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 present invention concerns a method of determining the amount of glycated haemoglobin HbA1c in a sample and a reagent kit which can be used in a method of determining the amount of glycated haemoglobin HbA1c in a sample. An enzymatic method has also been available for some time, in which a reaction of glycated haemoglobin with a diasts amino acid oxidoreductase FAOD is quantified.

In the enzymatic investigation—as moreover is also the xiasys with all other HbA1c methods—the first step is to haemolytically rupture the erythrocytes in the djasys sample to release the HbA1c contained therein. The released glycated haemoglobin is then brought into contact with a proteolytic agent to produce glycated haemoglobin degradation products. 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.

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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 hva1c oxidation step is the production of hydrogen peroxide H 2 O 2.

The amount of hydrogen peroxide produced in the above-mentioned oxidation step correlates with the amount of fructosylated amino acid or peptide. Accordingly the amount of hydrogen peroxide produced in this step is a measurement in respect of uba1c amount of HbA1c in the sample.

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.

In principle however it is also possible to correspondingly use any other analysis method for quantifying diasyz amount of hydrogen peroxide in a sample.

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In a given method of quantifying hydrogen peroxide, a reduced leuco dye is oxidised with hydrogen peroxide. That method however entails the difficulty that autooxidation of the leuco dye causes a non-specific blank value signal and an increase in the spectral background which causes difficulty in precise photometric measurement of the analyte signal. On the other hand leuco dyes have the advantage over other dyes that they usually have higher molecular absorption coefficients.

In addition for the major part leuco dyes have such high absorption maxima that optical influencing by interaction with constituents of the blood like for example bilirubin and haemoglobin can generally be disregarded. It will be appreciated that this also uba1c to the same diaxys as for the above-discussed leuco dyes, for the enzymes and other reagents used in HbA1c tests.

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 intermediate diasyd end products of the reaction to be ensured to achieve reliably reproducible results. Therefore the inventors of the present application set themselves the object of providing a method of determining the amount of glycated haemoglobin HbA1c in a sample and reagents which can be used in that respect, in which the chemical compounds essential to the reaction are present in sufficiently stable form.

Preferably the aim of the method according to the invention and the reagents according to the invention is to permit total haemoglobin determination at the same time as determining the amount of HbA1c. 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:.

Diasya most cases the sample will be a sample of diasts whole blood. The present invention however also embraces such samples diazys for example blood preserves, purified blood, whole blood lyophilisate, erythrocytes concentrate, pre-haemolysed blood samples, haemoglobin standard solutions, HbA1c standard solutions and standard dkasys which contain synthetic hab1c degradation products like for example synthetic HbA1c degradation products. Insofar as the HbA1c is already diayss free in the sample to be analysed method step a is not required.

In the case of standard solutions which contain synthetic haemoglobin or HbA1c degradation products method step b is in addition also not required. Insofar as viasys 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. Haemolysis of the erythrocytes can basically be effected with all mechanical, chemical or osmotic haemolysis means or methods, of which the man skilled in the art knows that they lead to complete haemolysis of the erythrocytes.

One means or method in accordance with the present invention is deemed to be haemolytically acting when it leads to dissolution of the erythrocytes by destruction of the cell membrane and transfer of the haemoglobin contained in the erythrocytes into the ambient medium. Examples hab1c haemolytically acting means or methods which are known to the man skilled in the art are ultrasound treatment or the addition of haemolytic detergents or strongly hypotonic salt solutions.

In the embodiments of the invention in which haemolytically acting detergents are used they can be selected from non-ionic, anionic, cationic and zwitterionic detergents, wherein the term detergent is used here to mean that this embraces substances which reduce the surface tension of a liquid or the interfacial tension between two phases.

Detergents are organic compounds which are made up of a nonpolar and a polar part, wherein the nonpolar part is at least an alkyl group or an alkylbenzene group and the polar diaasys is selected diasyys least hba11c an alcohol, ether, alcohol-ether, carboxyl, sulphonyl, sulphatyl or quaternary ammonium group.

