrier molecules, such as insoluble and soluble polymers.
The inventive antibody may be a polyclonal or monoclonal preparation. It may contain a mixture of a definite number of monoclonals. It can be obtained by commonly known methods, except that our novel T-5-G derivatives (conjugates) should be used, for instance as an immunogen to raise polyclonal antibodies or as an antigen for the screening of immune response or of cell lines (e.g. hybridomas) secreting the inventive antibody or various antibody libraries from which each individual antibody component can be isolated together with the corresponding coding sequence. Thus the techniques contemplated do also encompass antibodies obtained via selection by phage display techniques. Once obtained, the inventive antibody preparation may be modified by recombinant techniques.
The best mode at the priority date for obtaining the inventive antibody preparation is given in the experimental section and utilizes as the immunogen the conjugate between keyhole limpet hemocyanine (KLH) with T-5-G as defined in patent example 3 (reaction scheme 2) and selection of appropriate hybridoma clones as described in the experimental section. The best mode inventive antibody thus preferentially binds to T-5-G among various 5-HTOL compounds present in native samples.
The Inventive 5-HTOL Analogues
The T-5-G (i.e. 3-(2-hydroxyethyl)indole-5-O-&bgr;-D-glucopyranosiduronic acid) and its derivatives are novel in the sense that they never have been isolated before. Thus the broadest concept of this aspect of the invention is a 5-HTOL compound characterized in exhibiting the structure of 3-(2-hydroxyethyl)indole-5-O-&bgr;-D-glucopyranosiduronic acid, optionally derivatized at one or more OH in the carbohydrate part, i.e. at an alcoholic hydroxy group (positions 2, 3 and 4) or at the carboxy group (position 6) or at the 3-hydroxyethyl hydroxyl or at the indole nitrogen.
Derivatization at an alcoholic hydroxy group may be accomplished as normally is done for glucuronide derivatives, e.g. selective alkylation or acylation at positions 2, 3 and/or 4, either before or after
Akerblom Eva
Beck Olof
Borg Stefan
Brandt Ragnhild
Helander Anders
Alco Dia A.B.
Chin Christopher L.
Cook Lisa V
Liniak Berenato & White
Orzechowski Karen Lee
LandOfFree
5-hydroxytryptophol (5-HTOL) derivatives, antibodies,... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with 5-hydroxytryptophol (5-HTOL) derivatives, antibodies,..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and 5-hydroxytryptophol (5-HTOL) derivatives, antibodies,... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3085775