DK154859B - Method for determination of tumour-associated glyco-bonds and equipment for use during this method - Google Patents

Description

DK 154859 B

BACKGROUND OF THE INVENTION.

The present invention relates to a method for determining tumor-associated glycobinding (hereinafter referred to as TAG) in mammalian body fluid, more particularly TAG comprising glycoproteins, glycopeptides, glycolipids and / or carbohydrates with a particular specific end group, which TAG increases in the proliferation of undifferentiated cells, especially tumor cells or cancer cells.

Methods for diagnosing cancer are known by measuring a specific glycoprotein that is specifically formed in patients with cancer. Most of these methods utilize the antigenicity of the glycoprotein protein moiety; eg. known a method for the diagnosis of primary liver cancer by measuring alpha-foeto protein and a method for diagnosing cancer in a digestive organ, especially rectal cancer, by measuring CEA (Igaku no Åyumi (Progress in Medicine), 106, 5) .

Fifth Saturday Special Issue, pages 235-250 (1978)).

However, the utility of these diagnostic procedures is relatively limited.

Further studies have shown that the body fluid of a cancer patient contains TAG formed by undifferentiated cells (mainly 25 cancer cells) and delivered to the body fluid, and that such TAG is very different from the carbohydrates formed by differentiated cells (mainly normal cells) and delivered to the body fluid, with respect to the carbohydrate chain structure, carbohydrate chain length and the nature of the carbohydrate residues contained. It has also been found to determine the TAG level in a sample of body fluid by reacting the TAG in the sample with a lectin that can specifically combine with galactose (βΐ- * 3 or | 31- * 4) -N-acetyl -glucosamine and / or galactose (β1-> 3 or βΐΗΜ) -N-acetyl-galactosamine end groups (Danish Patent Application No. 369/80).

Given an ever stronger desire to 2

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obtaining methods for measuring the TAG level and diagnostic methods which are less limited in their utility and which have improved sensitivity, extensive studies have been carried out and as a result 5 have been found that TAG contains glycoproteins, glycopeptides , glycolipids and / or carbohydrates with N-acetyl-D-galactoseamine (hereinafter referred to as AG) or L-fucose end group to specifically associate with certain species of lectin (hereinafter, a lectin which may specifically combine with an AG end group, is termed AG-binding lectin, and a lectin that can specifically associate with an L-fucose end group will be designated L-fucose-binding lectin) and, therefore, by reacting TAG in body fluid. with a 15 AG-binding lectin or L-fucose-binding lectin (both lectins will sometimes be referred to as specific lectin), can detect cancer cells, control the degree of their proliferation, and become aware of their growth profile. or cancer diagnosis.

This is achieved by the method of claim 1.

Brief Description of the Invention

Therefore, the main object of the present invention is to provide a new method for determining the TAG level in body fluid. The invention therefore provides a method for determining the TAG level in a body fluid sample comprising reacting TAG in the sample with an AG-binding lectin or L-fucose-binding lectin to form a TAG lectin complex and measuring the amount of the TAG lectin complex or of unreacted lectin.

Brief description of the drawings.

FIG. Figures 1 and 2 are graphs showing a standard curve obtained by one embodiment of the method of the invention using competing turnover (Examples 1 (viii) and (ix)).

FIG. 3 and 3 'are graphs showing a calibration curve for one embodiment of the method

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3 of the invention using the competition process of Example 2 (ii).

FIG. Figure 4 is a graph showing the TAG level in healthy individuals and in patients with various cancers 5 determined using the method of the present invention using the competition process of Example 2 (iii).

FIG. Figure 5 is a graph showing the TAG level in healthy subjects and in patients with various cancers of Example 3 (ii).

FIG. 6 is a graph showing a calibration curve for another embodiment of the method of the invention using a sandwich process of Example 3 (iii).

FIG. 7 is a graph showing a calibration curve obtained by the method of Example 3 (iv) using PGM as the standard substance.

FIG. 8 (si), (b) and (c) are graphs showing the TAG level in samples determined using the calibration curve of FIG. 7th

FIG. 9 and 9 'are graphs showing a calibration curve for a further embodiment of the method according to the invention using a competition method of Example 4 (i).

FIG. 10 is a graph showing a calibration curve for a further embodiment of the method of the invention using a sandwich process of Example 4 (ii).

FIG. Figure 11 is a graph showing the TAG level in 30 healthy individuals and in patients with various cancers determined using the competition process of Example 4 (iii).

FIG. Figure 12 is a graph showing the TAG level in healthy subjects and in patients with various cancers determined using the method of Example 4 (iv).

FIG. 13 is a graph showing a calibration curve obtained by the method of Example 4 (v).

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FIG. 14 is a graph showing the TAG level in samples determined using the calibration curve of FIG. 13th

Detailed description of the invention.

In the method of the invention, various body fluids, including blood, tissue fluid, lymph, thoracic fluid, abdominal fluid, amniotic fluid, gastric juice, urine, pancreas, cerebrospinal fluid and saliva can be used.

Of these, blood in the form of serum or blood plasma is preferred. The amount of body fluid to be used for the determination is from approx. 0.01 to approx. 10 ml, preferably from 0.1 to 0.2 ml.

According to the invention, TAG is isolated from the body fluid, reacted with a specific lectin according to the invention, and the amount of the TAG-bound specific lectin or residual specific lectin is measured, or alternatively, a specific lectin is labeled and added directly to the body fluid and the TAG-bound labeled specific lectin. or unreacted labeled specific lectin is isolated and the amount of said TAG bound labeled specific lectin or unreacted labeled specific lectin is measured. In each of these methods, the TAG level of the body fluid can be determined.

The desired TAG can be isolated from the body fluid 25 by any of the extraction or separation methods usually used to obtain glycoproteins, glycopeptides, glycolipids and / or carbohydrates with AG or L-fucose end group. Among these methods are salting, precipitation, extraction, centrifugation, dialysis, molecular sieving and inactivation of enzymes. These methods can be used in combination.

More particularly, the desired fraction may be prepared by adding sulfosalicylic acid, trichloroacetic acid or zinc sulfate to serum or plasma, or by heating the serum or plasma, filtering the resulting precipitate to remove albumin, immunoglobulin, etc. and then dialyzing the residue.

In cases where a labeled specific 5 is used

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lectin, samples of body fluid, other than blood, can be used directly as test samples (hereinafter referred to as "samples"). However, to prevent denaturation of the samples and to accelerate the reaction with the specific lectin, proteins containing lower carbohydrates such as bovine serum albumin (BSA) are preferably added as protective proteins. A better result is obtained by adding an appropriate amount of protective protein to the sample after removal of albumin, immunoglobulin or the like. When the body fluid is blood, serum obtained by a known serum collection method or plasma obtained by a plasma collection method can be used as a sample using an anticoagulant such as heparin, EDTA, citric acid or the like. The particularly preferred sample is plasma collected and prepared using heparin as an anticoagulant. If the TAG level is relatively high as in a patient with ascites, these samples may be diluted with a suitable buffer solution to the desired extent.

Any AG-binding lectin or L-fucose-binding lectin can be used according to the invention if it is capable of binding specifically to glycoproteins, glycopeptides, glycolipids and / or carbohydrates with AG-25 or L-fucose end group. . Suitable examples of AG-binding lectin include Dolicho's bean (Dolichos biflorus), Maclura tomifera lectin, Helix pomatia lectin, lima bean (Phaseolus limensis) lectin, soybean (Glycine max) lectin and Bauhinia bean (Bauhinia purpurea) lec-30 tin. Suitable examples of the L-fucose-binding lectin include Lotus tetragonolobus lectin [Brt. j. Enp.

