Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-09-13
2002-06-04
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C422S067000, C435S025000, C435S970000
Reexamination Certificate
active
06399293
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention concerns a method for the determination of the ratio of glycated to non-glycated haemoglobin in a sample. The invention also concerns an analytical test element which is suitable for determining the ratio of glycated to non-glycated haemoglobin in a sample on the basis of the new method according to the invention.
The glycation of haemoglobin and serum proteins is increased in patients with diabetes mellitus. The increase depends on the glucose concentration and the period of incubation of the protein with glucose. In these cases the glycation of serum proteins, including haemoglobin, does not take place enzymatically but rather uncatalysed by chemical reaction of glucose with amino groups of the proteins. Experts assume that the concentration of a certain protein-glucose adduct reflects the glucose concentration over a particular period as well as the turn-over rate of the protein. Glycated haemoglobin is regarded as an indicator of the mean blood glucose concentration during the last two to three months before blood withdrawal and examination. Glycated serum protein reflects blood glucose concentration during a shorter time span. Hence the determination of glycated protein such as glycated haemoglobin (in particular HbA1c) or glycated serum protein is very important for the long-term glycaemic control of diabetes patients. In this connection the ratio of glycated haemoglobin to total haemoglobin in a blood sample is of particular relevance.
The content and determination of haemoglobin A1 (HbA1c) and of the medically equivalent glycated haemoglobin is an important medical criterion for the long-term metabolic control of diabetic patients. In ca. 80% of the glycated haemoglobin the glucose is located on the N-terminal valine of the &bgr; chain (so-called “HbA1c”) whereas the carbohydrate residues of the remaining glycated haemoglobin are located at other sites or other sugars apart from glucose have reacted with haemoglobin.
At least 5 methodologies are at present commonly used to determine glycated haemoglobin in blood. A brief description is given in the following:
1. Ion Exchange Chromatography With a Microcolumn
The ion exchange chromatography with the aid of a micro-column is described for example in J.Clin.Chem.Clin. Biochem., 19, 1097-1101 (1981), Lab.Med., 8, 74-78 (1984) and J.Am.Chem.Soc., 83, 1472-1478 (1961). In this method the haemoglobin of a haemolysed sample is bound to an ion exchanger and fractionally eluted. The labile aldimine form of HbA1 is degraded by an eliminator in the haemolysis reagent. Non-glycated haemoglobin (HbA0) and glycated haemoglobin (HbA1) are separated on the basis of the different charge of the two types of haemoglobin. The amounts of glycated and non-glycated haemoglobin are measured with a photometer at a wavelength of 415 nm. EDTA blood and heparin blood can be used in this method as a sample.
A disadvantage in determining glycated haemoglobin by ion exchange chromatography with a microcolumn is the high temperature and pH dependency and interference by labile precursors of glycated haemoglobin, non-glucose adducts of haemoglobin and other haemoglobin variants. Moreover this method can only be carried out in an analytical laboratory by trained staff.
2. Electrophoretic Method
The electrophoretic method for determining glycated haemoglobin is described for example in Clin.Chem., 34, 145-148 (1988) and Clin.Chem., 26, 1598-1602 (1980). The method separates HbA1 and HbA0 by the action of endoosmosis due to the different charges on glycated and non-glycated haemoglobins. The electrophoresis film is evaluated with a densitometer. EDTA blood and oxalate blood can be used as sample materials. Heparinized fresh blood cannot be used for this method.
Like the ion exchange chromatography with a microcolumn, labile precursors of glycated haemoglobin, non-glucose adducts and other haemoglobin variants interfere with the electrophoretic method. The electrophoretic method can also only be carried out in an analytical laboratory by trained staff.
3. Affinity Chromatography
The determination of glycated haemoglobin by means of affinity chromatography is based on the principle of separating glycated and non-glycated haemoglobin on an agarose column which contains covalently bound aminophenyl-boronic acid. The method is described for example in Diabetes, 33, 73-76 (1984), Fresenius Z.Anal.Chem., 317, 703-704 (1984), Diabetologia, 27, 56-58 (1984) and in Clin.Chim.Acta. 168, 81-86 (1987).
The trihydroxyaminophenylborane immobilized on the agarose column forms a complex with the ketoamine form of glycated haemoglobin so that this fraction is retained on the column. The non-glycated haemoglobin passes through the column without delay as a main fraction and is collected. The glycated haemoglobin is eluted from the column with buffer containing sorbitol and is also collected separately. The amount of glycated haemoglobin is calculated from the absorbances of the fractions.
The separation by affinity chromatography of glycated and non-glycated haemoglobin is characterized in that there is hardly any influence by pH and temperature and there are no interferences by the labile aldimine form, other haemoglobin variants or non-glucose adducts of haemoglobin. Haemolysates of fluoride and EDTA blood are suitable as sample material. The method comprises several process steps and can only be carried out in an analytical laboratory by trained staff.
4. Immunological Determination
The immunological determination of glycated haemoglobin is described for example in Klin.Lab., 39, 991-996 (1993). In this method the glycated haemoglobin (HbA1c) of the sample reacts with anti-HbA1c antibodies in a first reaction step. Since the HbA1c-specific epitope only occurs once in each &bgr; chain of the glycated haemoglobin, there is no aggregation of the antigen-antibody complex. Only after addition of a polyhapten which has several HbA1c-specific epitopes, do the molecules react with the excess antibodies from the first reaction and an insoluble immune complex is formed. This can be measured turbidimetrically. The turbidimetric signal is inversely proportional to the concentration of glycated haemoglobin in the sample.
A disadvantage of this method which comprises several processing steps, is that it can only be carried out in a specialized analytical laboratory.
5. Detection of Glycated Haemoglobin with the Aid of Boronic Acid-dye Conjugates
The detection of glycated haemoglobin with the aid of boronic acid-dye conjugates is described for example in EP-B 0 471 774, EP-B 0 557 357 and in U.S. Pat. No. 5,506,144 and U.S. Pat. No. 5,631,364. In this method a conjugate composed of phenylboronic acid and a blue dye, bind to the glycated sites of glycated haemoglobin after lysis of the erythrocytes. For this a reagent mixture containing the boronic acid-dye conjugate is added to the haemolysate after haemolysing the blood sample, the total amount of haemoglobin is precipitated and separated from the remainder of the sample by filtration. The boronic acid-dye conjugate is bound to the sugar residue of the glycated haemoglobin in this process. Non-bound dye is subsequently removed with the aid of a wash solution and the filtration medium is measured at wavelengths of 470 nm (for the total amount of haemoglobin) and 630 nm (for the blue dye which is bound via boronic acid to glycated haemoglobin).
A disadvantage of this method is that several steps are required for the procedure and in each of which the required liquids (sample, precipitation solution, wash solution) have to be exactly dosed.
The object of the invention is to eliminate the disadvantages of the prior art. In particular the object of the present invention is to provide a simple and rapid method and a corresponding analytical test element for the determination of the ratio of glycated to non-glycated haemoglobin in a sample.
The object is achieved by the subject matter of the invention as defined in the patent claims.
SUMMARY OF THE INVENTION
The invention concerns a method for the d
Horn Carina
Pachl Rudolf
Roche Diagnostics GmbH
Woodard, Enhardt, Naughton, Moriarity & McNett
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