Method for the Detection of Cancer

This invention relates to a process for immunologically assaying glycolipid in an organism. Recently there has been observed in many countries a very high mortality rate due to cancer. The expectation, need of detection, and treatment for cancer in its early stage have become progressively greater. Morphological tests such as imaging diagnostic technology and immuno-biochemical diagnostic technology, which is a noninvasive test carried out by collecting a small amount of humors, have been used to detect cancer in its early stage. The role of the immunobiochemical diagnostic technology has become increasingly important. The following have been attempted to detect tumor associated antigens, the number of which would be remarkably increased by the proliferation of cancer cells; to diagnose cancer immuno-biochemically, which has resulted in the finding of .alpha.-fetoprotein, carcinoembryonic antigen and the like; and the development of assay systems thereof which are clinically useful in practice. However, these markers are not satisfactory from the viewpoint of an early diagnosis of cancer. Therefore it has been eagerly desired to find a novel tumer associated antigen as well as to develop an assay system and find clinical application thereof.

Turning attention to glycolipid which is one of the constituents of cell membrane, we have investigated the relationship between glycolipid and cell adhesion by analyzing glycolipid in cancerous cells and examining the metabolism thereof. Consequently we have found that the difference in cell adhesion might be represented by the difference in glycolipid metabolism. In particular, a biosynthetic pathway of glycolipid via another glycolipid which is seldom observed in a normal cell has been found in a highly malignant cancerous cell devoid of cell adhesion. That is to say, four different glycolipids have been found to increase with the proliferation of cancer cells. These glycolipids have been identified as asialo GM.sub.1, asialo GM.sub.2, fuco GA.sub.1 and paragloboside as a result of various analyses. The structures of these glycolipids are as follows:

asialo GM.sub.1, Gal-GalNAc-Gal-Glc-Cer;

asialo GM.sub.2, GalNAc-Gal-Glc-Cer;

fuco GA.sub.1, ##STR1## and paragloboside, Gal-GlcNAc-Gal-Glc-Cer

wherein Gal represents galactose, Glc represents glucose, Fuc represent fucose, GalNAc represents N-acetylgalactosamine, GlcNAc represents N-acetylglucosamine and Cer represents ceramide.

We have found that these glycolipids may be available as novel cancer markers, since they would increase in human cancerous cells and would therefore be extremely useful in cancer diagnosis by determining their concentrations in humors. We have further established a simple process for immunologically assaying these glycolipids with high sensitivity and accuracy. It has been hitherto impossible to clinically determine such a low concentration of these glycolipids in humors.

An object of the present invention is to provide novel cancer markers.

Another object of the present invention is to provide a process for immunologically assaying glycolipid in an organism.

Other objects and advantages of the present invention will be apparent from the following description.

According to the present invention, glycolipids in an organism, such as asialo GM.sub.1, asialo GM.sub.2, fuco GA.sub.1 and paragloboside, are immunologically assayed by using respective antibodies specific thereto.

Methods of immunological assaying include a method based on a competitive reaction using a labelled antigen, a sandwich method using a labelled antibody and an immunometric assay using a labelled antibody.

Antibodies available for assaying asialo GM.sub.1, asialo GM.sub.2, fuco GA.sub.1 and paragloboside may be prepared by intracutaneously injecting a mixture of one of these glycolipids and a foreign high-molecular carrier such as bovine serum albumin, methylated bovine serum albumin or erythrocytic membrane protein as an antigen into an animal other than a human, such as a rabbit. According to the present invention, asialo GM.sub.1, asialo GM.sub.2, fuco GA.sub.1 and paragloboside in humors can be assayed by an immunological method using an antibody obtained as described above. Alternatively, it is possible to use a monoclonal antibody to assay the glycolipid.

The present invention will be illustrated more particularly, using asialo GM.sub.1 as an example.