Pathi., 34, 94 (1953)] and Ulex europus lectin [Boyd., W.C. and Sharpleigh. E., Blood, 9, 1195 (1954)].

Various enzymes, fluorescent materials and radioactive materials can be used as material for labeling the lectin according to the invention. Illustrative examples of enzymes include glucoamylase, glucose oxidase, peroxidase, alkali phosphatase, β-galactin 6

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tosidase and active fragment of hemoctapeptide, etc. Examples of the fluorescent materials include fluorescein, fluorescein isothiocyanate, rhodamine, dansyl chloride (ie 5-dimethylamino-1-naphthalene sulfonyl chloride), etc., and radioactive materials include e.g. radioactive iodine (e.g., ^ 25I, 131I, etc.), radioactive tritium, etc.

As will be described below, the specific lectin to be used according to the invention can be labeled with these labeling materials by a method usually used to label known proteins such as antigens or antibodies with enzymes, fluorescent materials or radioactive materials. .

The process of the invention is carried out as follows: first, a predetermined amount of body fluid or a TAG fraction is mixed with a labeled or unlabeled specific lectin and the mixture is heated to a temperature below 45 ° C, preferably between 20 ° C and 40 ° C. and most preferably between 20 and 40 ° C. The resulting TAG-bound labeled or unlabeled specific lectin or the unreacted labeled or unlabeled specific lectin can be isolated by conventional isolation techniques such as chromatography, electrophoresis, salting, fractionation, dialysis, gel filtration, adsorption or combinations thereof. Alternatively, agar gel, agarose gel or polyacrylamide gel may be used as separating agents (see Japanese Patent Application (OPI) No. 151263/80 (the term "OPI" as used herein means a "published unexamined Japanese patent application")).

More particularly, the unreacted labeled or unlabeled specific lectin can be isolated by adding to the reaction liquid an appropriate amount of a precipitant for a glycoprotein-bound specific lectin such as polyethylene glycol, saturated ammonium sulfate or rivanol (acrinol); followed by centrifugation or other means for removing the TAG bound tag or 7

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unlabeled specific lectin. Suitable conditions can be used for the centrifugation depending on the precipitant used if polyethylene glycol is used as the precipitant, preferably the centrifugation 5 is carried out at approx. 1000 G for 30 to 60 minutes.

The TAG bound labeled or unlabeled specific lectin can be readily separated from the unreacted labeled or unlabeled specific lectin using the difference in diffusion rate on an agar gel, agarose gel or polyacrylamide gel. In particular, if the reaction mixture is applied to a gel, the TAG bound specific lectin will not diffuse but remain on the surface of the gel while the unreacted specific lectin diffuses through the gel.

Accordingly, the TAG-bound specific lectin can be readily separated from the unreacted specific lectin.

The above gel can be prepared by a conventional method. Eg. an appropriate amount of agar, agarose or polyacrylamide is added to a diluent such as distilled water or dilute citric acid or 20-hydrochloric acid buffer solution (pH = about 7.5) and the mixture is heated at 60 to 80 ° C with gentle stirring to form a solution placed in a suitable container such as a test tube and allowed to cool until the solution coagulates into a gel.

The concentration of the gel is chosen depending on the size (e.g., molecular weight, stereospecific structure) of the unreacted lectin of the invention and the TAG bound labeled specific lectin. The gel usually has a concentration of 0.4 to 2.0% by weight, preferably from 0.7 to 1.0% by weight. If necessary or desired, the gel may contain a preservative. The gel thus prepared may have a flat surface, but a concave surface is preferred since the resulting complex does not adhere to the inner wall of the container.

The TAG level of the body fluid can be calculated from the amount of the isolated TAG bound labeled or unlabeled specific lectin or unreacted labeled or unlabeled specific lectin as measured by a conventional

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8 method.

Various methods can be used to measure the amount of unlabeled specific lectin that has not been consumed by the reaction. Preferably, a substance that reacts specifically with the specific lectin during agglutination or precipitation is added to the reaction liquid and the resulting specific change is observed visually or measured by photometric analysis. More specifically, the reaction liquid is serially diluted by the double dilution method with a diluent such as 0.15 M phosphate buffer or physiological saline solution and a predetermined amount of dilution is placed on a V-shaped or U-shaped slide or in a small test tube or the like. and a substance which causes a specific agglutination reaction with the specific lectin is added; the mixture is stirred and allowed to stand at a temperature below 45 ° C, preferably between 4 and 40 ° C, for a period of 30 minutes or longer, preferably between 60 and 90 minutes.

The final or maximum dilution rate at which agglutination takes place is determined. The maximum degree of dilution is defined as the agglutination value. All specific lectins useful in the present invention have substantially the same agglutination value.

An example of a substance that causes a specific agglutination reaction with the specific lectin is a glycoprotein with AG or L-fucose end group.

The AG binding lectin may be used Sephadex, 30 rubber, glass beads or the like coated with AG end group glycoproteins such as cytolipins K and R of human erythrocyte membrane, sulfated glycopeptide type A from porcine gastric mucosa, asialode derivative of active substance type A from human blood, mycine type A + from the submandibular membrane of pigs, asialo GM

Follisman antigenic substance. For the L-fucose-binding lectin, Sephadex, rubber, glass beads or the like are coated with L-fucose end glycoproteins.

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9a group such as active substance type Le and Le from human blood, sulfated glycoprotein type A from porcine gastric mucosa, sulfated glycoprotein active substance type H (0) from porcine gastric mucosa and human erythrocyte H 2 antigen.

When the labeled specific lectin is used, the TAG level can be measured by a suitable method selected according to the labeling agent for the specific lectin.

For example, When the specific lectin is labeled with an enzyme, the TAG level can be determined by measuring the enzymatic activity using an appropriate enzyme substrate for a colorimetric assay system or fluorescence assay system. If the label is a fluorescent material, the TAG level can be determined by measuring the fluorescence intensity, and if the label is a radioactive material, the TAG level can be determined by measuring the radioactivity. In this way, the amount of the TAG-bound labeled specific lectin or the unreacted labeled specific lectin can be measured.

A particularly convenient method of carrying out the above-described assay process of the invention is to use an assay equipment to determine the TAG level of body fluids such as blood plasma or serum.

For the purposes of the invention, an apparatus containing the specific lectin which can be specifically bound to TAG is used. A stabilizer and / or a preservative such as glycerol or bovine serum protein may be added to the reagent consisting of the specific lectin. The specific lectin reagent may be a lyophilized product or the equipment may contain a water-soluble or water-miscible solvent. Furthermore, the reagent consisting of specific lectin may contain a buffer solution to maintain its pH after reconstitution and / or a preservative and / or a stabilizer to prevent premature degradation of the sample. The buffer solution is not essential for the equipment, but if used, its dense pH value is preferably set from 6 to 7.8.

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10

The reconstitution agent preferably contains water, but some or all of the water may be replaced by a water-miscible solvent. Water-miscible solvents are well known to those skilled in the art? Suitable Examples 5 thereof include glycerol, alcohols, glycols and glycol ethers, but they are by no means limited thereto.

