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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Insofar as the haemoglobin or the HbA1c inherently contained therein or degradation products thereof are not already present in solution in the sample to be analysed they are put into a usually aqueous solution prior to or during step a.
In addition there is proposed a reagent kit for use in a method of determining the HbA1c in a sample, which is 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: The above-mentioned reagent kit can be used in a method of HbA1c determination in the following manner.
Alternatively for stabilising the leuco dye it is also possible to add thio compounds, more especially single ones or a plurality of thioalcohols, thioethers, thioketones or mixtures thereof.
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 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 of the beta chain of glycated haemoglobin.
The chelator concentration can be freely selected within the above-specified range in dependence on the amount of protease.
In certain embodiments of the present invention the molar ratio of chelator: In a special embodiment of the present invention the reagent kit comprises three reagent solutions having the following constituents: Stabilisation of the Unfolded Hemoglobin Preceding efficient unfolding of haemoglobin and also stabilisation of that unfolded form are of essential significance for accurate measurement of Hb and HbA1c.
An aliquot without stabiliser substance served in that case as a reference. In most cases the sample will be a sample of fresh whole blood.
Inherent colouring Diiasys at nm in mE separate batch. Stabilisation of the Leuco Dye with Phosphines To improve the stability of the leuco dye in a reagent matrix various water-soluble stable substances were checked in regard to their leuco dye-stabilising action. Preferably the stabiliser is used with a concentration in the range of 0.
The results shown here impressively demonstrate the storage capability of an inactivated but structurally stabilised thermolysin in a liquid reagent matrix over various temperatures and time and the reactivatability thereof.
The cleaved glycated peptide is then reacted by the FPOX, wherein hydrogen peroxide is produced upon cleaving of the glycated peptide into peptide and glucosone. For that purpose various combinations of concentrations of calcium ions and EDTA were added to a reagent base matrix 2, with a respective equal amount of the example protease thermolysin.
Those reagent 2 variations with respectively different haemolysis solution and reagent 1 were measured on a BM c. Stabilisation of the protease, shown in FIG. The measurement intervals however are very heavily dependent on the respectively employed analyser, photometer and so forth.
It is known from the state of the art that haemoglobin is unfolded to a certain degree by the reduction in pH-value in the haemolycate.
In accordance with the invention the above-described object 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:. A method for determining the amount of glycated haemoglobin HbA1cin which—if required—the erythrocytes in a sample are haemolysed, the haemoglobin that is then released—if required—is contacted with a proteolytic agent and the glycated haemoglobin degradation products obtained in this way or otherwise are quantified is disclosed.
The method according to claim 1, wherein quantification of the glycated haemoglobin degradation products is effected in method step c by oxidation thereof by means of fructosyl peptide oxidase or fructosyl amino acid oxidase with the production of hydrogen peroxide and by determining the hbq1c amount of hydrogen peroxide, wherein the amount of hydrogen peroxide is quantified on the basis of the colour reaction of a leuco dye in the presence of a peroxidase, and wherein the leuco dye is produced siasys a solution which for stabilisation of the dye contains a compound of the general formula IV: 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 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.
Precision Made Easy
In that case the signal of HbA1c determination was measured over a period of minutes on the analyser and respectively related to the freshly ascertained HbA1c value. In certain diasjs the concentration is in the range of 10 to 20 mol. In the embodiments in which haemolytically acting detergents are used they are preferably stored and used in the form of a 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.
That is achieved by suitable buffering of the haemolysis solution.
The reagent kit according to claim 11, wherein the solution R2 for stabilising 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.
In vivo, ex vivo and in vitro regenerated cartilage. 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 diwsys, 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 hbw1c protease and to put the haemoglobin into a measurable photometrically stable form.
In a specific embodiment of the invention the pH-value of the solution is in the range of 4. In an aspect of the present invention stabilisation diassy the leuco dye is effected in the method of determining the amount of HbA1c in a sample in that, in the method method steps a to c are performed, and quantification of the glycated haemoglobin degradation products is effected in method step c by oxidation thereof by means of FPOX or FAOX with the production of hydrogen peroxide and by determining the resulting amount of hydrogen peroxide, wherein the amount of hydrogen peroxide is quantified on the diasy of the colour reaction of a hba1d dye in the presence of a peroxidase, and wherein the leuco dye is produced in a solution which for stabilisation of the dye contains a compound of the general formula IV:.
Therefore, determination of the amount of HbA1c can ultimately be effected by quantifying the amount of hydrogen peroxide, for example on the basis of a colour reaction which is to be evaluated photometrically and which stoichiometrically correlates with the amount of hydrogen peroxide. The method according to claim 1, wherein an operation for determining the total haemoglobin concentration is carried out during or between method steps a -c.
At the present time 54 metalloprotease families are divided into 15 clans, wherein outstanding significance is attributed to the clan MA with 39 families. Table 2 B in contrast shows functionality of thermolysin in the presence of zinc ions with different ratios of calcium ions and EDTA. A method of determining the amount of glycated haemoglobin HbA1c in a sample, wherein the following method steps are performed: It is further pointed out that it is self-evident to the man skilled in the art that the embodiments by way of example hereinafter only serve to set forth by way of example the possible embodiments of the present invention, that are set out as examples of the invention.
As was already mentioned in the opening part of dkasys specification the principle of leuco dyes is based on the fact that detection of a substance is effected by way of the leuco dye converting from its colourless leuco starting form into a colour form by virtue of the presence of the substance to be detected.
HbA1c Calculator – HbA1c
A HbA1c calibration signals. Inter alia the following metalloproteases belong to the clan MA: Special Embodiments of the Reagent Kits In an embodiment of the invention described in the present application there is provided a reagent kit which includes at least the following three solutions in separate containers: 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.
An enzymatic method has also been available for some time, in which a reaction of glycated haemoglobin with a fructosyl amino acid oxidoreductase FAOD is quantified.