1. Assay of asialo GM.sub.1 by a competitive reaction:

Asialo GM.sub.1 to be assayed and a given amount of labelled asialo GM.sub.1 are allowed to competitively react with a given amount of antibody. Then the labelled asialo GM.sub.1 conjugated with the antibody is separated from the labelled asialo GM.sub.1 not conjugated with the antibody. The activity of the labelling agent in one or both of these fractions is determined to assay the asialo GM.sub.1. Alternately, it is possible in such an assay based on the competitive reaction to use a labelled oligosaccharide prepared by labelling the saccharide chain portion (oligosaccharide) of asialo GM.sub.1, instead of using the labelled asialo GM.sub.1 as the labelled antigen to assay asialo GM.sub.1, and allowing the labelled oligosaccharide to competitively react in the same manner as described above.

2. Assay of asialo GM.sub.1 by a sandwich method:

Asialo GM.sub.1 to be assayed is allowed to react with an insolubilized antibody prepared by conjugating an antibody with an insoluble material (solid phase) to form an antibody/asialo GM.sub.1 conjugate. The resulting conjugate is allowed to react with a labelled antibody prepared by labelling an antibody with a labelling agent to form a sandwich-like antigen/antibody conjugate, i.e. antibody/asialo GM.sub.1 /labelled antibody. Then the activity of the labelling agent on the obtained antigen/antibody conjugate is determined to assay the asialo GM.sub.1.

3. Assay of asialo GM.sub.1 by an immunometric method:

(1) Asialo GM.sub.1 in humors to be assayed is allowed to react with a given amount of a labelled antibody prepared by labelling an antibody with a labelling agent. Then unreacted labelled antibody is adsorbed by an immunoadsorbent prepared by conjugating asialo GM.sub.1 with an insoluble material (solid phase). The activity of the labelling agent on the immunoadsorbent is determined to assay the asialo GM.sub.1.

(2) Asialo GM.sub.1 to be assayed is allowed to react with a given amount of an antibody. Unreacted antibody is adsorbed by an immunoadsorbent prepared by conjugating asialo GM.sub.1 with an insoluble material (solid phase). Then, labelled anti-IgG antibody prepared by labelling an anti-IgG antibody against the antibody with a labelling agent is allowed to react with the antibody on the immunoadsorbent. Then the activity of the labelling agent of the labelled anti-IgG antibody conjugated with the immunoadsorbent is determined to assay the asialo GM.sub.1.

In these assay methods, a standard curve which has been previously drawn by carrying out the same procedure as described above by using standard asialo GM.sub.1 of known concentration is used to determine the amount of asialo GM.sub.1.

Table 1 shows the results of a recovery test by the present process. These recoveries were obtained by adding asialo GM.sub.1 of various known concentrations to three specimens. The recoveries ranged from 82 to 116%, indicating good results.

FIG. 7 shows the results of a dilution test by the present process. The dilution curves are linear, which indicates good results. The results of the recovery and the dilution tests suggest that the present process would be useful in assaying asialo GM.sub.1 accurately.

Asialo GM.sub.2, fuco GA.sub.1 and paragloboside may likewise be assayed by the aforementioned methods of (1), (2) and (3).

Various humors such as blood, fluid in the thoracic cavity, abdominal dropsy or urine may be used in the present process.

Labelling agents such as radioactive materials, enzymes or fluorescent materials may be used in the present invention.

An insolubilized glycolipid, i.e., immunoadsorbent, in which asialo GM.sub.1, asialo GM.sub.2, fuco GA.sub.1 or paragloboside is conjugated with an insoluble material (solid phase), is prepared by chemically conjugating one of these glycolipids with a solid phase or physically adsorbing the former by the latter. An insolubilized antibody in which an antibody against each glycolipid is conjugated with an insoluble material (solid phase), is prepared by the same way. Conventional solid phase such as cellulose, sepharose, glass or polystyrene may be used.

Thus, the present invention has made it possible to assay glycolipid in a trace amount in humors of patients afflicted with various diseases, including cancer, by taking advantage of an antigen/antibody reaction.