The amount of the specific lectin contained in the solvent or diluent is appropriately selected according to the agglutination value (defined as the final or maximum dilution of serially double diluted samples), the type of labeling agent or the substance to be determined, etc. .. Usually, the specific lectin is contained in an amount of approx. 0.01 to approx. 100 µg / ml, preferably 0.03 to 40 µg / ml. The above solution of the labeled or unlabeled specific lectin can be further diluted.

The object of the present invention can be achieved with further advantage by means of a competition process or a sandwich process as described below.

(1) Body fluid TAG to be measured (hereinafter sometimes referred to as the material to be measured) and a given amount of insoluble TAG or insoluble TAG-like material reacted competitively with the specific lectin that has been labeled with a labeling agent (hereinafter referred to as labeled specific lectin), and the insolubilized TAG or insolubilized TAG-like material bound to the labeled specific lectin is separated from the unbound labeled specific lectin and the activity of the labeling agent on each material is measured to determine the TAG level.

(2) The material to be determined and a given amount of TAG or TAG-like material which have been labeled with a labeling agent (hereinafter referred to as labeled TAG and labeled TAG-like material, respectively) are reacted competitively with a given amount of the specific lectin or insoluble specific lectin,

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11 and the tagged TAG or tagged TAG-like material bound to the specific lectin or unresolved specific lectin is separated from the unbound tagged TAG or unbound tagged TAG-like material and the activity of the tagging agent on each material is measured to determine the TAG level.

(3) The material to be determined is reacted with the insolubilized specific lectin to form a complex of TAG and the insolubilized specific lectin, and the complex is reacted with a given amount of the labeled specific lectin and that of the labeled specific lectin bound complex is separated from the unbound labeled specific lectin, and the activity of the labeling agent on each material is measured to determine TAG level.

In the present specification, the term "TAG-like material" means a carbohydrate derivative having an AG or L-fucose end group. Examples include carbohydrates down-AG end group such as sulfated glycopeptide type A from porcine gastric mucosa, cytolipins K and R from human erythrocyte membrane, human blood asialode derivative, mycine type A + from pig submandibular membrane, asialo GM 2 and Follisman antigenic or carbohydrate derivatives with L-fucose end group cL b 25 such as human blood type Le and Le active substance, sulfated glycoprotein type A from porcine gastric mucosa, sulfated glycoprotein active substance type H (O) from porcine gastric mucosa and human erythrocyte antigen.

The insolubilized TAG, insolubilized TAG-30 material and the insolubilized specific lectin can be prepared by chemical or physical reaction between TAG, TAG-like material or the specific lectin and an insoluble carrier. Examples of such an insoluble carrier are cellulose powder, Sephadex, Sepharose, polystyrene, filter paper, carboxymethyl cellulose, ion exchange resin, dextran, plastic film, plastic tube, nylon, glass beads, silk, polyamine-methyl-vinyl ether-maleic acid, maleic acid, maleic acid, maleic acid, maleic acid, maleic acid,

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12-maleic acid copolymer, etc. The insolubilization can be carried out by a process whereby a covalent bond is formed [i.e. a diazop process, a peptide process (e.g., an acid amide-derived process, a carboxychloride resin process, a carbodiimide resin process, a maleic anhydride-derived process, an isocyanate-derived process, a cyano bromide-activated polysaccharide process, a cell process , a process using a condensing agent, etc.), an alkylation process, a carrier-bonding process using a cross-linking agent such as glutaraldehyde, hexamethylene isocyanate, etc., a carrier-bonding process according to Ugi reaction and the like]; an ion-bonding process using such a carrier as ion-exchange resin; and a physical adsorption process using porous glass such as glass beads as a carrier. Of these, the cyanobromide-activated polysaccharide process and the carrier-bonding process using a crosslinking agent are preferred within the process of forming a covalent bond. According to the cyano-bromide-activated polysaccharide process, insolubilized TAG, insolubilized TAG-like material or insolubilized specific lectin can be obtained by reacting TAG, etc., with a 10 to 1000 times the amount of a cyanobromide-activated carrier in a suitable solvent 25 at 0 to 40 ° C, preferably at 20 to 30 ° C, for 2 to 4 hours.

The insolubilized TAG, insolubilized TAG-like material and the insolubilized specific lectin can also be prepared by a radiation-induced polymerization process. That is, an aqueous dispersion of a polymerizable monomer containing TAG, TAG-like material or the specific lectin is prepared, and the dispersion is irradiated with light or ionizing radiation to polymerize the monomer 35 to form a polymeric matrix of TAG, TAG. similar material or the specific lectin. Such aqueous dispersion is prepared by dispersing a hydrophobic polymerizable monomer (A) in a 0.1 to 5 wt.

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13 solution of a water-soluble polymer (B), disperse hydrophilic polymerizable monomer (C) in an aqueous solution, or disperse a mixture of the hydrophobic polymerizable monomer (A) with a hydrophilic polymerization bar monomer (C) in a 3 to 20% by weight aqueous saline solution, or disperse the hydrophobic polymerizable monomer (A) in an aqueous solution containing 0.01 to 5% by weight of a surfactant (D). When the dispersion thus prepared is irradiated with light 10 or ionizing radiation, the polymerizable monomer present as a dispersion phase is polymerized to form a polymeric matrix of TAG, TAG-like material or the specific lectin. If desired, the matrix can be formed into a sheet or into particles in a suitable manner.

As specific examples of the hydrophobic polymerizable monomer (A) may be mentioned glycidyl methacrylate, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, trimethylolpropane tri-20 methacrylate, polyethylene glycol-200 dimethacrylate, di-propylene glycol dimethacrylate, 1,4-butylene glycol dimethacrylate, 1,6-hexanglycoldimethacrylat, methoxydiethylenglycoldimethacrylat, methyl methacrylate, ethyl methacrylate, butyl methacrylate and the corresponding acrylates thereof.

In general, any water-insoluble monomer which can be polymerized by light or other radiation can be used. As special examples of the hydrophilic polymerizable monomer (C), mention may be made of 2-hydroxyethyl methacrylate, methoxytetraethylene glycol methacrylate, methoxypolyethylene glycol-400-methacrylate, methoxypolyethylene glycol-1000-methacrylate, polyethylene glycol-400-dimethacrylate, polyethylene glycol-400-dimethacrylate, N-vinyl-2-pyrrolidone, etc. and the corresponding acrylates thereof. In general, any water-soluble monomer that can be polymerized by light or other radiation can be used.

As special examples of the water-soluble

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14 polymers (B) include polyvinylpyrrolidone, polymethacrylic acid, polyacrylic acid, polyvinyl alcohol, hydroxypropylcellulose, gum arabic, etc.

Specific examples of the surfactant (D) are sodium lauryl sulfate, potassium oleate, sodium oleate, sorbitan monolaurate, sorbitan monostearate, sorbitan monooleate, propylene glycol monolaurate, oleic acid and sodium dodecylbenzenesulfonate. However, any surfactant which can retain the polymerizable monomer or TAG, TAG-like material or the specific lectin dissolved in the polymerizable monomer in its micelle structure can be used.

TAGs labeled with radioactive material, TAGs similar to those labeled with radioactive material and specific lectin labeled with radioactive material can be prepared by introducing a radioactive iodine atom such as 125 131 I or I into TAG, TAG-like material or specific lectin. The introduction of radioactive iodine is carried out by ordinary iodination processes, e.g. an oxidative iodination process using chloramine, T [Nature, 194, p. 495 (1962); Biochem. J., 89, pp. 114 (1963)].