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In certain embodiments the haemolytically acting detergents used are selected for example from the following: In the embodiments in which haemolytically acting detergents are used they are preferably stored and used in the form of a haemolysis solution. In principle all proteolytically acting means known to the man skilled in the art fall to be considered like for example proteases, wherein a means in accordance with the present invention has a proteolytic effect when it leads to cleaving of proteins by xiasys of the peptide bonds.

In the embodiments in which the proteolytic agent is a protease, it in principle can be selected from all proteases obtained recombinantly from eukaryotes or prokaryotes or obtained endogenously from organisms or organism constituents from serine, threonine, cysteine, asparagine, metal or unknown type, like for diqsys acrosin, aminopeptidase B, bromelain, calpain I, carboxypeptidase A, cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin K, chymotrypsin, collagenase, dipeptidyl peptidase 4, dispase, elastase, factor IIa, factor Xa, ficin, gpr-endopeptidase, HIV-protease, kallikrein, MBTPS1, bromelain, papain, pepsin, plasmin, prepilin type IV peptidase, prolyl-oligopeptidase, proteinase K, proteasom, renin, seccretases alpha- beta- and gamma-secretasethermolysin EC 3,4,24,27thrombin, trypsin, urokinase, protease N from Bacillus sp.

In certain embodiments of the invention there can be a related advantage in using proteases which specifically cleave HbA1c.

In general the present invention however does not require any specificity of the protease in regard to differentiation between glycated and non-glycated haemoglobin. In that respect therefore it is also possible to use such proteases which do not specifically distinguish between HbA1c and non-glycated haemoglobin. In many embodiments it may even be explicitly desired for a protease to be used, which does not act specifically in a corresponding fashion.

In many aspects of the present invention there is no need for the protease used to lead to given degradation products. 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. Determining the amount of HbA1c can basically be effected by all quantification procedures, known to the man skilled in the hba1x, for the glycated haemoglobin degradation products produced in method step blike for example riasys an HPLC dlasys or enzymatic determination, as was diaxys hereinbefore.

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 jba1c 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.

In accordance with a first aspect diasye above-described object of the invention is attained in that there is proposed a method of determining the hbx1c of HbA1c in a sample, in which—insofar as required—method steps a to c are performed. In accordance with the present aspect of the invention the proteolytically acting agent used in method step b is activated in that at least two different solutions are brought into contact with the HbA1c, wherein the one solution has a pH-value in the range of 1 hb1ac 8, and contains.

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: In a specific embodiment of the invention the pH-value of the solution is in the range of 4.

At the present time 54 metalloprotease families are divided into 15 clans, wherein outstanding significance is attributed to the clan Diiasys with 39 families. Just 19 of those 39 families can be associated with the so-called neutral zinc metalloprotease. The other metalloprotease are manganese- or cobalt-dependent. Inter alia the following metalloproteases belong to the clan MA: If proteases are in their active form over a prolonged period there is the risk that significant proportions yba1c the enzyme are destroyed by self-digestion.

HbA1c Calculator – HbA1c

In that respect temporary inactivation of the enzymatic activity of proteases is frequently wanted if self-digestion of the enzyme is to be avoided.

The problem of self-digestion of proteases is already known per se in the state of the art. To resolve that problem the state of the art proposed for example for the protease thermolysin removing zinc from the theremolysin by chelators, in particular SH group-containing reagents, or by a simple excess of EDTA.

As calcium or magnesium ions are of essential significance for the stability of the protein structure of the protease the use of an excess of chelator consequently causes destabilisation of the protease protein. 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.

The chelator concentration can be freely selected within the above-specified range in dependence on the amount of protease. In an embodiment the solution contains 0. In another embodiment the solution contains 0. The amount of calcium or magnesium ions can also be freely selected in the above-mentioned ranges of concentration. The metal ions can be used in the reagent solution employed in any suitable salt form as long as the selected salt form affords the required amount of dissolved metal ion in the batch like for example chlorides, nitrates, sulphates, formiates and acetates.