The present process for assaying asialo GM.sub.1, asialo GM.sub.2, fuco GA.sub.1 or paragloboside exhibits excellent reproducibility, high sensitivity and accuracy with simple procedures. Therefore, it is remarkably useful as a daily clinical test method. As shown in FIGS. 4, 5 and 6, using asialo GM.sub.1 as an example, assay of these glycolipids in humors by the process of the present invention is extremely effective in the detecting of cancer in its early stage and the examination of the progress of treatment.

FIG. 1 is a standard curve obtained by an immunometric assay with a radioisotope by using standard asialo GM.sub.1 solutions of known concentrations. In the figure, the abscissa refers to the asialo GM.sub.1 concentration (ng/ml), while the ordinate refers to the count (.times.10.sup.3 cpm). By using the standard curve, the asialo GM.sub.1 concentration of a specimen can be determined by reading the concentration against the count thereof.

FIGS. 2 and 3 are standard curves of asialo GM.sub.2 and paragloboside respectively, obtained by an immunometric assay using an enzyme.

FIG. 4 shows plots of the asialo GM.sub.1 concentration in serum of cancer patients assayed by the process of the present invention by comparison with those of benign disease patients and normal subjects. In this figure, the abscissa refers to the asialo GM.sub.1 concentration (ng/ml).

FIG. 5 shows plots of the asialo GM.sub.1 concentration in serum of cancer patients that have been assayed by the process of the present invention and classified by the sites of cancer, i.e., thyroid gland, lung, breast, liver, bile duct, gallbladder, pancreas, gullet, stomach, kidney and colon.

FIG. 6 shows changes in the asialo GM.sub.1 concentration in serum of lung cancer patients (survival and dead) with the passage of time during a chemical treatment.

FIG. 7 shows results of a test in which three specimens were assayed by the process of the present invention, with a dilution varied as indicated in the figure.

To further illustrate the present invention, and not by way of limitation, the following examples will be given.

1. Preparation of antigen (glycolipid)

(1) Purification of asialo GM.sub.1

2.0 g of a glycolipid fraction of bovine brain was hydrolyzed in the presence of 1N formic acid for 1 hour at 100.degree. C. After the completion of the hydrolysis, an asialo glycolipid fraction was separated by DEAE-Sephadex A-25 column chromatography. Sephadex is a type of cross-linked dextrangel; DEAF-Sephadex is a sephadex one anion exchanger. The crude asialo glycolipid thus obtained was further purified by column chromatography with Iatrobeads which are made up of a type of silica gel particle (a product of Iatron Co., Ltd.) in a solvent gradient of CHCl.sub.3 /CH.sub.3 OH/H.sub.2 O of 80:20:0.5 and CHCl.sub.3 /CH.sub.3 OH/H.sub.2 O of 50:45:3.5.

Thus approximately 600 mg of purified asialo GM.sub.1 was obtained from 2.0 g of the bovine brain glycolipid.

(2) Purification of asialo GM.sub.2

500 mg of the bovine brain GM.sub.1 was dissolved in an acetate buffer solution (pH 4.5). 200 mg of sodium periodate was added and the mixture was allowed to stand in the dark for 10 hours at 4.degree. C. Then a few drops of ethylene glycol was added and the mixture was dialyzed overnight. After adjusting the pH to 8.0 with a borate buffer solution and adding 380 mg of NaBH.sub.4, the mixture was allowed to stand for 12 hours at 0.degree. C. Acetic acid was added to cease the reaction and to adjust the pH value to less than 4, and the mixture was dialyzed again. H.sub.2 SO.sub.4 was added in a sufficient amount to give a concentration of 0.1N. Then the mixture was allowed to react for 1 hour at 80.degree. C., dialyzed against distilled water and lyophilized. The powder thus obtained was dissolved in CHCl.sub.3 and purified by Iatrobeads (a product of Iatron Co., Ltd.) column chromatography. Thus, approximately 100 mg of purified asailo GM.sub.2 was obtained.