Hereby such iodination is carried out in a suitable solvent [e.g. a buffer solution of pH 6-8, preferably 0.2 M phosphate buffer solution (pH 7)] at about room temperature for 5 to 60 seconds in the presence of chloramine T. Radioactive iodine and chloramine T are preferably used in amounts of 1 to 5, respectively. mCi and 10 to 100 nanomoles per nanomol tyrosine contained in TAG, 30 TAG-like material or specific lectin. The thus-labeled TAG, TAG-like material or specific lectin is isolated and separated in the usual manner and, if necessary, stored in lyophilized form.

Enzyme-labeled TAG, enzyme-labeled TAG-like material and enzyme-labeled specific lectin can be prepared by a known coupling process [e.g. B. F Erlanger et al., Acta. Endocrinol. Suppl., 168, 206 (1972) and M.H.

Karol et al., Proc. Night. Acad. Sci. United States, 5T 713 (1967)].

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15

That is, TAG, TAG-like material or specific lectin is reacted with an enzyme in buffer solution with pH

4-6 (e.g., 1 mM acetate buffer solution (pH 4.4)) at room temperature for 2 to 5 hours in the presence of an oxidizing agent such as NalO 4 followed by reduction with NaBH 4 or the like. The enzyme is used in an amount of 1 to 3 moles per ml. moles of TAG or the like. The oxidizing agent is used in an amount of 100 to 300 moles per liter. moles of TAG or the like, and the reducing agent is used in an amount of 1 to 2 moles.

Fluorescent material labeled TAG, fluorescent material labeled TAG-like material and fluorescent material labeled specific lectin are prepared by reacting TAG, TAG-like material 15 or specific lectin with a known fluorescent material such as fluorescein isothiocyanate (FITC) or tetramethyl rhodamine isothiocyanate ( RITC) in water or physiological saline solution at pH 6-8 at 0 ° C to room temperature, preferably at room temperature, for 1/2 to 3 hours (fluorescent antibody process; Ikagaku Jikkenho Koza, No. 4, p. 263-270). The fluorescent material is preferably used in an amount of 1/50 of TAG or the like.

The determination process according to the invention in the form of the competition or sandwich process will be described below.

In the two processes, the reactions are carried out in a suitable solvent at 45 ° C or lower, preferably at 4 to 40 ° C, and most preferably at 20 to 40 ° C. As a solvent, those which do not adversely affect the reaction of TAG or TAG-like material with specific lectin such as water, physiological saline and buffer solutions of pH 6 to 7.8 (e.g. 0.1 to 0.3 M tris) are preferred. hydrochloric acid buffer solution (pH about 7.5), 0.1 M phosphate buffer solution (pH about 7.4), etc.). The reactions are carried out for 5 to 40 hours, preferably 15 to 25 hours.

The resulting TAG (or TAG-like material)

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16) bound to the specific lectin can be separated from the unbound specific lectin or unbound TAG (or TAG-like material) in known manner. When insoluble TAG (or TAG-like material) or insoluble specific lectin is used, the solid phase is simply separated from the liquid phase by centrifugation, filtration or decanting. In other cases, chromatography, electrophoresis, salting, fractionation, dialysis, gel filtration, adsorption or combinations thereof may be used, or a separation process may be employed using agar gel, agarose gel or polyacrylamide gel as described in Japanese Patent Application (OPI) No. 151263 / 80th

The activity of the labeling agent for the product thus separated can be measured by a suitable method selected from the techniques already described according to the nature of the labeling agent. The measured activity can be used to determine the TAG level of the sample.

As described above, the present invention makes it possible to determine the TAG level in body fluid in an advantageous manner. The particular TAG level can be used to diagnose cancer at any stage, and the invention is particularly useful for detecting cancer at an early stage. Furthermore, in the present method, the glycine binding is determined so that the method, in comparison with the usual methods, most of which use antibodies to measure the protein portion (α ^ -photoprotein, CEA, etc.), can be used to diagnose a wider range of 30 cancers such as malignant lymphadenosis, malignant lymphoma, chorionic epithelioma malignum, liver cancer, gallbladder cancer, pancreatic cancer, lung cancer, gallbladder cancer, thyroid cancer, multiple myeloma, stomach cancer, breast cancer, colon carcinoma, ovarian cancer, ovarian cancer, ovarian cancer prostate cancer, liposarcoma, malignant melanoma, uterine cancer and primary stomach sarcoma. The process is specific according to the specific specification used.

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17 tin. If AG-binding lectin is used, the method of the invention is particularly useful in diagnosing cancer diseases arising from undifferentiated cells, such as malignant lymphadenosis, malignant lymphoma, and chorionic epithelioma malignum. Furthermore, the method for diagnosing cancer diseases using AG-binding lectin or L-fucose-binding lectin according to the invention is advantageous in that this method shows a significantly reduced cross-reactivity 10 with diseases other than cancer, e.g. hepatic induration, hepatitis, gastric ulcer, diabetes mellitus, colitis, etc., in comparison with conventional cancer disease diagnostic methods using the α 2 -photoprotein, CEA and the like. The conventional diagnostic methods very often give positive results for hepatic diseases especially hepatic induration, acute and chronic hepatitis and the like and cause cancer diagnoses to be largely confused with other diagnoses. In contrast, the diagnostic method 20 of the present invention shows a low cross-reactivity with hepatic diseases and thus provides accurate cancer diagnoses.

Furthermore, the present method enables the determination of carbohydrates and carbohydrate derivatives (e.g., glycopeptides, glycoproteins, glycolipids, glycoterpenes, and glycosteroids) by the AG or L-fucose end group.

The process of the invention will be described in more detail below with reference to the following examples.

Example 1 (i) Activation of Peroxidase.

5 mg of peroxidase (from horseradish) was dissolved in 1 ml of a 0.3 M aqueous sodium hydrogen carbonate solution.

To the resulting solution was added 0.1 ml of a 0.1 M ethanolic fluorine nitrobenzene solution and after gentle stirring for 1 hour at room temperature, 0.1

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18 ml of a 0.06 M NalO 4 solution followed by gentle stirring for 30 minutes at room temperature. To the reaction mixture was added an additional 1 ml of 0.16 M ethylene glycol and the resulting solution was gently stirred at room temperature for 1 hour. Then, the solution was dialyzed against 0.01 M carbonic acid sodium hydrogen carbonate buffer solution (pH 9.5) at 4 ° C for an entire day and night.

(ii) Method for labeling lectin with peroxidase.

10 (Dolichos bean lectin peroxidase): 5 mg of Dolichos bean lectin was dissolved in 3 ml of the activated peroxidase obtained according to (i) and gently stirred at room temperature for 2 to 3 hours to obtain reaction. 5 ml of NaBH 3 was added and reacted at 4 ° C for 3 hours. Then, this solution was dialyzed against a 0.1 M tris-hydrochloric acid solution (pH 7.4) for 1 day and 1 night and subjected to Sephadex G 150 gel column chromatography (eluent: 0.1 M tris-hydrochloric buffer solution, pH 7.4) for 20 of gel filtration. Each fraction was measured by OD2gQ and OD403 fictions with peaks for OD2go ° 9 OD403 ^ lev collected.

(iii) Method for labeling lectin with peroxidase.

(Lotus tetragonolobus lectin peroxidase): 5 mg of Lotus tetragonolobus lectin was dissolved in 3 ml of the activated peroxidase obtained according to (i) and the solution was gently stirred at room temperature for 2 to 3 hours. 5 mg of NaBH 3 was added to the solution and allowed to stand at 4 ° C for 3 hours.

