Chemistry: analytical and immunological testing – Hemoglobin – myoglobin – or occult blood
Patent
1993-07-12
1996-04-09
Snay, Jeffrey R.
Chemistry: analytical and immunological testing
Hemoglobin, myoglobin, or occult blood
436161, 436166, 436175, 436177, 436536, 436815, 436824, 436 64, 436539, G01N 3372
Patent
active
055061443
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a ligand binding assay for the assessment of glycated blood proteins (GBPs).
It has long been known that many glucoproteins in body tissues and fluids occur as a result of non-enzymatic reactions of body proteins with sugars. These non-enzymatically glycosylated proteins are referred to herein as glycated proteins.
As mammalian tissue appears to contain no enzyme capable of reversing the glycation reaction, the extent to which any given protein is glycated is essentially dependent on
Accordingly, unlike direct measurements of glucose concentration in body tissue or body fluid samples (e.g. blood, plasma or urine), which give information only about the glucose concentration at the time of sampling, the degree of glycation of a protein provides an indication of the body's control of glucose concentration averaged over a longer period of time.
In patients with unstabilized diabetes mellitus, the degree of protein glycation is frequently several times higher than in normoglycemic patients, and as a result, several GBP assays have been proposed as screens for diabetes mellitus or as means by which a patient's medium to long term control over blood glucose levels may be evaluated.
Such assays have been proposed for glycated haemoglobin (GH) and glycated serum albumin (GSA) in particular as the relatively long lifetimes of these proteins provide an indication of the long and medium term control of blood glucose and as these proteins are both abundant and relatively prone to glycation. Thus GSA and GH levels reflect the body's control over blood glucose during the previous 1-3 weeks and 1-3 months respectively.
GBP assays generally rely on the separation out of the GBP from a body fluid or tissue sample, on the binding to the GBP in such a sample of a detectable label, e.g. a chromophore, a fluorophore or a radiolabel, or on a chemical degradation of the GBP in such a sample, e.g. oxidation of the fructoseamine moiety in alkaline conditions in the presence of a redox indicator. Such assays and their advantages and disadvantages were reviewed by Schleicher et al in J. Clin. Chem. Clin. Biochem. 27: 577-587 (1989).
Prior art methods are known which involve separation of the glycated protein from the non-glycated protein by means of ion exchange chromatography. This was the method first proposed for GH assays and is the clinical method most commonly used. However, it is expensive and time consuming and results are influenced by small temperature variations.
Several further assays have involved the use of boronic acid derivatives to isolate or label the glycated proteins in a sample. It has long been known that while boronic acids form esters with carbohydrate moieties having cis-diol residues, such as glycated proteins, enzymatically formed glycoproteins do not form such esters. Thus chemically immobilized boronic acids have been proposed for use in isolation of glycated proteins by affinity chromatography and the use of such materials to quantify the glycated fraction of hemoglobin was proposed for example by Dean et al in GB-A-2024829.
The use of such columns however is expensive and time-consuming and for GBPs other than GH Schleicher (supra) warns that the degree of binding of the glycated protein is very sensitive to the conditions under which chromatography is effected.
The reaction in the liquid phase of glycated proteins with chromphore or fluorophore labelled boronic acids has also been proposed as the basis for an assay for glycated proteins, in particular GH. Thus Schleicher in DE-A-3720736 proposed an assay relying on one of the following three principles for measurement of the total glycated protein present in a sample: when bound to a glycated protein, label when bound to a glycated protein, or acid bound to glycated protein after removal of excess unbound labelled boronic acid, e.g. using activated charcoal.
None of those methods however can be used for the quantitation of a specific GBP. In addition the reagents proposed by Schleicher have rather low absorption coefficients a
REFERENCES:
patent: 4268270 (1981-05-01), Gabbay et al.
patent: 4701418 (1987-10-01), Katopodis
patent: 4861728 (1989-08-01), Wagner
patent: 5242842 (1993-09-01), Sundrehagen
Schleicher, E. et al., "Protein Glycation: Measurement and Clinical Relevance" J. Clin. Chem. Clin. Biochem., vol. 29, 577-580 (1989).
Hayashi, Y. et al., "Fluorometric Measurement of Glycosylated Albumin in Human Serum" Clin. Chim. Acta, vol. 149, 13-19 (1985).
Ohe et al., Clinica Chimica Acta, 169, 1987, 229-238.
Axis Biochemicals AS
Snay Jeffrey R.
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