(3) Purification of fuco GA.sub.1

30 g of powder prepared by lyophilizing rat abdominal dropsy liver cancerous cells AH7974 F was extracted with 500 mg of a solvent mixture (CHCl.sub.3 :CH.sub.3 OH:H.sub.2 O=10:5:1). The extract was filtered and the residue was extracted with the same amount of a solvent mixture (CHCl.sub.3 :CH.sub.3 OH=1:1) and then with a solvent mixture (CHCl.sub.3 :CH.sub.3 OH=1:2). The extracts were combined and solvents were distilled off in vacuo. The residue was again dissolved in a solvent mixture (CHCl.sub.3 :CH.sub.3 OH:H.sub.2 O=15:30:4) and subjected to DEAE-Sephadex A-25 column chromatography to remove acidic lipids.

The neutral glycolipids fraction containing fuco GA.sub.1 thus obtained was acetylated with a mixture of acetic anhydride and pyridine (2:3) and subjected to Florisil (an activated magnesium silicate) column chromatography to separate a glycolipid fraction from other phospholipids. Then the glycolipid fraction was deacetylated, dialyzed and lyophilized. The powder thus obtained was dissolved in CHCl.sub.3 and purified by subjecting to Iatrobeads (a product of Iatron Co., Ltd.) column chromatography to obtain approximately 5 mg of purified fuco GA.sub.1.

(4) Purification of paragloboside

Erythrocytic membrane was obtained by centrifuging 30 l of bovine blood to separate erythrocytes, hemolyzing the erythrocytes with 10 times as much 0.5% acetic acid solution and centrifuging at 8,000 rpm. The erythrocytic membrane was treated with 5 to 10 times as much acetone and dehydrated. Then it was extracted with successive, 20 times as much portions of CHCl.sub.3 :CH.sub.3 OH (2:1), CHCl.sub.3 :CH.sub.3 OH (1:1) and CHCl.sub.3 :CH.sub.3 OH (1:2). The extracts were combined and concentrated in vacuo. The extract was dissolved in 2 liters of a solvent mixture (CHCl.sub.3 :CH.sub.3 OH=2:1) and mixed thoroughly with 500 ml of 0.9% NaCl. The upper layer was taken out, dialyzed and lyophilized. The powder thus obtained was subjected to Iatrobeads column chromatography to separate sialoylparagloboside composed of paragloboside conjugated with sialic acid. The obtained material was treated with 1N formic acid for 1 hour at 80.degree. C. to deconjugate the sialic acid. Then the powder obtained by dialyzing and lyophilizing was dissolved in CHCl.sub.3 and purified by Iatrobeads column chromatography to obtain approximately 50 mg of purified paragloboside.

2. Preparation of glycolipic antibody

(1) Preparation of anti-asialo GM.sub.1 anti-serum

Two mg of asialo GM.sub.1 and 4 mg of bovine serum albumin were suspended in 1 ml of distilled water. To the suspension, 1 ml of Freund's complete adjuvant was added to emulsify. Two ml portions of the emulsion was injected into a rabbit intracutaneously three times at two-week intervals. Two weeks after the final injection, whole blood was collected. Antibodies against asialo GM.sub.2, fuco GA.sub.1 and paragloboside might be prepared in the same manner as described above.

(2) Purification of anti-asialo GM.sub.1 antibody

100 ml of the anti-asialo GM.sub.1 anti-serum as prepared in Example 2(1) was salted out with a 50% saturated solution of ammonium sulfate. The precipitate was dissolved in 100 ml of a phosphate buffer solution (pH 7.2) and further salted out with a 20% saturated solution of ammonium sulfate to remove fibrinogen. The supernatent was salted out with a 35% saturated solution of ammonium sulfate to obtain a .gamma.-globulin fraction. This fraction was dissolved in a small amount of a phosphate buffer solution (pH 7.2) and subjected to Sephadex G-200 column chromatography (2.5.times.110 cm). The obtained chromatogram indicated two peaks (small one is IgM fraction; the other is IgG fraction). The IgG fraction was dialyzed against distilled water and lyophilized.

50 mg of the IgG fraction thus obtained was dissolved in 30 ml of a phosphate buffer solution (pH 7.2) and poured into a column containing 10 ml of AH-Sepharose 4B conjugated with an oligosaccharide, the saccharide chain portion of asialo GM.sub.1. Sepharose is a water soluble spherical form of an agarose gel. Then the column was washed with 120 ml of a phosphate buffer solution (pH 7.2) and eluted with 100 ml of 3.0M NaSCN. The elution was carried out at a rate of 10 ml/hour and 5 ml portions of fractions were collected.