The reaction liquid was dialyzed against a 0.1 M tris-hydrochloric acid buffer solution (pH 7.4), for a whole day and a whole night and subjected to gel filtration by Sephadex G 150 gel column chromatography (eluent: 0.1 M tris-hydrochloric acid buffer solution, pH 7.4). Each fraction was measured at OD2gQ and OD ^^, and fractions with peaks for OD2sq ° 9 odao3 ^ ev were collected.

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(Iv) Process for the preparation of insoluble lectin.

15 g of CNBr-activated agarose was suspended in 3 liters of 0.001 N hydrochloric acid and, after standing for 30 minutes, washed with 1 liter of 0.1 M sodium hydrogen carbonate (pH 8.5) on a glass filter. A total amount of approx. 50 ml of activated agarose. This was suspended in 200 ml of 0.1 M sodium hydrogen carbonate (pH 8.5) and 5 ml of a 0.01 M 10 phosphate buffer solution (pH 7.7) containing 50 mg was added.

Dolichos bean lectin followed by reaction at room temperature for 2 hours with occasional stirring. After completion of the reaction, the reaction solution was washed on a glass filter and the reaction product was added to 200 ml of a 1 M monoethanolamine solution (pH 8.5) and reacted for 2 hours at room temperature. The reaction product was then washed on a glass filter. In the above procedures, the washing was carried out with 1 liter of 0.1 M acetic acid buffer solution (containing 20 0.5 M NaCl) and 1 liter of 0.1 M boric acid buffer solution (containing 0.5 M NaCl) alternately three times. .

(v) Process for Preparation of Insoluble Lectin.

15 g CNBr-activated agarose was suspended 25 in 3 liters of 0.001 N hydrochloric acid and, after standing for 30 minutes, washed with 1 liter of 0.1 M sodium hydrogen carbonate (pH 8.5) on a glass filter. A total amount of approx. 50 ml of activated agarose. This was suspended in 200 ml of 0.1 M sodium bicarbonate 30 (pH 8.5) and 5 ml of 0.01 M phosphate buffer solution (pH 7.7) containing 50 mg of Lotus tetragonolobus lectin followed by reaction with room temperature for 2 hours with occasional stirring. After completion of the reaction, the reaction solution was washed on a glass filter and the reaction solution was added to 200 ml of 1 M monoethanolamine solution (pH 8.5) and reacted for 2 hours at room temperature. Then, the reaction product was washed on a glass filter. In the foregoing,

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In 20 ways, washing was performed with 1 liter of 0.1 M acetic acid buffer solution (containing 0.5 M NaCl) and 1 liter of 0.1 M boric acid buffer solution (containing 0.5 M NaCl) alternately three times.

(Vi) Process for producing TAG-like material.

1 g of swine gastric mucosal glycoprotein (hereafter abbreviated to PGM) was suspended in 100 ml of a 0.05 M phosphate buffer solution 10 (pH 7.0) and a 1 N aqueous NaOH solution was added dropwise for setting the pH to 11.

After stirring for 30 minutes at room temperature, the mixture was centrifuged for 10 minutes at 3000 rpm and the supernatant was adjusted to pH 7.0 with 15 IN HCl followed by centrifugation for 10 minutes at 3000 rpm. was dialyzed against 10 liters of a 0.01 M phosphate buffer solution (pH 7.0) overnight to obtain purified TAG-like material (pure PGM).

(Vii) Process for preparing labeled TAG-like material.

(a) Labeling with an enzyme (PGM peroxidase): 4 mg of horseradish peroxidase (HRPO) (0.1 µM) was dissolved in 1 ml of distilled water. To this was added 0.2 25 ml of 0.1 M NalO 2 and, after stirring at room temperature for 20 minutes, the solution was dialyzed against 1 mM acetic acid epuffer solution (pH 4.4) for a full day and night to remove unreacted NalO 2. To this dialyzed reaction solution was added ca. 60 µl 0.2 M hydrogen carbonate buffer solution (pH 9.5) to adjust the pH of the solution to 9.0. Then, to this solution, 0.6 ml of PGM (10 mg / ml) dissolved in a 0.01 M hydrogen carbonate buffer solution (pH 9.5), which was mixed for 2 hours at room temperature, was immediately added and 0 was added. 1 ml of a 4 mg / ml NaBH 4 solution in distilled water followed by standing at 4 ° C for 2 hours. This solution was further dialyzed against a 0.01 M phosphate buffer solution (pH 7.2) for a full day

DK 154859 B

21 and a whole night and purified using Sephadex G-200 (1.5 x 150 cm) to obtain pure PGM peroxidase (PGM-POX). The gel effluent was collected in 5 ml portions, the absorption of which was measured by and OD ^ q ^.

(B) Isotope labeling (- ^^ I-PGM).

125 PGM were labeled with I according to an oxidative iodination process using chloramine T.

10 µg PGM was dissolved in 50 µl of a 0.2 M phosphate buffer solution (pH 7.0), and 10 µl of 1mCi Na 2 2 µl (carrier-free; New England Nuclear Co.) and 50 µg / 100 µl were added. chloramine T solution in a 0.2 M phosphate solution and after mixing at room temperature for 30 seconds, 100 µg / 100 µl was added

Na9 S2 Or solution in a 0.2 M phosphate buffer solution.

Then 1 mg of Na I was added and mixed. The I-PGM thus obtained was purified on Sephadex G-50 125 (1 x 30 cm). The I-PGM thus prepared had a radioactivity of approx. 1 to 2 µCi / µg.

(viii) Determination procedure in 125 20 0.1 ml of I-PGM (100 ng 0.17 µCi corresponding to about 2.4 x 10 cpm) obtained according to (vii), 0.1 ml of the pure standard PGM (0, 1 ug / ml, 0.2 ug / ml, 0.5 ug / ml, 1 ug / ml, 2.5 ug / ml, 5 ug / ml and 10 ug / ml) obtained according to (vi), 0.1 dichloromethane (10 µg / ml) and 0.2 ml of a 0.05 M phosphoric acid buffer solution (0.15 M NaCl, 0.1% BSA: 0.02% NaN 2) were mixed in a 10 x 75 mm glass tube and incubated at 25UC for 1 hour. After completion of the reaction, 0.1 ml of anti-DBA rabbit serum (DBA = Dolichos bean lectin, made by EY Laboratory; 10 times diluted solution) was added to the 125 I-PGM bound to DBA and 125 I-PGM bound to DBA. , and after incubation at 25 ° C for 1 hour, the reaction solution was centrifuged at 4 ° C for 30 minutes at 125,000 rpm. The radioactivity of the precipitate (I-35 PGM bound to DBA) was counted to produce a standard curve ( Figure 1). As can be seen from the results thus obtained, the percentage of bound material (B / T = bound / total) was usually 20 to 25%, and

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22, 50% inhibition was achieved at a concentration of 0.06 µg / ml.

(ix) Method of Determination: 125 0.1 ml of I-PGM (100 ng 0.17 yCi corresponding to about 2.4 x 10 cpm) obtained according to (vii), 0.1 ml of the 5 pure standard PGM (0 , 1 ug / ml, 0.2 ug / ml, 0.5 ug / ml, 1 ug / ml, 2.5 ug / ml, 5 ug / ml and 10 ug / ml) obtained according to (vi), 0, 1 ml of Lotus tetragonolobus lectin (10 µg / ml) and 0.2 ml of a 0.05 M phosphoric acid buffer solution (0.15 M NaCl, 0.1% BSA: 0.02% NaN 2) were mixed in a 10 x 75 mm glass tube and incubated at 25 ° C for 1 hour.

