Chemistry: analytical and immunological testing – Involving immune complex formed in liquid phase
Patent
1990-11-01
1993-09-07
Housel, James C.
Chemistry: analytical and immunological testing
Involving immune complex formed in liquid phase
436 66, 436161, 436166, 436175, 436177, 436815, 436824, 436539, 436 67, G01N 3372
Patent
active
052428427
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The present invention relates to a ligand binding assay for specific assessment of glycosylated hemoglobin.
Proteins in solution in body fluids are continually subject to glycosylation processes. Glucose reacts with the proteins by non-enzymatic reactions to form glycoproteins, and in many cases the level of glycoprotein formation is proportional to the glucose concentration in the body fluid in question. For proteins not initially synthesized as glycoproteins, the fraction of a protein present in glycosylated form is therefore a function of
the life-time of the protein in the organism and
the glucose concentrations to which the protein has been exposed.
Unlike measurements of glucose concentrations in blood, plasma or urine, which only give information about the glucose concentration at the time of sampling, the amount of a protein present in glycosylated form gives an indication of the organism's control of glucose concentration during longer periods of time.
Erythrocytes (red blood cells) have a mean lifetime of approximately 120 days and contain large amounts of haemoglobin. The fraction of erythrocyte haemoglobin in glycosylated form is thus a good measure of the control of the disease in patients with diabetes mellitus, and is a function of the glucose concentrations in the blood of the patient in the weeks prior to the blood sampling.
In clinical practice numerous different methods have been used to measure the glycosylated haemoglobin fraction in order to quantitatively evaluate the long-term control of blood glucose in patients with diabetes mellitus. The main methods which are in clinical use are:
1. Separation of glycosylated and non glycosylated haemoglobins by means of ion-exchange chromatography. This was the first method proposed and still is the clinical method most commonly used. High costs and time-consuming manufacturing methods together with time-consuming performance of the separations are drawbacks of the method, and the results are influenced by small temperature variations.
2. Use of boronic acid derivatives to specifically isolate the glycosylated haemoglobin fraction. It has long been known that boronic acid moieties bind to carbohydrate moieties having cis-diol residues, (Boeseken J., Advances in carbohydrate chemistry 4, 189-210, 1949. Solms J. and Deuel H., Chimica 11, 311, 1957) and this property can be used as the basis for an affinity chromatographic seperation. Thus, for example, boronic acid residues have been chemically immobilized on solid phases such as agarose, for isolation of glycoproteins, carbohydrates and nucleotides (Hageman, J and Kuehn, G., Anal Biochem. 80: 547, 1977.). Columns of such material have also been used to quantify the glycosylated fraction of haemoglobin (Dean P. D. G. & al, UK patent application GB-A-2024829). Such columns are time-consuming and expensive to make and slow to run: Haemolysate must be passed through the column, the different fractions must be collected and the volumes of the fractions must be corrected, before the haemoglobin content of the different fractions can be measured and the fraction of haemoglobin in glycosylated form can be calculated.
3. Electrophoretic separation. Glycosylation of haemoglobin alters the electric charge of the protein, and this may be used to separate the glycosylated and non-glycosylated fractions electrophoretically, following which the different fractions may be quantitated for example by reflectometry. This method is also time-consuming and expensive.
In addition to the glycosylation of haemoglobin within erythrocytes, glycosylation of proteins in serum takes also place at an elevated rate in patients suffering from diabetes mellitus. However, since the different serum proteins have different half-lives in the body, and most of these half-lives are significantly shorter than that of haemoglobin, measurements of glycosylated serum proteins are only used for short to intermediate term retrospective monitoring of the regulation of the disease. Different colorimetric methods
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Axis Research AS
Housel James C.
Snay Jeffrey R.
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