Downloads. Package insert Spanish Lot: – Expiry: 05/ Ed. 8; Package insert French Lot: – Expiry: 05/ Ed. 8; Package insert . Summary [1, 2]. Hemoglobin A1c (HbA1c) is a glycated hemoglobin which is formed by the non-enzymatic reaction of glucose with native hemoglobin. HbA1c FS*. Diagnostic reagent for quantitative in vitro determination of hemoglobin A1c (HbA1c) in whole blood on. DiaSys respons® Order Information.
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The stabiliser used according to the invention can be a single chemical compound of the above-indicated kind a compound which is covered by one of formulae III or III or a mixture of two or more chemical compounds of the above-indicated kind. In these embodiments selectively one of the reagent solutions also contains an SH group-trapping agent like for example NEM, wherein the SH group-trapping agent is contained in one of the reagent solutions in which the leuco dye is not contained.
The peroxidase already introduced into the composition by way of the first reagent solution R1in the presence of the resulting hydrogen peroxide, causes oxidation of the leuco dye towards its coloured oxidation form. The addition of the SH group-trapping agent should accordingly occur at latest immediately prior to carrying out the HbA1c detection reaction. The man skilled in the art will therefore readily understand that all other embodiments which have the features or combinations of features according to the invention as recited in the claims also fall within the scope of protection of the invention.
The method according to claim 1, wherein the at least one stabiliser is a phosphatidylcholine of the general formula I wherein R 1 and R 2 are selected from completely unsaturated or singly or multiply unsaturated straight-chain or branched-chain fatty acid residues of a chain length in the range of C8 to C That is achieved by suitable buffering of the haemolysis solution.
In that respect the inventors of the present application have shown that SH groups which cause disturbance in the above-mentioned detection reaction must be blocked as much as possible. Besides the above-described method the present invention also proposes reagent kits for use in a method of determining the amount of HbA1c in a sample, which are characterised in that they comprise at least two different solutions in separate containers, wherein the at least two different solutions are respectively of such a composition that the various aspects of the invention described in detail hereinafter are implemented.
Besides stabilisation of the protease the inventors of the present invention also set themselves the object of being able to unfold the haemoglobin contained in a sample, including HbA1c, as greatly as possible, and stabilise it in that unfolded form in order for example to permit digestion of the utmost efficiency of the haemoglobin by a protease and to put the haemoglobin into a measurable photometrically stable form.
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AmE in that respect corresponds to the difference in respect of Calibrator Level 2 in relation to Calibrator Level 1 in mE. The inventors of the present invention however found a way in which, with a very low pH-value, extremely rapid and efficient unfolding and subsequent stabilisation of the unfolded haemoglobin can be achieved.
Examples of haemolytically acting means or methods which hba1cc known to the man skilled in the art are ultrasound treatment or the addition of haemolytic detergents or strongly hypotonic salt solutions. In certain embodiments of the invention the pH-value is in the range of 2 to 3. The fructosyl amino acid or fructosyl peptide is oxidised by the activity of the enzyme fructosyl amino acid oxidase FAOX or the enzyme fructosyl peptide oxidase FPOXwherein a result of that oxidation step is the production of hydrogen peroxide H 2 O 2.
The combination of a substance in accordance with formula IV with a further thio compound from the group of thioketones, thioethers or thioalcohols is particularly suitable as optimum stabilisation of the leuco dye over a long period of time up to 24 months can be ensured when using the respectively individual redox potentials and the different stability and reactivity of the specified thio compounds in aqueous solutions.
For that purpose various combinations of concentrations of calcium ions and EDTA were diays to a reagent base matrix 2, with a respective equal amount of the example protease thermolysin.
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In addition there is proposed a reagent kit for use in a method of determining the HbA1c in a sample, which hba1 characterised in that the reagent kit includes at least two different solutions in separate containers, wherein the one solution has a pH-value in the range of 1 to 8 and contains the above-mentioned components i to iiiwherein the molar ratio of chelator: Ca 2 and chelator: In another embodiment the solution contains 0.
The reagent kit according to claim 11, wherein the solution R2 for hbba1c the leuco dye contains at least one thio compound, wherein the at least one thio compound is preferably selected from thiodiglycol, thiomalic acid, thionicotinamide, thio-NAD and mixtures thereof. Those proteolytically produced degradation products include the fructosyl valine Fru-Val and fructosyl valine histidine Fru-Val-His or even longer-chain functosyl peptides, which are cleaved from the amino-terminal end of the beta chain of glycated haemoglobin.
In a specific embodiment of the invention the pH-value of the solution is in the range of 4. In a special embodiment of the present invention the reagent kit comprises three reagent solutions having the following constituents: In an embodiment of the present invention the various reagents used in HbA1c determination are brought together in the form of the following solutions which are provided in separate containers: A comprehensive explicit representation of all conceivable combinations of features is dispensed with here only for the sake of brevity and readability of the description.