After completion of the reaction, 0.1 ml of anti-Lotus tetragonolobus lectin rabbit serum (prepared by E.Y.

Laboratory; 10 times diluted solution) to it to 125

Lotus tetragonolobus lectin bound I-PGM and the 125 I to Lotus tetragonolobus lectin not bound I-PGM, and after incubation at 25 ° C for 1 hour, the reaction solution was centrifuged at 4 ° C for 30 minutes at 125,000 rpm. the precipitate (I-PGM bound to Lotus tetragonolobus lectin) was counted 20 to produce a standard curve (Fig. 2). As can be seen from the results thus obtained, the percentage of bonded material (B / T) was usually 20 to 25% and 50% inhibition was achieved at a concentration of 0.6 µg / ml.

(X) Preparation of insoluble TAG-like material.

(Preparation of PGM-insoluble disks):

An excess of PGM was added to 100 ml of a 0.01 M phosphate buffer solution (pH 7.0) to prepare a suspension. To this was added a 0.01 N NaOH solution to adjust the suspension pH of approx. 11 followed by centrifugation at 3000 rpm. for 20 minutes to obtain the supernatant. To this supernatant, 0.03 N HCl was added dropwise to adjust the pH to 7.0 and centrifuged again at 3000 rpm. for 20 minutes. The supernatant was dialyzed against a 0.01 M phosphate buffer solution (pH 7.0) to prepare a PGM solution. What

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23 regarding the carbohydrate content and protein content of the solution, the hexose content was measured at 5 to 7 mg / ml according to a phenol-sulfuric acid method using glucose as standard, and the protein content was measured to be 5 1 to 2 mg / ml using BSA as standard. The PGM solution was subjected to the radiation-induced polymerization below.

The radiation-induced polymerization was carried out as follows: Hydroxyethyl methacrylate (HEMA) (used as a monomer) was mixed with the above-described PGM solution at a mixing ratio of 33:67 and the resulting mixture was placed in a 1 cm x 15 to 20 cm glass tubes and quickly lyophilized to -70 ° C

g or less. Then it was irradiated with 1 x 10 15 rad gamma rays to polymerize the monomer.

The polymer bar was cut into slices each 10 mm thick.

(xi) Preparation of insoluble TAG-like material.

20 g (dry weight) CNBr-activated Sepharose 4B

(manufactured by Pharmacia AB) was suspended in 3 liters of 0.001 N hydrochloric acid and, after standing for 30 minutes, washed with 1 liter of 0.1 M sodium hydrogen carbonate (pH 8.5) on a glass filter to obtain ca. 50 ml activated 25 Sepharose. This was suspended in 200 ml of 0.1 M sodium bicarbonate (pH 8.5) and 5 ml of a 0.01 M phosphate buffer solution (pH 7.7) containing 50 mg PGM followed by reaction at room temperature for 2 hours with occasional stirring.

After completion of the reaction, the reaction solution was washed on a glass filter and the reaction product was added to 200 ml of a 1 M monoethanolamine solution (pH 8.5) followed by reaction at room temperature for 2 hours. Then, the reaction mixture was washed on a glass filter with 1 liter of a 0.1 M acetic acid buffer solution (containing 0.5 M NaCl) and 1 liter of a 0.1 M boric acid buffer solution (containing 0.5 M NaCl) alternately 3 times.

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(Xii) Process for the preparation of insoluble TAG-like material (PGM spheres): 10,000 Polystyrene spheres of 5 6.4 mm diameter manufactured by Precision Plastic Co., Ltd., U.S.A. was washed with a dilute solution of (b) synthetic soap (Mamalemorr®, manufactured by Lion Co.,

Ltd.) at a concentration of 1.5 ml / liter of distilled water and then with distilled water. Further, after being immersed in 0.5 M aqueous NaOH solution for 3 days, the beads were carefully washed until the pH of the wash liquid was about 10%. 6. The 10,000 balls thus washed were added to 2.5 liters of a 35 (w / v)% PGM solution in 50 mM acetic acid buffer adjusted to pH 4.5 with 10 N NaOH, rotated at ca. 10 rpm for 24 hours, filtered and washed with 8 liters of distilled water 4 times. Then, the beads were added to 2.5 liters of glutaraldehyde solution with a final concentration of 1 (vol / vol)% in 50 mM sodium 20 phosphate buffer (pH 7), rotated at 10 rpm. for 2 hours, filtered and washed with distilled water in the same manner as described above. The 10,000 balls thus treated were added to 2.5 liters of 1 M glycine solution in 50 mM sodium phosphate buffer (pH 7.0), rotated at 10 rpm. for 2 hours, filtered and washed with distilled water in the same way as above, followed by drying at 37 ° C overnight to obtain PGM beads. The surface area of the spheres was determined by the Orcinol® SO 2 method (M. Schonenberger, et al., 30 Z. Physiol. Chem., 309, 145 (1957)) and the result obtained was 2.7 ± 0.2 µg PGM /ball.

Example 2 (i) Preparation of test samples:

5 ml of blood samples were taken from patients 35 with various cancers, non-malignant patients and healthy subjects using heparin (500 units) treated syringes and

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The 25 valves were centrifuged at 2000 rpm. for 10 minutes. Experimental samples were prepared by the supernatant.

(ii) Measurement: To 0.1 ml of each of the test samples prepared according to (i), an equal volume of 10 µg / ml of Dolichos bean lectin peroxidase in 0.2 ml of 0.15 M phosphate buffer solution was added and added. one PGM-insoluble disk; the mixture was well stirred and left at 20-37 ° C for 24 hours. After careful washing of the PGM-insolubilized slice in the reaction liquid, the activity of the Dolichos bean lectin peroxidase bound to the slice was measured from OD4g2 by the enzymatic activity assay, using as the sucrate and orthophenylenediamine as colorant. A calibration curve was obtained by replacing the test samples with a standard material (PGM) of varying concentrations as shown in FIG. 3 and 3 ", wherein the values of TAG-D are indicated respectively as a hexose-reduced level of PGM and N-acetylgalactose-amine-reduced level of PGM.

(iii) Results:

The results were obtained using a standard curve shown in FIG. Third

25 As shown in FIG. 4 all healthy individuals had a TAG-D value of approx. 0.1 n mol / ml.

Example 3 (i) Preparation of agarose gel:

An agarose (product of Iwai Kagaku Co., Ltd.) 30 was suspended in 0.01 M tris-hydrochloric acid buffer solution (pH 7.5) to give a concentration of 1 (w / w 6%). The suspension was heated. at 70-80 ° C to form a solution to which 0.01 (w / v) thimerosal was added The solution was partitioned in reagent glass in 1 ml amounts and left at room temperature to prepare agarose gels with a concentration of 1 (wall t / wall i)%.

DK 154859 B

(Ii) Measurement:

Two test samples (each of 200 µl) were placed in two test tubes to which 50 µl of peroxide-labeled Dolichos bean lectin (3.5 µg / ml lectin in 0.1 M Tris-hydrochloric acid buffer solution with pH) was added. value of approx.

7.5). Each mixture was stirred lightly and left at 20-30 ° C for one hour.