The specific activity of the eluted anti-asialo GM.sub.1 antibody was 2.sup.5 to 2.sup.6 complement-fixed titer/10 to 20 .mu.g protein/ml.

The anti-asialo GM.sub.1 antibody was desalted and concentrated by using a PM 30 Membrane (Amicon).

It was possible to use the AH-Sepharose 4B column conjugated with oligosaccharide of asialo GM.sub.1 with washing with a phosphate buffer solution (pH 7.2) repeatedly until NaSCN was consumed.

100 mg of asialo GM.sub.1 was dissolved in 20 ml of a mixture of methanol and hexane (4:3) and ozone was passed through the solution for 30 min. The suspended lipid-like materials were removed by centrifugation. An aqueous solution of sodium carbonate was added and the mixture was allowed to stand for 18 hours at room temperature (pH 10.5 to 11). Then it was centrifuged at 10,000 rpm for 20 min and Dowex 50 (H.sup.+ form) was added to the supernatant to remove sodium ions. Approximately 40 mg of oligosaccharide was obtained by purifying with Sephadex G-25 column chromatography.

Five mg of the obtained oligosaccharide and 20 mg of NaBCNH.sub.3 were dissolved in 20 ml of distilled water. The resulting solution was mixed with 20 ml of AH-Sepharose 4B and refluxed for 5 hours. After cooling, the AH-Sepharose 4B reacted with oligosaccharide was centrifuged and washed with distilled water several times.

The AH-Sepharose 4B conjugated with oligosaccharide of asialo GM.sub.1 thus obtained was equilibrated with a phosphate buffer solution (pH 7.2) and used to purify the antibody.

3. Preparation of anti-rabbit .gamma.-globulin antibody

One mg of rabbit .gamma.-globulin was dissolved in 2 ml of a physiological saline solution. Two ml of Freund's complete adjuvant was added to the solution to emulsify. The emulsion was injected into a goat intracutaneously five times at two-week intervals. Two weeks after the final injection, whole blood was collected to obtain anti-serum.

1. Assay of asialo GM.sub.1

100 .mu.l of standard asialo GM.sub.1 or a serum sample, 100 .mu.l of oligosaccharide/tyrosine/.sup.125 I and 100 .mu.l of anti-asialo GM.sub.1 antibody were introduced into a test tube and incubated. Then 500 .mu.l of anti-rabbit .gamma.-globulin was introduced to the tube and further incubated.

After centrifuging at 3,000 rpm for 20 min, the supernatant was removed and the radioactivity of the precipitate was determined.

1-1 Preparation of the saccharide chain portion of asialo GM.sub.1

100 mg of asialo GM.sub.1 was dissolved in 20 ml of a mixture of methanol and hexane (4:3). Then ozone was passed through the solution for 30 min. The suspended lipid-like materials were removed by centrifugation. An aqueous solution of sodium carbonate was added thereto and the mixture was allowed to stand for 18 hours at room temperature (pH 10.5 to 11). Then it was centrifuged at 10,000 rpm for 20 min and Dowex 50 (H.sup.+ form) was added to the supernatant to remove sodium ions. Approximately 40 mg of oligosaccharide was obtained by purifying with Sephadex G-25 column chromatography.

1-2 Preparation of a tyrosine derivative of oligosaccharide of asialo GM.sub.1

Five mg of the oligosaccharide of asialo GM.sub.1 as prepared in II (b) and 20 mg of NaBCNH.sub.3 were dissolved in 20 ml of distilled water. To this mixture, 5 mg of diaminohexane was added and refluxed for 5 hours. After cooling, the reaction liquor was concentrated in vacuo and subjected to Sephadex G-25 column chromatography to purify oligosaccharide conjugated hexylamine.