A reagent kit for use in a method of determining the amount of glycated haemoglobin HbA1c in a sample, wherein eiasys reagent kit includes at least the following solutions in separate containers: In accordance with the invention the above-described hba11c is attained by various aspects which are described in detail hereinafter and which usually have the common denominator that they comprise a method of determining the amount of HbA1c in a sample, in which the following method steps are performed:.
In the above-described method of determining the amount of HbA1c in a sample reagents are frequently used, which contain SH groups like for example the above-mentioned thio compounds.
These various haemolysis solution variations with and without additive were measured with respectively identical reagent 1 and reagent 2 on a clinical-chemical analyser Hitachi reagent compositions were used as specified in 1b correspondingly modified as described in the Tables.
In certain embodiments the haemolytically acting detergents used are selected for diazys from the following: Hga1c measurement intervals however are very heavily dependent on the respectively employed analyser, photometer and so forth. Accordingly in certain embodiments of the invention the leuco dye is produced in a solution which for stabilisation of diassy dye contains at least one compound of the general formula I like for example TCEP and at least one thio compound like for example thiodiglycol.
Insofar as is desired, the method according to diays invention can also be carried out in such a way that the total haemoglobin concentration is determined during or between method steps a -c. Examples of chelators for divalent metal ions, that are suitable in accordance with the present invention, are acetylacetone, nitrilotriacetic acid NTAethylenediamine, ethylenediamine tetraacetate EDTAN- 2-hydroxyethyl -ethylenediamine-N.
Now a respective different stabilizer in respectively identical concentrations was added to the specified reagent base matrix without stabiliser. The haemoglobin which is unfolded in accordance with the present invention therefore remains in solution in contrast to denatured haemoglobin.
The inventors of the present application found that this specific amount of the respective divalent metal ions is sufficient to reoccupy the sites in the protein structure of the protease, diays are required for activation of the protease, in spite of the simultaneously present significant amounts of chelator and calcium or magnesium ions.
The method according to claim 7, wherein the solution containing the leuco dye for stabilisation of the dye contains at least one thio compound. The method hba1d to claim 1, wherein an operation for determining the total haemoglobin concentration is carried out during or between method steps a -c.
However, as diqsys already mentioned hereinbefore, there is the risk that the haemoglobin is denatured, agglutinated and precipates, as can also be effected for example by adding trichloroacetic acid. In an diazys embodiment the SH group-trapping agent, for example NEM, is contained in a reagent solution which is kept separately from the reagent solution and is added, in which there is a leuco dye stabilised with thioalcohols.
In many embodiments of the invention however a protease is specifically used, whose proteolytic activity leads to the release of fructosyl valine histidine or fructosyl valine from the amino-terminal end uba1c the beta chain of glycated haemoglobin. In an embodiment of the invention described in the present application there is provided a reagent kit which includes at least the djasys three solutions in separate containers: Stabilisation of the protease, shown in FIG. That is a bar to the use of the protease for liquidly stable reagents with a uniform quality requirement over prolonged periods of time.
The method according to claim 8, wherein the at least one thio compound is selected from thiodiglycol, thiomalic acid, thionicontinomide, thio-NAD and mixtures thereof. Hbac1 increase dlasys nm extinction in relation to time is in that case a measurement in respect of conversion of the leuco form fiasys the dye into its coloured variant and hbac a measurement in respect of oxidative destabilisation of the leuco form.
A further object that the inventors of the present application set themselves is stabilisation of the leuco dye in the methods of determining the amount of HbA1c in a diasye, in which such a leuco dye is used.
A plurality of thio compounds were checked in respect of their dye-stabilising action upon use in combination with TCEP.
At the same time however it is also of not immaterial importance for the stability of the starting material which is to be to be investigated as well as that of the diassy and end products of the reaction to be ensured to achieve reliably reproducible results.
Nerolidol, Terpene, and Terpene Deriviative Synthesis. More specifically one of the objects of the present invention is also that of combining together as many as possible of the reagents used in the HbA1c determination operation into as few as possible combined reagent solutions.
The inventors of the present application found that effective stabilisation of leuco dyes can be achieved by the addition of compounds of the general formula I without the actual detection reaction in the present case detection of hydrogen peroxide in the presence of the enzyme peroxidase being adversely effected or damaged thereby.
The other metalloprotease are manganese- or cobalt-dependent. The cleaved glycated peptide is then reacted by the FPOX, wherein hydrogen peroxide is produced upon cleaving of the glycated peptide into peptide and diasyz.
Preferably the zwitterionic detergent is selected from at least one chemical compound which is covered by the following general formula III.