To one sample (sample A) was added 250 µl of 8 (w / v)% solution of polyethylene glycol (mol weight = 10 6,000) in 0.1 M tris-hydrochloric acid buffer solution, and to the other sample (sample B) 250 µl 0.1 M tris-hydrochloric acid buffer solution was added and both mixtures were gently stirred. The two samples were left at 20-30 ° C for 30-60 minutes and centrifuged with a swing-15 rotor at 1000 G for 40 to 60 minutes. The supernatant (50 µl) was poured into 2 ml of physiological saline solution, and. the mixture was carefully stirred. To each mixture was added 500 µl of a solution of peroxidase substrate (referred to below as substrate liquid) and the mixture was left in a dark room at 20-30 ° C for 30 minutes. The substrate liquid was prepared by adding ortho-phenylenediamine and aqueous hydrogen peroxide to 0.1 M citrate-phosphate buffer solution to obtain final concentrations of 6% and 0.1%, respectively. The substrate liquid is preferably kept at 4 ° C until used. The enzyme reaction was quenched by the addition of 1 ml of 2N hydrochloric acid. The color change was assessed by measuring the absorbance at 492 nm with a spectrophotometer.

The value (c) obtained by subtracting the absorbance (a) of sample A from the absorbance (b) of sample B was plotted as the amount of TAG-bound lectin. The result is shown in FIG. 5, wherein the circles indicate the healthy individuals, the numbers (1), and (2) indicate patients with liver cancer, and (3) indicate patients with malignant lymphadenopathy. The samples having a higher value (c) than the samples from the healthy individuals indicate a higher plasma TAG level and suggest that cancerous cells had formed in the hosts.

DK 154859 B

(Iii) Sandwich Procedure: 200 µg of DBA agarose was added to 100 µl of a 0.05 M phosphate buffer solution (pH 7.0) dissolved in 1 to 10 µg / ml PGM and incubated at 5 µl. 25 ° C for 1 hour with stirring. After washing the reaction solution 3 times with a 0.05 M phosphate buffer solution (pH 7.0), 6 µg of DBA labeled with peroxidase obtained according to Example 1- (ii) and 100 µl of a 0.05 M phosphate buffer solution (pH 7) were added. 0) followed by 10 incubation at 25 ° C for 1 hour with stirring. After centrifugation at 3000 rpm. for 10 minutes, the precipitate was recovered and washed three times with 0.05 M phosphate buffer solution (pH 7.0).

60 mg of orthophenylenediamine was dissolved in 20 ml of 15 0.2 M Mcllevein buffer (pH 5.8), and to the resulting solution was added a final concentration of 0.02 drum / volume (%) and the mixture was stirred to form of a coloring agent.

Into a test tube were placed 2 ml of physiological saline solution and 500 µl of the staining agent, as well as the washed ball followed by incubation at room temperature for 30 minutes. The enzymatic reaction was then quenched with 1 ml of 3 N HCl. Absorbance was measured at 492 nm.

The results thus obtained are shown in FIG. 6th

(Iv) Competition Procedure: 100 µl of a sample (test sample obtained according to Example 2- (i)) was placed in a test tube to which was added 500 µl of 0.3 M tris-HCl buffer (pH 7, 4) containing finally 0.22 (w / v)% gelatin, 5 mM CaCl 2, and 5 mM MgC 2. One PGM sphere (insolubilized TAG-like material prepared according to Example 1- (xii)) and 100 µl lectin peroxidase (the lyophilized labeled DBA prepared according to Example 1- (ii) at a concentration of 1 mg / l of the above-described tris. HCl buffer was added to the sample and after stirring the mixture was incubated for 48 hours at 4 ° C. The reaction mixture was removed using an aspirator and the sphere was washed with 2 ml of physiological saline solution followed

DK 154859 B

28 by removing the washing liquid using an aspirator. This washing operation was repeated 3 times.

60 ml of orthophenylenediamine was dissolved in 20 ml of 0.2 M Mcllevein buffer (pH 5.8) and 5% of the resulting solution was added to a final concentration of 0.02 (vol / vol)%. and the mixture was stirred to form a staining agent.

Into a test tube were placed 2 ml of physiological saline solution and 500 μΐ of the staining agent as well as the washed sphere, followed by incubation at room temperature for 30 minutes. The enzymatic reaction was then quenched with 1 ml of 3 N HCl. The optical density of the reaction mixture was measured at 492 nm. At the same time, the absorbance was measured in the same way except that the sample was replaced with different concentrations of a standard material (PGM) to produce a calibration curve (Fig. 7). Furthermore, TAG was determined in the test samples obtained in Example 2- (i) using the calibration curve. The results obtained are shown in Figures 20 8 (a), 8 (b) and 8 (c).

Example 4 (i) Competition Procedure:

A round slice (insoluble TAG-like material prepared according to Example 1- (x)) was placed in 50 µl of Lotus tetragonolobus lectin-bound peroxidase (labeled lectin prepared according to Example 1- (iii)) and 200 µl of the sample (PGM of various concentrations) and incubated at 25 ° C for 20 hours. Then, the slice was washed with PBS and placed in 2.0 ml of an aqueous brine solution and 0.5 ml of a peroxide semi-material was added thereto, followed by incubation at 25 ° C for 1 hour. Then 1.0 ml of 3 N hydrochloric acid was added and the absorbance measured at 492 nm. At the same time, the absorbance was measured in the same way except that the sample was exchanged with different concentrations of a standard material (PGM) to produce a calibration curve (Figures 9 and 9 'wherein the TAG values are given respectively as

DK 154859B

29 a hexose-reduced level of PGM and an L-fucose-reduced level of PGM).

(ii) Sandwich Method: 200 µg Lotus tetragonolobus lectin agarose was added to 100 µl of a 0.05 M phosphate buffer solution (pH 7.0), dissolved in 1 to 10 µg / ml PGM and incubated at 25 µl. ° C for 1 hour with stirring. After washing the reaction solution 3 times with a 0.05 M phosphate buffer solution (pH 7.0), 6 µg of 10 peroxidase-labeled Lotus tetragonolobus obtained according to Examples 1- (iii) and 100 µl of a 0.05 M phosphate buffer solution were added. (pH 7.0) followed by incubation at 25 ° C for 1 hour with stirring. After centrifugation at 3000 rpm. for 10 minutes, the precipitate was recovered and washed 3 times with a 0.05 M phosphate buffer solution (pH 7.0).

60 mg of orthophenylenediamine was dissolved in 20 ml of 0.2 M Mcllevein buffer (pH 5.8), and H 2 O 2 was added to the resulting solution to a final concentration of 0.02 (vol / vol)% and the mixture was stirred to form a staining agent.

2 ml of physiological saline solution and 500 ml of the staining agent and the washed ball were placed in a test tube and incubated at room temperature for 30 minutes. The enzymatic reaction was then quenched with 1 ml of 3 N HCl. The absorbance was measured at 492 nm. At the same time, the absorbance was measured in the same way except that the sample was replaced with different concentrations of a standard material (PGM) 30 to produce a calibration curve shown in FIG. 10th

(iii) Determination:

To 0.1 ml of each of the test samples prepared according to Example 2- (i) was added an equal volume of 10 µg / ml Lotus tetragonolobus lectin peroxidase in 0.2 ml of 0.15 M phosphate buffer solution and one PGM. - insoluble slice and the mixture was stirred well and left at 20 to 37 ° C for 24 hours

DK 154859 B

30

After careful washing of the disk with insolubilized PGM in the reaction liquid, the activity of the Lotus tetragonolobus lectin peroxidase bound to the disk was measured by the OD4g2 process to determine enzymatic activity using substrate and orthophenylenediamine as a staining agent. A calibration curve was obtained by replacing the test samples with a standard material (PGM) of various concentrations. The curve is shown in FIG. 11, wherein the values of 10 TAG-D are indicated as a hexose-reduced PGM level. results:

As shown in FIG. 11 all healthy subjects had a TAG-D value of less than 3 nM / ml.