An N-hydroxysuccinimide ester was prepared from 10 mg of tyrosine in the presence of N-hydroxysuccinimide and dicyclohexylcarbodimide. To this compound, 5 mg of oligosaccharide conjugated hexylamine prepared above was added and the mixture was allowed to react for 24 to 72 hours at room temperature. Approximately 6 mg of a tyrosine derivative of oligosaccharide of asialo GM.sub.1 was obtained by purifying with Sephadex G-25 column chromatography.

1-3 .sup.125 I labelling of oligosaccharide

20 .mu.l of 1 mCi of Na.sup.125 I, 25 .mu.l of a 0.5M phosphate buffer solution (pH 7.4), 1.5 .mu.g of oligosaccharide of asialo GM.sub.1 incorporating tyrosine and 25 .mu.l of Chloramine T (1 mg/ml) were introduced into a test tube and allowed to react for 30 sec with shaking. Then 100 .mu.l of sodium pyrosulfite (1 mg/ml) was added to stop the reaction.

Free .sup.125 I was removed by Sephadex G-25 column chromatography to obtain oligosaccharide/tyrosine/.sup.125 I.

1. Assay of asialo GM.sub.1

100 .mu.l of standard asialo GM.sub.1 or a serum sample and 100 .mu.l of a 0.1M borate buffer solution (pH 8.6) were introduced into an assay plate. An anti-asialo GM.sub.1 antibody bead was added to the mixture and incubated. Then the reaction liquor was removed and washed with a physiological saline solution twice. 200 .mu.l of anti-asialo GM.sub.1 antibody-.sup.125 I was added thereto and incubated. After removing the reaction liquor and wasing with a physiological saline solution three times, the head was transferred to a counting tube to determine the radioactivity.

1-1 Preparation of solid-phase anti-asialo GM.sub.1 antibody

500 polystyrene beads were introduced into a 100 ml bottle with a lid. Then 2 .mu.g/bead of anti-asialo GM.sub.1 antibody and 0.16 ml/bead of a 0.1M phosphate buffer solution (pH 7.8) was added thereto and the mixture was sealed and rotated overnight. Then it was washed with a 0.9% NaCl solution five times and dried to a desiccator to obtain a solid-phase antibody.

1-2 .sup.125 I labelling of anti-asialo GM.sub.1 antibody

20 .mu.l of 1 mCi of Na.sup.125 I and 25 .mu.l of a 0.5M phosphate buffer solution (pH 7.5) were introduced into a test tube treated with silicone. Then 50 .mu.l of an anti-asialo GM.sub.1 antibody solution and 25 .mu.l of Chloramine T (3 mg/ml) were added thereto. After thoroughly shaking for 20 to 30 sec to allow to react, 100 .mu.l of sodium metabisulfite (3 mg/ml) was added to stop the reaction. To the reaction liquor 25 .mu.l of potassium iodide (50 mg/ml) and 100 .mu.l of a bovine serum alubumin solution (5%) were added. Free .sup.125 I was removed by gel filtration with Sephadex G-25 to obtain .sup.125 I labelled anti-asialo GM.sub.1 antibody.

1-3 Fluorescein labelling of anti-asialo GM.sub.1 antibody

Five mg of anti-asialo GM.sub.1 antibody was dissolved in 1 ml of a 0.1M Tris-HCl buffer solution (pH 8.0). To this solution, an saline solution containing 50 .mu.g of fluorescein isothiocyanate (FITC) was added with stirring. Then 1N NaOH was added slowly with stirring to adjust the pH to 9.5 and stirring was continued for additional 1 hour at room temperature to allow to react. After the completion of the reaction, the reaction liquor was dialyzed against a 0.01M phosphate buffer solution (pH 7.6) overnight at 4.degree. C. and gel-filtered with Sephadex G-25 to obtain a labelled antibody.

