Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-11-02
1997-12-16
Knode, Marian C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 792, 436518, 544258, 530405, G01N 33535, G01N 33547, C07D47504, C07K 200
Patent
active
056984081
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/EP94/00632 filed Mar. 3, 1994, published as WO94/21636 Sep. 29, 1994.
Neopterin and dihydroneopterin are predominantly produced and secreted by monocytes/macrophages of primates (and to a lesser degree by lower mammals) after activation by interferons and lipopolysaccharides. Increased neopterin levels can be determined in body fluids of patients with diseases which involve activation of the cellular immune system.
The determination of neopterin levels has proven to be diagnostically useful, e.g. prognostically for transplant rejection and HIV infection or in screening for otherwise unrecognized infectious diseases of blood donors in order to increase the safety of blood transfusions. The measurement of neopterin may be accomplished by various methods, e.g. by high pressure liquid chromatography (HPLC) with fluorescence detection, radioimmunoassay, thin layer chromatography, etc. Dihydroneopterin can be determined as neopterin after an oxidation step (with iodine or manganese dioxide) at low pH using known procedures or alternatively as dihydroneopterin by HPLC with electrochemical detection.
Tetrahydrobiopterin (BH.sub.4) is the cofactor of essential enzymes, e.g. the aromatic amino acid hydroxylases (synthesis of the neurotransmitters dopamine and hydroxytryptamine) and the nitric oxide synthetases. Therefore, its determination could be valuable. Biopterin itself is not present in body fluids; however, it is quantitatively obtained from BH.sub.4 by acidic oxidation and can be determined by various methods, like neopterin above.
Non-radioactive immunoassays have also been described in the literature, however hitherto no enzyme immunoassay for the determination of neopterin in serum which is easy to perform (in comparison to HPLC) and which provides correct results has been introduced into the market.
A radioimmunoassay for the determination of pterins has been described in DE-A-30 25 226, wherein the spacer group between the amino group in position 2 and the label group is a straight-chain or branched alkylene group having from 1 to 6 carbon atoms. These immunoassays are not sufficiently sensitive to be suitable for performing routine determinations of neopterin or biopterin with reliable results.
The immunoassays according to EP-A-0 370 960 comprising a hexanoic acid group as the spacer group, also lead to unsatisfactory results.
Thus, it is the object of the present invention to provide substances in order to prepare substantially more reliable immunoassays, esp. non-radioactive assays to measure neopterin levels in body fluids. Extensive investigations resulted in the unexpected finding that such immunoassays can be developed, if the spacer group between the pterin ring and the label group is a .omega.-aminobutyl-carboxamidopropyl group linked through the propyl group to the amino group at C.sup.2 ("N.sup.2 ") or to the N.sup.3 of neopterin or biopterin.
Accordingly, under a first aspect the present invention relates to pterin derivatives having the general formula I Neopterin and Biopterin have the formula ##STR2## The nomenclature used herein has the following meanings: N.sup.3 : nitrogen at position 3 of the pterin ring ##STR3## wherein R.sub.l represents a group --(CHOH).sub.2 --CH.sub.3 or --(CHOH).sub.2 --CH.sub.2 OH, group --(CH.sub.2).sub.3 CONH(CH.sub.2).sub.4 --NH--R.sub.4, wherein R.sub.4 represents hydrogen or a usual label group for immunoassays or usual coating/support materials for solid phase immunoassays or immunogenic groups for the preparation of antibodies.
Examples of the "usual label groups" are radioactive labels, enzyme labels, fluorescence labels or chemiluminescence labels.
Examples of "support materials" for solid phase immunoassays are microtiter plates, polystyrene tubes or beads, to which the "usual coating materials" are bound covalently or by absorption. Examples of the "usual coating materials" are proteins, such as serum albumins, transferrin, immunoglobulins, or synthetic polypeptides, e.g. poly(phenylalanine-lysine), etc. However, com
REFERENCES:
Rautenberg et al, "New Competitive Enzyme Immunoassay for the Determination of Neopterin in Human Serum" (in German), Klin. Lab. 39, 503-510(1993).
Sugimoto et al., "An Enzyme Immunoassay for Neopterin and Biopterin Using a Microtitre Plate Linked with N2-(3-Aminopropyl) Derivatives of Pteridines", Pteridines 2, 141-146 (1990).
B.R.A.H.M.S. Diagnostica GmbH
Knode Marian C.
Wortman Donna C.
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