(iv) Determination: 15. Two of the test samples (each of 200 μΐ) prepared according to Example 2- (i) were placed in two test tubes and 50 μΐ of the peroxidase-labeled Lotus tetragonolobus lectin (3.5 μg / ml lectin) was added. in 0.1 M tris-hydrochloric acid buffer solution having a pH of about 7.5) prepared according to Examples 1- (iii).

Each of the mixtures was gently stirred and left at 20 to 30 ° C for one hour. To one sample (sample A) was added 250 μΐ of an 8 (w / v)% solution of polyethylene glycol (mole weight = 6000) in 0.1 M tris-25 hydrochloric acid buffer solution, and to the other sample (sample B) 250 µΐ of 0.1 M tris-hydrochloric acid buffer solution was added and both mixtures were gently stirred. Both samples were left at 20-30 ° C for 30 to 60 minutes and centrifuged with a rotary rotor at 1000 G for 40 to 30 60 minutes. The supernatant (50 μΐ) was decanted into 2 ml of physiological saline and the mixture was stirred carefully. To each of the mixtures was added 500 μΐ of a solution of peroxidase substrate (hereinafter referred to as substrate liquid) and the mixture was left in a dark room at 20-30 ° C for 30 minutes. The substrate liquid was prepared by adding orthophenylenediamine and aqueous hydrogen peroxide to 0.1 M citrate buffer solution to a final concentration of

DK 154859 B

31 show 6% and 0.1%. The substrate liquid is preferably kept at 4 ° C until used. The enzymatic reaction was stopped by adding 1 ml of 2N hydrochloric acid. The color change was assessed by measuring the absorbance at 5 492 nm with a spectrophotometer. The value (c) obtained by subtracting the absorbance (a) of sample A from the absorbance (b) of sample B was plotted as the amount of TAG-bound lectin. The result is shown in FIG. 12, where the circles indicate the healthy persons, the numbers (1) 10 and (2) indicate patients with stomach cancer, and (3) indicate patients with breast cancer. The samples having a higher value (c) than the samples from the healthy individuals indicate a higher plasma TAG level and suggest that cancerous cells have formed in the hosts.

(V) Competition procedure: 100 μΐ of a sample (test sample obtained according to Example 2- (i)) was placed in a test tube to which was added 500 μΐ 0.3 M tris-HCl buffer (pH 7.4 ) with a s lut concentration of 0.22 (w / v)% gelatin, 20 5 mM CaCl 2 and 5 mM MgC 2. One PGM sphere (insoluble TAG-like material prepared according to Example 1- (xii)) and 100 µl lectin peroxidase (the lyophilized labeled DBA prepared according to Example 1- (iii) at a concentration of 1 mg / l in the tris-25 HCl buffer described above) was added to the sample and after stirring the mixture was incubated for 48 hours at 4 ° C. The reaction mixture was removed using an aspirator and the sphere was washed with 2 ml of physiological saline solution followed by removal of the washings using an aspirator. The wash operation was repeated 3 times.

60 mg of orthophenylenediamine was dissolved in 20 ml of 0.2 M Mcllevein buffer (pH 5.8), and H2O2 was added to the resulting solution to a final concentration of 0.02 (vol / vol)% and the mixture was stirred to form a staining agent.

Into a test tube were placed 2 ml of physiological saline solution and 500 μΐ of the staining agent and the

DK 154859B

32 washed sphere, then incubated at room temperature for 30 minutes. The enzymatic reaction was then quenched with 1 ml of 3 N HCl. The optical density of the reaction mixture was determined at 492 nm. At the same time, the absorbance was measured in the same way except that the sample was replaced with different concentrations of a standard material (PGM) to produce a calibration curve (Fig. 13). Furthermore, TAG in the test samples obtained according to Example 2- (i) was determined using the calibration curve. The results obtained are shown in FIG. 14th

Claims (15)

A method for determining the level of tumor-associated glycobinding (TAG) in a body fluid sample, characterized by reacting TAG in a body fluid sample with an N-acetyl-D-5-galactosamine (AG) -binding lectin or L-fucose-binding lectin to form a TAG lectin complex and measure the amount of the TAG lectin complex or of unreacted lectin. 2. A process according to claim 1, characterized in that the reaction between the TAG and the lectin is carried out by competing the reaction of the body fluid TAG to be measured and a certain amount of insoluble TAG or insoluble TAG-like material with a certain amount of labeled lectin, separates the insolubilized TAG or insolubilized TAG-like material bound to labeled lectin and unbound lectin and measures both labeling activity. Method according to claim 1, characterized in that the reaction between the TAG and the lectin is carried out by competing reactively the body fluid TAG to be measured and a certain amount of labeled TAG or labeled TAG-like material with a certain amount. lectin or insolubilized lectin, separates the labeled TAG or labeled TAG-like material bound to lectin or to insolubilized lectin and unbound labeled TAG or labeled TAG-like material and measures both labeling activity. Method according to claim 1, characterized in that the reaction between TAG and the lectin is carried out by reacting the body fluid TAG to be measured with an insoluble lectin to form a TAG-insoluble lectin complex, reacting this complex with a specific amount of labeled lectin, the complex and unbound labeled lectin bound to the labeled lectin differ from each other and measure both labeling activity. DK 154859 B Method according to claim 1, 2, 3 or 4, characterized in that it further comprises separating TAG from said body fluid sample and reacting said separated TAG with said lectin. Process according to claim 2 or 4, characterized in that said labeled lectin is a lectin labeled with an enzyme, a fluorescent substance or a radioactive substance. The method of claim 6, characterized in that a protective protein is added to said body fluid. Method according to claim 1, 2, 3 or 4, characterized in that the body fluid is blood, tissue fluid, lymph, thoracic fluid, ascites, amniotic fluid, gastric juice, urine, pancreas, cerebrospinal fluid or saliva. Method according to claim 8, characterized in that said body fluid is blood serum or blood plasma. Process according to claim 6, characterized in that the enzyme is glucoamylase, glucose oxidase, peroxidase, alkali phosphatase or hemeoctapeptide or active fragment thereof. Process according to claim 6, characterized in that the fluorescent substance is fluorescein, fluorescein isothiocyanate, rhodamine or dansyl chloride. Process according to claim 6, characterized in that the radioactive substance is radioactive iodine or tritium. A method of diagnosing cancer, characterized by measuring the level of tumor-associated glycobinding in body fluid from a patient by the method of any one of claims 1, 2, 3 and 4 and comparing the level thus measured with the level of a patient. person with normal health. Equipment for determining the TAG level in a body fluid, characterized in that it comprises AG-binding lectin or L-fucose-binding DK 154859B lectin as a specific agglutinating agent for TAG. Equipment according to claim 14, characterized in that the lectin has been lyophilized and that the equipment further contains a reconstitution reagent which contains a water-based or water-miscible solvent.