Chemistry: analytical and immunological testing – Biospecific ligand binding assay
Patent
1993-02-19
1994-10-04
Nucker, Christine M.
Chemistry: analytical and immunological testing
Biospecific ligand binding assay
436514, 436516, 436541, 436808, 435 71, 435 18, 435775, 422 61, G01N 3353, G01N 33564
Patent
active
053526163
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method of assessing the concentration of a subset of analyte variants within a group of different analyte variants, and to test kits and reagent compositions for use in such a method.
Biological molecules such as enzymes, antigens and other biologically important proteins frequently occur as a population of variants which, although sharing a common biological function or property e.g. antigenicity or enzymic activity, differ slightly in their structure. This variance in structure may be due to differences in primary, secondary or tertiary structures in the amino acid sequences of proteins or polypeptides or to differences in the carbohydrate moieties or the lipid composition of the molecule. Although a common characterising property or function between the variant molecules in a given population is retained, such differences in their structure often serves to alter other properties of the variants for example size, charge, isoelectric point (pI) which in turn alters their behaviour in different fractionation and separation systems and may be used as the basis for separating one or more such variants in a variant mixture.
Enzymes frequently occur as variants e.g. isoenzymes and many other biological proteins are now known to exist in two or more variant forms, frequently differing in the extent of glycosylation of the protein or in the carbohydrate composition. The relative concentrations of such variants in a given body tissue or fluid are generally constant but may be disturbed in certain diseases or pathological states or as a result of other disturbances to the body. Thus, for example the ratio of glycosylated to non-glycosylated haemoglobins is known to increase in the serum of patients suffering from diabetes. Similarly certain structural proteins of analytical interest e.g. myoglobins may have slight structural differences in different organs and may be released into the bloodstream following cell damage resulting from disease or injury.
Thus, by measuring the levels of the different variants in the blood or other body tissue or fluid a diagnosis or assessment of the state of a disease or cellular damage can be made.
Of particular importance in this regard is the assessment of the levels of different variants of the protein transferrin in the serum. Transferrin generally occurs in sialylated forms i.e. carrying three or more sialyl residues. However in chronic alcohol abusers, desialylated transferrin i.e. transferrin carrying two or less sialyl residues, is relatively increased in content compared to non-alcoholics e.g. from a normal level of about 1-3% to about 6-25% of the total transferrin content of the patient's serum. Desialylated transferrin, often termed carbohydrate deficient transferrin (CDT), is now generally regarded as a clinically reliable marker for chronic alcoholism. However present methods of transferrin variant quantitation are time consuming and require advanced biochemical equipment. Thus, in the absence of a simple and convenient test for the desialylated transferrin marker, chronic alcohol abuse is still presently diagnosed on the basis of clinical history, information from the patients themselves regarding their alcohol consumption, and a number of laboratory tests, all of which have limited sensitivity and specificity. Blood alcohol level is a reliable measure only when block is sampled within 24 hours after alcohol consumption. All present clinical chemistry tests have sensitivities and specificities too low to reliably identify all alcohol
The microheterogeneity of transferrin and its correlation with alcohol abuse were first detected in 1980. (Stibler H, Sydow O, Borg S. "Quantitative estimation of abnormal microheterogeneity of serum transferrin in alcoholics". Pharmac. Biochem. Behav, 1980, 13 Suppl. 1,47-51). The clinical importance of this microheterogeneity has been further investigated, and isoelectric focusing and chromatofocusing methods for the quantitative separation of the different transferrin variants have been studied (Vest
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Axis Biochemicals AS
Dubrule Chris
Nucker Christine M.
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