Chemistry: analytical and immunological testing – Including sample preparation – Stabilizing or preserving
Patent
1993-10-01
1996-07-30
Chan, Christina Y.
Chemistry: analytical and immunological testing
Including sample preparation
Stabilizing or preserving
435 71, 435 24, 435963, 436518, 436536, 436 8, 436826, G01N 128, G01N 3368, G01N 33543
Patent
active
055411169
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a method for the stabilisation of endogenous, physiologically active peptides in human whole blood, serum or plasma samples before and/or during the determination of the concentration of these peptides by immunodiagnostic or physical assay methods.
Peptides have important biological functions as hormones and biomodulators. Owing to their high biological activity, their half-life in the body is very limited, and the various physiologically active peptides have different deactivation and degradation mechanisms corresponding to the biological function of these peptides. A review of a number of physiologically important peptides or peptide hormones and their deactivation in the human body is to be found, for example, in the article by Hugh P. J. Bennett and Colin McMartin in Pharmacological Reviews, Vol. 30, No. 3, pages 247 to 292. This article states that the half-life of peptides in the blood is influenced by many different factors, including the absorption or binding by the tissue, degradation by specific tissue regions or organs and degradation or conversion in the blood or plasma.
If it is intended to determine the concentration of such short-lived peptides in the blood of experimental animals or in particular of human patients, this short life must be borne in mind and if necessary countermeasures taken.
To determine the concentration of biological molecules, such as peptides, in the blood, it is first necessary to take a blood sample, from which a serum sample or plasma sample is then obtained in a known manner and is then used in the assay method for determining the concentration of the particular biological molecule. Although the concentration of various biological molecules may also be measured by means of physical methods, such as chromatographic methods, spectroscopic methods or separation, such as dialysis or ultrafiltration, biological molecules in the blood are as a rule measured by means of various immunodiagnostic methods, owing to the small amounts in which they occur and owing to the high accuracy of determination required in clinical investigations. In these methods, the serum sample or plasma sample, together with the further substances required for the particular immunodiagnostic method (labelled or unlabelled, suspended or immobilised antibodies, labelled tracer molecules, buffer) are incubated with one another for a certain time.
Usually, a considerable .time elapses between taking of the sample and obtaining serum or plasma samples and the actual measurement of biological molecules. In the case of sensitive endogenous peptides, however, endogenous biological degradation of the peptides in the sample may occur in the period between taking of the sample or sample preparation and the actual determination, and the endogenous biological degradation of the peptides may continue even during the period of incubation of the sample during the assay procedure, so that, depending on the degree of degradation, the assay method measures not the required physiological peptide concentration which was present at the time of blood withdrawal but another peptide concentration whose magnitude is dependent on the method and duration of storage, the serum plasma isolation and the incubation. If only the complete, undegraded peptide is recognised in the assay method, values which are too low are therefore obtained. There are, however, immunodiagnostic assay methods in which possible degradation products of a certain peptide are better recognised than the peptide itself, so that, in the case of degradation of the required peptide, concentrations which are higher than the actual concentrations may be measured.
However, incorrect measurements may lead to incorrect clinical interpretations of the measurements.
In order to counteract the danger of the degradation of endogenous peptides, which has been known in principle for a long time, attempts are known to have been made to prevent such degradation by the presence of EDTA or of individual proteolysis inhibitors. It is also known that t
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B.R.A.H.M.S. Diagnostica GmbH
Chan Christina Y.
Grun James L.
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