JPH03115863A - Determination of glycolipid by immunoassay - Google Patents

Abstract

PURPOSE:To measure glycolipid with high accuracy by competitively reacting glycolipid in body fluids and labelled glycolipid labeled with a marker with an anti-glycolipid antibody and measuring the activities of the markers in labeled glycolipids bonded and not bonded to the antibody. CONSTITUTION:Glycolipids such as asialo-GM1,GM2, fuco-GA1 and paragloboside increase with the canceration of cells. These glycolipids are immunologically measured according to the following method. For example, asialo-GM1 to be determined and a definite amount of labeled-asialo-GM1 in body fluids are competitively reacted with a definite amount of an antibody. Subsequently, labeled asialo-GM1 bonded to the antibody and labeled asialo-GM1 not bonded to the antibody are separated and the activity of the marker in one of those fractions or the activities of the markes in both fractions is measured to determine asialo-GM1. As the body fluids, blood, abdominal dropsy fluid in the thoracic cavity or urine are used and, as the marker, radioactive substance, enzyme and fluorescent substance are used.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a method for immunologically measuring glycolipids in a biological sample.

More specifically, the present invention relates to a method for immunologically quantifying glycolipids asialoGM1, asialoGM1, 7coGA, and valagloboside in a biological sample using antibodies specific to each.

[Background technology]

Cancer has become the leading cause of death in Japan, and expectations and necessity for early detection and treatment of cancer are increasing. For early detection, there are morphological examinations such as imaging methods and immunobiochemical diagnostic methods, which are non-invasive tests that collect a small amount of body fluid, but immunobiochemical diagnostic methods are gaining an increasingly important position. It has reached the point where it occupies the majority. To date, great efforts have been made to search for tumor-associated antigens, which are substances that increase significantly as cells become cancerous, as immunobiochemical diagnostic methods for cancer.
toprotein) and carcinoembryonic antigen (carcinoembryonic antigen).
The discovery of embryonic antigens and the development of methods for measuring them are one of the major achievements.
It is used clinically.

However, from the perspective of early detection of cancer, these markers are currently far from being sufficient, and the discovery of new tumor-related antigens, the development of their measurement methods, and their clinical application are desperately needed. There is.

The present inventors focused on glycolipids, which are one of the cell membrane components,
As a result of studying the relationship between glycolipids and cell adhesion through glycolipid analysis and metabolic studies of cancer cells, we were able to express differences in cell adhesiveness as differences in glycolipid metabolism. Especially in highly malignant cancer cells that have lost cell adhesion, normal mN1
A biosynthetic pathway for glycolipids was discovered that goes through glycolipids, which is rarely seen in the human body. That is, the present inventors learned that there are four different types of glycolipids that increase with cell canceration, and as a result of identifying the glycolipids through various analyses, these were found to be asialo GM1, asialo GM1, asialo GM1
, Fuco GA.

and balagloboside. Their structures are shown below.

Asialo GM.

Ga1-GalNAc-Gal-Glc-Cer Asialo Ge1f GalNAc -Cal -Glc -Cer Fuco GA
.

Gal-Ga1NAc-Ga! -Glc -Ce
ruc Balagloboside Ga1-GlcNAc-Gal-Glc-CarGal
Nigalactose Glc + Glucose Fuc: Fucose Ga1NAc: N-acetylgalactosamine G
lcNAc: N-acetylglucosamine Car
:Ceramide The present inventors discovered that these glycolipids also increase in human cancer cells and are extremely useful for cancer diagnosis by measuring their concentration in body fluids, which leads to the discovery that these glycolipids can be used as new cancer markers. It was revealed that. Furthermore, because the concentration of these glycolipids in body fluids is extremely low, it has been impossible to quantify them in minute quantities for clinical diagnosis.
The present inventors have succeeded in establishing a highly sensitive, accurate, and easy-to-operate immunoassay method for these glycolipids according to the present invention.

[Disclosure of the invention]

The present invention relates to Asialo GM1, Asialo GM1, Fuco GA
It is based on an immunoassay method that utilizes an antigen-antibody reaction using antibodies specific to , and valagloboside. That is, the present invention provides methods (1), (2), and (2) below.