1-4 Peroxidase labelling of anti-asialo GM.sub.1 antibody

Five mg of peroxidase was dissolved in 1 ml of a 0.3M sodium hydrogencarbonate buffer solution (pH 8.1). After adding 0.1 ml of a 1% 1-fluoro-2,4-dinitrobenzene (FDNB) in ethanol solution, the mixture was allowed to react for 1 hour at room temperature. Then 1 ml of 0.06M sodium periodate was added and the mixture was allowed to react for 30 min at room temperature. Subsequently 1 ml of 0.16M ethylene glycol was added and the mixture was further allowed to react for 1 hour at room temperature. Then it was dialyzed against a 0.01M sodium carbonate buffer solution (pH 9.5) overnight at 4.degree. C. Five mg of anti-asialo GM.sub.1 antibody was added and the mixture was allowed to react for 2 hours at room temperature. Then 5 mg of sodium borohydride was added and the mixture was allowed to stand overnight at 4.degree. C. Then it was dialyzed against a 0.01M phosphate-buffered sodium chloride solution overnight at 4.degree. C. and gel-filtered with Sephadex G-200 to obtain a labelled antibody.

1. Assay of asialo GM.sub.1 by an immunometric assay with a radioisotope

100 .mu.l of standard asialo GM.sub.1 or a serum sample and 100 .mu.l of a diluted solution of anti-asialo GM.sub.1 antibody were introduced into an assay plate and incubated (the first reaction). Then a bead conjugated with asialo GM.sub.1 was added to the mixture and further incubated (the second reaction). The bead was washed with 0.9% NaCl four times and incubated with 200 .mu.l of anti-rabbit .gamma.-globulin antibody-.sup.125 I (the third reaction). The bead was washed with 0.9% NaCl four times and transferred to a counting tube to determine the radioactivity.

An example of standard curve thus obtained and the results of assay of asialo GM.sub.1 in blood of cancer patients are shown in FIGS. 1, 4, 5 and 6.

1-1 Preparation of insolubilized asialo GM.sub.1 (immunoadsorbent)

Polystyrene beads were introduced in a beaker of an appropriate size to form one or two layers of the beads and washed with pure water five times. 0.2 ml/bead of an asialo GM.sub.1 solution was added and the mixture was allowed to stand for 60 to 90 min at room temperature. After removing the asialo solution by decantation, the residue was washed with a phosphate-buffered sodium chloride solution (PBS) four times and stored in PBS at 4.degree. C.

Insolubilized asialo GM.sub.2, insolubilized fuco GA.sub.1 and insolubilized paragloboside were prepared in the same manner as described above.

1-2 .sup.125 I labelling of anti-rabbit .gamma.-globulin antibody

20 .mu.l of 1 mCi of Na.sup.125 I and 25 .mu.l of a 0.5M phosphate buffer solution (pH 7.5) were introduced into a test tube treated with silicone. Then 50 .mu.l of an anti-rabbit .gamma.-globulin antibody solution and 25 .mu.l of Chloramine T (3 mg/ml) were added. After thoroughly shaking for 20 to 30 sec to allow to react, 100 .mu.l of sodium metabisulfite (3 mg/ml) was added to stop the reaction. To the reaction mixture, 25 .mu.l of potassium iodide (50 mg/ml) and 100 .mu.l of a bovine serum albumin solution (5%) were added. Free .sup.125 I was removed by gel-filtration to obtain a .sup.125 I labelled anti-rabbit .gamma.-globulin antibody.

2. Assay of asialo GM.sub.2 by an immunometric assay with an enzyme

100 .mu.l of standard asialo GM.sub.2 and 100 .mu.of a diluted solution of anti-asialo GM.sub.2 antibody were introduced into an assay plate and incubated. Then a bead conjugated with asialo GM.sub.2 was added and the mixture was further incubated. After washing the bead with a 0.01M phosphate-buffer sodium chloride solution (PBS) five times, 200 .mu.l of peroxidase conjugated anti-rabbit .gamma.-globulin antibody was added to the reaction mixture and incubated. After washing the bead with PBS five times, 225 .mu.l of an enzyme substrate, which consisted of 0.05% hydrogen peroxide and 0.08% 5-aminosalicyclic acid (9:1) and was adjusted to pH 6.0 by NaOH, was added. After incubating the reaction mixture, the absorbance was determined at 450 nm.