The molar ratio of chelator: In certain embodiments of the present invention the molar ratio of chelator: In many embodiments the ratio of chelator: In other embodiments the ratio is inn the range of 1: 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, that are required for activation of the protease, in spite of the simultaneously present significant amounts of chelator and calcium or magnesium ions.

The metal ion content of the activation solution can be freely selected in the specified range depending on the respective embodiment involved. 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. 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: Ca 2 and chelator: The above-mentioned reagent kit can be used in a method of HbA1c determination in the following manner.


Firstly—insofar as is required—sample preparation is effected, in which whole blood is mixed with the haemolysing solution. The first reagent solution R1 is then added to the haemolysate resulting therefrom, the result of this being that the FPOX contained in the first reagent solution already breaks down endogenous fructosyle peptides possibly present, but not the terminal fructosylated peptides which are relevant to HbA1c determination as they are not yet released.

After the reaction is substantially concluded photometric determination of the total haemoglobin content of the pre-incubated haemolysate is effected. Actual incubation is then effected, in which the second reagent solution R2 is added to the pre-incubated haemolysate. The protease apo enzyme contained therein for example thermolysin apo enzyme is activated by the additional divalent metal ions for example zinc ions already contained in the composition by way of the haemolysis solution and cleaves inter alia N-terminal glycated peptide from the beta chain of the haemoglobin.

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. 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 actual HbA1c determination operation can then be effected by photometric measurement for example very quickly for example 10 to 30 seconds, depending on the nature of the measuring instrument after addition of the second reagent solution R2that is to say immediately before the FPOX-induced reaction occurs, and once again at a later time for example 2 to 15 minutes, depending on the respective nature of the measuring instrument after the addition of R2, that is to say after conclusion of the hydrogen peroxide-induced oxidation of the leuco dye.

Ultimately, determination of the HbA1c content is effected in consideration of the difference between the two measurements and by formation of the quotient from the contents of HbA1c and the previously determined total haemoglobin. The measurement intervals however are very heavily dependent on the respectively employed analyser, photometer and so forth.

Accordingly only one direct measurement after 2 to 15 minutes would also be possible. 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.

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 the conventional methods of determining the amount of HbA1c unfolding is effected at a pH-value of about 5. A greater reduction in the HbA1c value however has the disadvantage that the haemoglobin treated in that way is severely denatured and agglutinated and precipitates in that form so that it is no longer available in a suitable form for digestion with a protease.

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. In the embodiments with multiply unsaturated fatty acid residues they are preferably doubly, trebly or quadruply unsaturated and in certain embodiments independently of the degree of saturation the fatty acid residues are selected from those with a chain length in the range of C8 to C22 or those with a chain length in the range of C16 to C22 for example 1, 2-dioleoyl-sn-glycerophosphocholine.

In the embodiments which are present in the form of copolymers they include, besides the units with the above-specified general formula IIthey include further methacrylate units which are esterified with aliphatic or aromatic residues. Preferably the zwitterionic detergent has exactly two functional groups of opposite charges so that the molecule overall is electrically neutral.

Preferably the zwitterionic detergent is selected from at least one chemical compound which is covered by the following general formula III. Preferably R is selected from an alkyl residue of a chain length in the range of C8 to C 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.

Preferably the stabiliser is used with a concentration in the range of 0. In the cases in which the stabilizer includes a zwitterionic detergent which has a haemolytic action or comprises one or more haemolytically acting zwitterionic detergents various variants are possible: After the addition of the haemolysis solution the pH-value of the haemolysate corresponds to that of the haemolysis solution.

That is achieved by suitable buffering of the haemolysis solution. The constantly low pH-value of the haemolysate in conjunction with the stabilising detergents has the result that the unfolded haemoglobin can be quickly and efficiently broken down.

The reduction of the pH-value into the range of 1 to 3 leads to very rapid and strong unfolding of the haemoglobin. However, as was 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.