Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-05-20
2003-02-04
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007940, C435S028000, C435S975000, C436S518000, C436S536000, C436S811000, C436S827000, C530S387700, C530S388250, C530S389300, C530S391100, C530S391300, C530S396000
Reexamination Certificate
active
06514716
ABSTRACT:
BACKGROUND AND SUMMARY OF THE INVENTION
This invention relates to the determination of the terminal sialic acid residues in human transferrin. One embodiment of the invention relates to the quantification of terminal sialic acid residues in transferrin contained in samples of human body fluids, and in particular to an interpretation of changes in the chemical structure of transferrin such as are caused, for example, by regular and excessive alcohol consumption.
There are a number of indicators used in medicine to diagnose alcohol abuse or addiction. With the help of so-called “state markers”, it is possible to estimate alcohol consumption, different markers serving to estimate consumption over a short period of time (eg, blood alcohol level) or over longer periods (in particular various hepatic enzymes). A detailed review of alcoholism and suitable markers is contained in M. Soyka (publisher, Biological Markers for Alcoholism, Chapman and Hall, (1995)).
An abnormal transferrin variant has proved to be a suitable marker with a relatively high degree of specificity. The scientific studies published so far have shown that this molecular modification takes the form of a lack or a reduction in the number of sialic acid residues in the transferrin molecule.
Human serum transferrin is a glycoprotein with a relative molecular weight of about 77,000 g/mol. It consists of a single peptide chain of 679 amino acids, to which two oligosaccharide chains consisting of N-acetyl glucosamine, mannose and galactose are attached. These carbohydrate chains have two to three antennae, to the ultimate unit of which (galactose) a sialic acid residue is bound.
Regular alcohol abuse impairs the mechanism by which sialic acid is transferred to transferrin, and as a consequence there are increased numbers of transferrin isomers with fewer than the usual four-to-six terminally bound sialic acid residues (desialicized transferrin, sialic-acid deficient transferrin, sialic deficient transferrin, SDT)
According to Jong et al. (1980), the most common tetrasialotransferrin isomer (
FIG. 1
) is made up of:
4 terminal N-acetyl sialic acid residues, 4 galactose residues, 8 N-acetyl glucosamine residues and 6 mannose residues in each oligosaccharide side-chain of the transferrin molecule.
Methods described in the prior art are based mainly on the separation of differently glycosylated transferrin derivatives by way of their charge, eg, by way of isoelectric focusing or chromatographic techniques. Familiar methods include:
1. The isoelectric focusing method Electrophoretic separation of isotransferrins according to their isoelectric points (Pl 6.1-5.1). (Helena Stibler and Stefan Borg, Pharmacol. Biochem. Behav. 1980, 13, Suppl. 1, 47-51).
2. The chromatographic method by means of ion exchanger (Helena Stibler et al., Clinical and Experimental Research Sept./Oct. 1986, Vol. 10, No. Pages 535-543).
3. The HPLC method Saturation of the transferrin with an iron salt, followed by extraction on a column and subsequent densitometric determination. (Strahler et al., J. of Chromatography 266, 1983, 281-289).
4. Isocratic HPLC method Based on the separation of isotransferrins by means of cationic buffers. (Joustra Marius et al., Patent No. EP 0 172 217 B1), (HPLC techniques according to Jan Jeppson et al., Clin. Chemistry 39/10; 2115-2120 (1993).
5. Turbidimetric method of the company AXIS Likewise based on saturation of the isotransferrin by means of iron salt or a heterogeneous immunoassay with separation on a column followed by a turbidimetric measurement. (Patent: WO 99119983 A 911226).
6. The immunoenzymatic EIA method involving a Pharmacia conjugation Uses a monoclonal antibody after saturation of the isotransferrin with an iron salt and column separation (O. Märtenson et al.).
7. Pharmacia's RIA method: A chromatographic method using an ion exchanger after saturation of the isotransferrin with an iron salt and quantitative determination by means of radio-immunoassay.
A The above-mentioned methods, however, are not yet satisfactory for the user. The likelihood of false diagnoses and the very complicated and time-consuming test procedures are particularly problematical.
The prior art also describes a class of proteins or glycoproteins referred to as lectins, which bind to certain carbohydrate configurations and to glycoproteins which carry such carbohydrate configurations. On account of their specificity for certain carbohydrates, such lectins have been used, eg, for the blood-group-specific agglutination of erythrocytes.
The object of this invention was to provide a method allowing rapid and uncomplicated determination of terminal sialic acid residues in human transferrin.
This object is established according to the invention by a method of determining terminal sialic acid residues in human transferrin contained in a sample fluid, said method being based on a sandwich principle and being characterized in that the sample fluid is incubated with a first receptor which binds specifically to human transferrin, the thus-formed complex is separated from the sample fluid and incubated with a second receptor which binds specifically to terminal sialic acid residues in human transferrin, the second receptor being bound to a marker or having the ability to bind thereto, and the complex comprising the first receptor, human transferrin and the second receptor is determined by means of the marker.
DETAILED DESCRIPTION
It is generally preferable, in keeping with familiar and time-tested technology, to use an anti-transferrin antibody as the first receptor. It is of advantage if the first receptor is a polyclonal anti-transferrin antibody. Antibodies of this kind are commercially available (eg, Sigma, No. T2027). However, there are other receptors specific to human transferrin which are suitable too, provided they do not significantly impair the binding of the second receptor to the sialic acid residues. It would be conceivable, for example, to use human transferrin receptor.
The second receptor is preferably a lectin, the most preferred of which is the lectin Sambucus nigra; its production has been described by Brokert et al. (Biochem. J. 221, 103-109 (1984)).
According to one embodiment of the method of the invention, the first receptor is bound to a solid phase, as a result of which, during incubation, the transferrin becomes attached to the solid phase. According to another embodiment, however, the first receptor can also be present in solution; in this case the receptor is able to bind to a solid phase by means of a specific pair of binding agents, one of which is bound to the solid phase and the other to the receptor. Suitable pairs of binding agents are known to those versed in the art.
It is expedient to use a wall of a reaction vessel, such as a sample tube, a microplate or a cuvette as solid phase. The solid phase can also consist of a particulate material such as polystyrene or magnetic beads.
In the same way, the second receptor can either be bound directly to the marker or else be capable of binding thereto by way of a specific pair of binding agents. According to a preferred embodiment, the specific binding agents are biotin and streptavidin or avidin, with the second receptor typically carrying the biotin and the marker the streptavidin/avidin.
The sample fluid is incubated with the first receptor and the second receptor for a period of 10 to 60 minutes in each case, preferably 20 to 40 minutes, and at a temperature of 10 to 40° C. A 30-minute incubation period at room temperature provides good results.
Basically, all detection techniques commonly used in connection with immunoassays are suitable for determining the marker on the second receptor. Persons versed in the art are familiar with such techniques, and they do not need to be explained in detail here. They include, in particular, enzymatic techniques, ie, techniques in which the marker is an enzyme which is quantified by determining a substrate. Enzymes commonly used for such purposes include, eg, phosphatases such as alkaline phosphatase, oxidases, perox
Chin Christopher L.
Fulbright & Jaworski L.L.P.
Padmanabhan Kartic
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