■ A method for quantifying glycolipids such as asialo GM1, asialo GMt, fucoGA, or paragloboside in body fluids, in which the glycolipid in the body fluid to be quantified and the glycolipid corresponding to the glycolipid are detected using a labeling agent. The labeled glycolipid is subjected to a competitive reaction with an anti-glycolipid antibody, and then the labeled glycolipid to which the antibody is bound and the labeled glycolipid to which the antibody is not bound are separated, and one of these fractions or 1. An immunological measurement method for glycolipids in body fluids, the method comprising quantifying glycolipids by measuring the activities of both labeling agents.

■ A method for quantifying glycolipids asialoGIJ1, asialoGM, fucoGAI, or paragloboside in body fluids, in which the glycolipid in the body fluid to be quantified and the M chain portion of the glycolipid corresponding to the glycolipid are used. A labeled oligosaccharide obtained by labeling a certain oligosaccharide with a labeling agent is subjected to a competitive reaction with an anti-glycolipid antibody, and then the labeled oligosaccharide to which the antibody is bound and the labeled oligosaccharide to which the antibody is not bound are separated, 1. A method for immunologically measuring glycolipids in body fluids, which comprises quantifying glycolipids by measuring the activity of a labeling agent in one or both of the fractions.

■ Asialo CM, a glycolipid in body fluids
M2, a method for quantifying fuco CA1 or valagloboside, in which an insoluble substance is used as a carrier, and an anti-glycolipid antibody of the glycolipid to be quantified is bound to the insoluble antibody, and the said glycolipid in the body fluid to be quantified is Glycolipids are reacted and then reacted with a labeled anti-glycolipid antibody labeled with a labeling agent. After that, the labeled anti-glycolipid bound to the glycolipid on the insolubilized antibody
1. An immunological measurement method for glycolipids in body fluids, which comprises quantifying glycolipids by measuring the activity of a labeling agent for glycolipid antibodies.

As the above immunoassay method, a method using a competitive reaction using a labeled antigen and a sandwich method using a labeled antibody are used.

The antibodies used to quantify asialo GMt, asialo GM1, seven GAI, and balagloboside target these glycolipids with carrier polymers foreign to these glycolipids, such as bovine serum albumin, methylated bovine serum albumin, and red blood cell membrane proteins. It can be produced by intravenous injection into non-human animals, such as domestic rabbits, as an antigen. In the present invention, the antibodies thus obtained are used to quantify asialo GM1, asialo GM1, fucoGA, and valagloboside in body fluids by immunological methods. Hereinafter, the method of the present invention will be specifically explained using Asialo GA as an example.

1. Quantification of asialo GM using competitive reaction The asialo GM to be quantified and a certain amount of labeled asialo GM are subjected to a competitive reaction with a certain amount of antibody, and then combined with the antibody l; Labeled asialo CM, and labeled asialo GM not bound to the antibody.

and the activity of the labeling agent in one or both of these fractions is measured to quantify Asian f:lGMI. Using a competitive reaction using such a labeled antigen, in the quantitative method, a competitive reaction was performed in the same way using a labeled oligosaccharide labeled with the sugar chain moiety (oligo) of asialo GM1 instead of labeled asialo GM. Asialo GM can also be quantified.

2. Asialo GM using the sandwich method.

Asialo GM to be quantified is reacted with the insolubilized antibody, which is an antibody bound to an insoluble substance (carrier), to form an antibody-asialo GM complex, and this complex is labeled with the antibody with a labeling agent. React the antibody, antibody-Asialo GM,-
A sandwich type antigen-antibody complex of labeled antibodies is produced, and the activity of the II marker on the antigen-antibody complex is measured to quantify asialo GM.

In these quantitative methods, standard Asialo GM whose concentration is known in advance is used instead of the body fluid specimen.

The amount of asialo GM is determined from a standard curve drawn using the same procedure.

The results of the recovery test using this method are shown in Table I. Table 1
This shows the results of a recovery test conducted by adding known concentrations of Asialo GM to three types of specimens.

Good results were obtained with a recovery rate of 82% to 116%.

Further, FIG. 1 shows the results of a dilution test using the powder method. The dilution curve showed linearity and good results were obtained. It can be seen from the results of these recovery tests and dilution tests that the powder method is a highly accurate method for quantifying Asialo CM.

Asialo GM2, FucoGA, and Balagloboside can also be quantified in the same manner as Asialo GM by the above method.

The body fluids targeted in the method of the present invention include various body fluids, such as blood, pleural effusion, ascites, urine, and the like.

