Chemistry: molecular biology and microbiology – Maintaining blood or sperm in a physiologically active state...
Patent
1996-07-26
1998-08-04
Achutamurthy, Ponnathapura
Chemistry: molecular biology and microbiology
Maintaining blood or sperm in a physiologically active state...
435 4, 435 71, 435962, C12Q 100, C12Q 137, G01N 3348, G01N 3353
Patent
active
057891494
DESCRIPTION:
BRIEF SUMMARY
This application claims benefit of international application PCT/EP94/03962, filed Nov. 29, 1994.
The invention relates to a method for the determination of osteocalcin in human serum or plasma and instruments and sample vessels for carrying out this method. The method according to the invention may also be referred to as a method for stabilization of endogenous osteocalcin in human serum or plasma samples, since it is based on prevention of the degradation of the endogenous osteocalcin which is normally to be observed during the time between taking of the sample and measurement of the osteocalcin.
Human osteocalcin, which is also referred to as vitamin K-dependent bone protein (BGP)!, is a specific peptide component of the bone matrix. The peptide consists of 49 amino acids and has the following amino acid sequence: Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala-Pro-Val-Pro-Try-Pro-Asp-Pro-Leu-Glu-Pro-Ar g-Arg-Gla-Val-Cvs-Gla-Leu-Asn-Pro-Asp-Cys-Asp-Glu-Leu-Ala-Asp-Arg-Ile-Gly-P he-Gln-Glu-Ala-Tyr-Arg-Arg-Phe-Tyr-Gly-Pro-Val.sup.49
The peptide is synthesised by the osteoblasts and a small part of the osteocalcin formed enters the blood. Certain valuable conclusions about the metabolism of the bone are possible through the determination of the concentration of this osteocalcin in the blood, and, for additional information, reference may be made to the article by Lian, J. B. and Gundberg, C. M. in: Clinical Orthopaedics and Related Research, 226, pages 267 to 291 (1988). According to this, the determination of the osteocalcin concentration in human serum or plasma may provide useful information for the diagnosis and therapy of metabolic disorders of the bone.
Various methods have been developed for measuring the osteocalcin concentration. Thus, according to methods known per se, the determination of the osteocalcin concentration is possible by an immunodiagnostic route, for example by the method according to U.S. Pat. No. 4,438,208 and the improved embodiment of such a method, described in German Patent 3,833,936 of the Applicant.
EP 0 557 663 A1 relates to an immunodiagnostic assay method wherein a very special monoclonal antibody for osteocalcin is used, which antibody recognizes certain conformations which are typical for the so-called non-carboxylated or under-carboxylated osteocalcin. Non-carboxylated or under-carboxylated osteocalcin is disguished over carboxylated osteocalcin by the fact that the initial GLU residues in the positions 17, 21 and 24 of the osteocalcin molecule are not or not completely carboxylated to form GLA residues. The different degree of carboxylation causes differences in the conformations of the two forms.
In order to be able to measure with a "conformational" antibody as used in the assay also the total amount of osteocalcin, i.e. to measure also the carboxylated osteocalcin present, a calcium salt is added to the sample, because it was found that in the presence of a calcium salt the conformation of the carboxylated osteocalcin changes so that the specific antibody recognizes also the carboxylated osteocalcin and binds to it for measurement purposes exactly as to the non-carboxylated or under-carboxylated osteocalcin. EP 0 557 663 A1 does not address the problem of stabilising osteocalcin during the period between the taking of the sample and the later immunodiagnostic determination.
Regardless of the method by which the osteocalcin concentration is determined, it is essential for a reliable and clinically relevant determination of the osteocalcin in the sample that the osteocalcin concentration measured reflects the concentration at the time of taking of the blood. Concentration changes in the period between taking of the blood and actual concentration measurement lead to useless results being obtained.
It is already known that endogenous osteocalcin in blood samples undergoes degradation (cf. Lian and Gundberg (1988) loc. cit and Coleman et al. (1988) Eur. J. Cancer Clin. Oncol. 24, 1211-1217). As a result of this, "falsely low" osteocalcin concentrations in the serum or plasma are found,
REFERENCES:
patent: 4410506 (1983-10-01), Price et al.
patent: 4438208 (1984-03-01), Deftos et al.
patent: 5168041 (1992-12-01), Bergmann
Tracy, et al: "Comparison of Monoclonal and Polyclonal Antibody-Based Immunoassays for Osterocalcin: A Study of Sources of Variation in Assay Results" Journal of Bone and Mineral Research, vol. 5, No. 5, 1990, pp. 451-461.
Coleman, et al: Osteocalcin: a Potential Marker of Metastatic Bone Disease and Response to Treatment, European Journal of Cancer & Clinical Oncology, vol. 24, No. 7, pp. 1211-1217, Jul. 1988.
Diaz Diego, et al: "Six Osteocalcin Assays Compared", Clinical Chemistry, vol. 40, No. 11, 1994, pp. 2071-2077.
Banfi, et al: "In Vitro stability of Osteocalcin", Clinical Chemistry, vol. 40, No. 5, 1994, pp. 833-834.
Bergmann Andreas
Weckermann Renate
Achutamurthy Ponnathapura
B.R.A.H.M.S. Diagnostica GmbH
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