An example of standard curves thus obtained is shown in FIG. 2.

2-1 Peroxidase labelling of anti-rabbit .gamma.-globulin antiboy

Five mg of peroxidase was dissolved in 1 ml of a 0.3M sodium hydrogencarbonate buffer solution (pH 8.1). Then 0.1 ml of a 1% 1-fluoro-2,4-dinitrobenzene (FDNB) in ethanol solution was added to the solution and allowed to react for 1 hour at room temperature. Further 1 ml of 0.06M sodium periodate was added and the mixture was allowed to react for 30 min at room temperature. Subsequently 1 ml of 0.16M ethylene glycol was added and the mixture was allowed to react for 1 hour at room temperature. Then it was dialyzed against a 0.01M sodium carbonate buffer solution (pH 9.5) overnight at 4.degree. C. After adding 5 mg of anti-rabbit .gamma.-globulin antibody, the mixture was allowed to react for 2 hours at room temperature. Then 5 mg of sodium borohydride was added and the reaction mixture was allowed to stand overnight at 4.degree. C. It was dialyzed against a 0.01M phosphate-buffered sodium chloride solution overnight at 4.degree. C. and gel-filtered with Sephadex G-200 to obtain a labelled antibody.

3. Assay of paragloboside by an immunometric assay with an enzyme

100 .mu.l of standard paragloboside and 100 .mu.l of a diluted solution of anti-paragloboside antibody were introduced into an assay plate and incubated. Then a bead conjugated with paragloboside was added and the reaction mixture was further incubated. After washing the bead with a 0.01M phosphate-buffered sodium chloride solution (PBS) five times, 200 .mu.l of peroxidase conjugated anti-rabbit .gamma.-globulin antibody was added and incubated. After washing the bead with PBS five times, 225 .mu.l of an enzyme substrate, which consisted of 0.05% hydrogen peroxide and 0.08% 5-aminosalicyclic acid (9:1) and wash adjusted to pH 6.0 by NaOH, was added. After incubating the reaction mixture, the absorbance was determined at 450 nm.

An example of standard curves thus obtained is shown in FIG. 3.

3-1 Peroxidase labelling of anti-rabbit .gamma.-globulin antibody

Five mg of peroxidase was dissolved in a 0.3M sodium hydrogencarbonate buffer solution (pH 8.1). To this solution, 0.1 ml of a 1% 1-fluoro-2,4-dinitrobenzene (FDNB) in ethanol solution was added and allowed to react for 1 hour at room temperature. Then 1 ml of 0.06M sodium periodate was added and the mixture was allowed to react for 30 min at room temperature. Subsequently, 1 ml of 0.16M ethylene glycol was added and the mixture was allowed to react for 1 hour at room temperature. Then it was dialyzed against a 0.01M sodium carbonate buffer solution (pH 9.5) overnight at 4.degree. C. After adding 5 mg of anti-rabbit .gamma.-globulin antibody, the mixture was allowed to react for 2 hours at room temperature. Five mg of sodium borohydride was added and the reaction mixture was allowed to stand overnight at 4.degree. C. Then it was dialyzed against a 0.01M phosphate-buffered sodium chloride solution overnight at 4.degree. C. and gel-filtered with Sephadex G-200 to obtain a labelled antibody.

4. Fluorescein labelling of anti-rabbit .gamma.-globulin antibody

Five mg of anti-rabbit .gamma.-globulin antibody was dissolved in 1 ml of a 0.1M Tris-HCl buffer solution (pH 8.0). To this solution, a saline solution containing 50 .mu.g of fluorescein isothiocyanate (FITC) was slowly added with stirring. Then 1N NaOH was slowly added to the solution with stirring to adjust the pH to 9.5 Stirring was continued for additional 1 hour to allow to react. After the completion of the reaction, the mixture was dialyzed against a 0.01M phosphate buffer solution (pH 7.6) overnight at 4.degree. C. and gel-filtered with Sephadex G-25 to obtain a labelled antibody.

The obtained labelled antibody may be used as a labelled compound in an immunometric assay system.