Chemistry: analytical and immunological testing – Involving diffusion or migration of antigen or antibody
Reexamination Certificate
2001-09-14
2004-04-06
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving diffusion or migration of antigen or antibody
C435S007100, C435S287100, C435S287200, C435S810000, C435S962000, C435S973000, C435S975000, C436S161000, C436S177000, C436S178000, C436S512000, C436S518000, C436S524000, C436S528000, C436S529000, C436S531000, C436S534000, C436S808000
Reexamination Certificate
active
06716641
ABSTRACT:
The present invention relates to a new dipstick assay for detecting and quantifying the content of an analyte in a sample. The assay is particularly useful for example in the diagnosis and monitoring of alcoholism by the detection of asialo transferrin or carbohydrate free transferrin (CFT).
Many biological proteins exist in two or more variant forms, frequently differing in the extent of glycosylation of the protein or in the carbohydrate composition per se. Other forms of variation may be in the lipid content or composition of the molecule, or even differences in the primary, secondary or tertiary structures of the protein. 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. The ratio, for example, of glycosylated to non-glycosylated haemoglobins is known to increase in the serum of patents suffering from diabetes. Similarly, some structural proteins for example, 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 of a protein in the blood or body fluid of interest, a diagnosis or assessment of a disease or cellular damage can be made.
Serum transferrin is a glycoprotein with a molecular weight of about 80 kD which comprises a single polypeptide chain with two N-linked polysaccharide chains. These polysaccharide chains are branched and each chain may terminate in either two or three antennae, each with terminal sialic acid residues.
Wong and Regoeczi, in Int. J. Peptide Res. (1977) 9:241-248, reported that human transferrin was naturally heterogeneous, occurring in variant forms with different levels of sialylation. Until recently, there were generally believed to be six such variants, the pentasialo, tetrasialo, trisialo, disialo, monosialo and asialo transferring. Many researchers working in the field of chromatographic analysis of transferrins have however reported that the levels of monosialo transferrin are very low.
The asialo, monosialo, disialo and trisialo variants are often referred to collectively within the field as carbohydrate-deficient transferrin or CDT.
In the normal healthy individual, the tetrasialo variant appears to predominate; however it has been reported that the asialo, monosialo, disialo and, to some degree the trisialo variants, ie. CDT, occur in elevated levels in the blood of alcoholics (see van Eijk et al. (1983) Clin Chim Acta 132:167-171, Stibler (1991)Chin Chim 37:2029-2037 and Stibler et al. in “Carbohydrate-deficient transferrin (CDT) in serum as a marker of high alcohol consumption”, Advances in the Biosciences, (Ed Nordmann et al), Pergamon, 1988, Vol. 71, pages 353-357).
CDT has been shown to be an effective marker for—alcohol consumption, in particular for detecting and monitoring chronic alcohol consumption. Monitoring of blood alcohol level is reliable only when blood is sampled within 24 hours of alcohol consumption and conventional tests (for example, quantitation of &ggr;-glutamyltransferase or measurement of mean corpuscular volume) cannot reliably be used to screen for heavy alcohol intake in patients with liver disease.
Early investigations showed that loss of the sialic acid residues correlated with changes in the isoelectric point (pI) of the transferrin molecules, for example, asialotransferrin exhibits a pI of 5.9, disialotransferrin exhibits a pI of 5.7 and so on. Recognition of the fact that the CDT profile of alcohol abusers differs from that of abstainers or normal users, combined with the identification of the relative amounts of each CDT isoform on the basis of pI, has led to the development of several diagnostic assays for CDT which are described in the patent and scientific literature.
A chromatography assay using anionic ion exchange of the sample to allow asialo, monosialo and disialo CDTs to be eluted whilst the “normal” tetra and pentasialo variants are retained on the column, is disclosed in U.S. Pat. No. 4,626,355 (Joustra) of Pharmacia AB.
An assay using isoelectric focussing and immuno-fixation techniques has been proposed in Dumon et al., (1996) Clin. Biochem. 29(6): 549-553 to assess disialotransferrin content of a sample.
However, these prior art methods for CDT analysis rely on differences in the pI or charge of the different transferrin isomers. Such methods tend to rely upon relatively complex procedures.
Traditionally, it has been thought that CDT arises from a loss of the terminal sialic acid residues of the carbohydrate side chains and it is upon this that the various prior art pi or charge based assays have been predicated (namely, that a loss of a charged sugar moiety would alter the charge and pH of the isoform as a whole).
However, recent studies (for example by Landberg et al. (1995) Biochem. Biophys. Res. Comm. 210(2): 267-274), have shown, by releasing the N-glycans from each isoform of transferrin and analysing them by high-pH anion exchange chromatography, that contrary to this understanding, the existence of disialo and asialo-transferrins appears rather to be correlated with the loss of one or both of the entire carbohydrate chains respectively from the transferrin polypeptide. This “deglycosylation” is not yet fully understood.
The carbohydrate chains may be bi or triantennary and hence each carbohydrate chain in its normal state will carry two or three sialic acid residues, one at the terminus of each antenna. It may be that the carbohydrate chains are cleaved from the transferrin molecules at their base in a single step process, i.e. at an asparagine molecule in the amino-acid backbone of the protein, leaving no sugar residues at that particular glycosylation site. Alternatively, individual or multiple sugar residues may be sequentially lost from transferrin molecules resulting in a gradual loss of carbohydrate content. It is also possible that the CDT transferrin molecules are never properly glycosylated in the first place due to aberrant enzymatic glycosylation processes.
To date, the prior art has favoured the idea that either measurement of all of the CDT variants ie. asialo, monosialo, disialo and trisialo transferrin, or at least two or more CDT variants was necessary to make a meaningful clinical evaluation, or that measurement of the disialotransferrin on its own was necessary.
Contrary to this trend in the art, the Applicants developed and described in WO99/00672 a new type of assay for detecting carbohydrate free transferrins (CFTS). This assay is based on the principle that the presence of transferrin isoforms which are completely devoid of carbohydrate, ie. carbohydrate free transferrin (CFT), is a strong indicator of alcoholism in the absence of any knowledge of the prevalence of any other CDT variants (ie. monosialo, disialo or trisialotransferrin variants). The assay is robust, simple and quick to perform, and readily amenable to automation or compatible with existing routine clinical diagnostic laboratory procedures. This is achieved by separating the carbohydrate-containing transferrins from a sample by contacting them with a carbohydrate-binding ligand and detecting and measuring the carbohydrate-free transferrin contained in the separated, non-binding fraction. However, it was not previously proposed to produce this assay in the form of a dipstick.
Dipsticks represent a solid phase format in common usage in the diagnostic field and indeed form the basis of many home testing kits e.g. home pregnancy testing kits. Many types of dipstick assay have been proposed in the prior art and many of these rely on the principles of the binding affinity of an analyte for a particular binding partner. Many different combinations of analyte:binding partner have been successfully employed in dipstick assays, and many different techniques have been devised which allow visible results to be obtained.
For example, WO 94/15215 of Medix Biochemica discloses a
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Nguyen Bao-Thuy L.
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