Chemistry: analytical and immunological testing – Hemoglobin – myoglobin – or occult blood – Glycosylated hemoglobin
Patent
1997-01-24
1999-03-02
Ceperley, Mary E.
Chemistry: analytical and immunological testing
Hemoglobin, myoglobin, or occult blood
Glycosylated hemoglobin
G01N 3372
Patent
active
058770259
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a method for carrying out an assay for glycated proteins, such as glycated haemoglobin.
The level of glycated haemoglobin in blood is a quantity which is used routinely to assess diabetic patients. High levels of circulating glucose lead to high percentages of glycated proteins. For example, in a non-diabetic person, the percentage of haemoglobin molecules that have been glycated (i.e. to which glucose has bound non-enzymatically) is 4.5-8.0 percent. In a diabetic patient, the levels are much higher (unless treatment is given) and can in severe cases be as much as 20 percent. The level of glycated haemoglobin can therefore be used to monitor treatment.
Virtually all existing methods for the determination of glycated haemoglobin in blood samples depend upon the separation of glycated haemoglobin using for example chromatography, electrophoresis, or solid phase reagents with washing step, prior to measurement.
For example, Wilson et al. (Clin Chem 39/10, 2090-2097 (1993)) describe an assay method for glycated haemoglobin in which the glycated haemoglobin is labelled with a soluble polyanionic affinity reagent, and the anionic complex is then captured with a cationic solid-phase matrix. In this method, the amount of glycated haemoglobin bound to the solid phase matrix is then determined by measuring the quenching by the haemoglobin of the static fluorescence from an added fluorophore.
EP-A-455225 (Nacalai Tesque, Inc.) discloses a method for determining the percentage of glycation of a particular protein. First, the protein is separated from the sample which contains it by preferentially binding it to an antibody which is fixed to a solid support. The support is then washed with labelled boronic acid, which binds to the glycated site on the protein. The liquid and solid phases are separated, and the amount of boronic acid bound to the protein in the solid phase can be measured, thereby allowing the number of glycated protein molecules to be calculated.
U.S. Pat. No. 4,861,728 (Becton, Dickinson & Co) describes a method of determining the percentage of glycosylated haemoglobin in blood. In this method, the total haemoglobin is separated from the liquid phase by binding it to a dipstick, and then the glycosylated haemoglobin is reacted with a dihydroxyboryl reagent conjugated to a fluorescent dye. The absorption of incident light by the dye provides a measure of the amount of glycated haemoglobin. This can be compared with the total amount of haemoglobin, which can be calculated by measuring the absorption of incident light at the absorption wavelength for haemoglobin.
A similar method is described in WO 90/13818 (Axis Research AS). In this method, the glycosylated haemoglobin-containing sample may be haemolysed to liberate any cell bound haemoglobin. Signal-forming molecules conjugated to dihydroxyboryl residues are then reacted preferentially with the glycosylated haemoglobin, the total haemoglobin is separated from the sample, and the amount of glycosylated haemoglobin is measured by measuring the amount of signal-forming molecules.
WO 93/18407 (Abbot Laboratories) is concerned with a method of measuring the amount of glycated haemoglobin in a sample, by reacting the total haemoglobin with a fluorescent marker, and measuring the fluorescent quenching due to the total haemoglobin. The glycated haemoglobin is then separated from the sample by standard methods (ion capture or solid phase separation), and the fluorescent quenching due to it is measured. The two quenching measurements give a percentage of the total haemoglobin which is glycated. The specific binding agent for glycated haemoglobin which is employed is coupled to a latex particle or to polyacrylic acid, in order to achieve the separation of glycated and non-glycated haemoglobin which is essential to the operation of the method.
Separation assays of this kind are time consuming and thus expensive to carry out in practice.
A non-separation assay for glycated albumin is described by Yukiko Hayashi et al. (Clinica Chimica Acta
REFERENCES:
patent: 4861728 (1989-08-01), Wagner
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K. Yoshimoto et al., Chemical Abstract of Japanese patent 07063735, Mar. 1995.
E. Schleicher et al., Clinical Chemistry, vol. 36, No. 6, pp. 992-993, No. 0193, Jul. 1990.
Hirsch, R. E., Methods in Enzymology 232, 231-246 (1994).
Wilson, D. H., et al, Clin. Chem. 39/10, 2090-2097 (1993).
Hayashi, Y., et al., Clinica Chimica Acta, 149, No. 1, 13-19 (1985).
Rockey, et al., Ophthalmic Research, vol. 14, No. 6, 416-427 (1982).
Blincko Stuart J.F.E.
Edwards Raymond
Ceperley Mary E.
The London Chest Hospital NHS Trust
The Royal Hospital of St Bartholomew
The Royal London Hospital
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