Examples of the labeling agent include radioactive substances, enzymes, fluorescent substances, and the like.

Insolubilized antibodies, in which antibodies against asialo GM1, asialo GM1, fuco GA, and valagloboside are bound to an insoluble substance (carrier), are produced by chemically binding or physically adsorbing these antibodies to the carrier. be done.

As the carrier, commonly used carriers can be used, examples of which include cellulose, sepharose,
Examples include glass and plastics such as polystyrene.

As mentioned above, the present inventors were the first in the world to develop a method for the microquantification of glycolipids that utilizes antigen-antibody reactions, and have used this method to determine the amount of these substances in body fluids in various patients including cancer patients. For the first time, it became possible to measure the concentration.

Asialo GM1 by the method of the present invention,
The measurement of fucoGA+ and valagloboside has excellent reproducibility, high sensitivity and accuracy, and is easy to operate, making it an excellent daily clinical testing method. Quantification of these glycolipids in body fluids by the method of the present invention is extremely useful for early detection of cancer and determination of treatment progress, as shown in Figures 2, 3, and 4 using Asialo GIJ as an example. Useful.

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example I Preparation of antigen and antibody 1. Purification of antigen (M lipid) (1) 2.0 g of purified bovine brain glycolipid fraction of A7 allo GM was incubated at 100°C in the presence of IN formic acid.
, hydrolyze for 1 hour. After completion of hydrolysis, the asialoglycolipid fraction was separated by DEAE-Sephadex A-25 column chromatography. The obtained crude asialoglycolipid was dissolved in a solvent gradient (CHCffa/CHsOH/H
,0,80:20:0.5-CHIJ,/CI
It was further purified by tsOI (/u,o, 50:45:35) column chromatography using Yatrobeads.

From the above, about 600 mg of purified Asialo GM was obtained from 2.0 g of bovine brain glycolipids.

(2) Asialo GM! Purification method: 50011 g of bovine brain GMl was added to an acetate buffer (pH 4, 5).
) dissolves in Add sodium periodate 200119 and leave in the dark at 4°C for 10 hours. 2.3 drops of ethylene glycol was added and dialyzed overnight. Adjust pH to 8 with borate buffer
．． Set to 0 and NaBH.

Add 1g of 380I and leave at 0°C for 12 hours. After stopping the reaction with acetic acid and adjusting the pH to 4 or less, dialysis is performed. Add H2SO to 0.1N and heat at 80℃ for 1
Dialysis is performed on distilled water that has been reacted for a period of time, and freeze-dried. After dissolving this powder in CHCQ, it was purified by Yatrobeads column chromatography to obtain purified Asialo GM of about 100 my! I got it.

(3) FucoGAI (7) Purified rat ascites liver cancer A) CH of 50011I2 for 30 g of powder obtained by freeze-drying 17974F cells (,Q3:
Extracted with a solvent of cllsOH:LO (10:5:l). The same amount of CH was added to the residue obtained by filtering the extract.
C4s: CH, 0H (1: l) ji and CH
(4,: CHIOH: H,O (1:2)). After combining the extracts, the solvent was removed under reduced pressure.
Acidic lipids were removed by DEAE/Sephadex A-25 column chromatography.

The obtained neutral glycolipid fraction containing FucoGA was acetylated (acetic anhydride:pyridine-2:3), and the glycolipid fraction was separated from other phospholipids using 70 lysyl column chromatography. The glycolipid fraction is then deacetylated, dialyzed, and freeze-dried. This powder was dissolved in CHCQs and purified by Yatrobeads column chromatography to obtain about 511 g of purified Fuco GA.

(4) Purification of Balagloboside After separating red blood cells from 30α bovine blood by centrifugation and hemolyzing them with 10 times the amount of 0.5% acetic acid, 8.0
Centrifugation was performed at 0 rpm to obtain red blood cell membranes. The red blood cell membrane was treated with 5 to 10 times acetone to dehydrate it. Next, 20 times the amount of CHCl2. : CH, OH (2: I
), CHCl13:CH30H (1: l), C
I (434C)! The mixture was extracted sequentially with 30H (1:2') 2°C, and the extracts were combined and concentrated under plate pressure. This extract was mixed with 2Q CHCQ3:CHIOH (2:1)
(7) After dissolving L fS in the solvent, 500 mDI) 0-
9% NaCQ was added and mixed well, and the upper layer was taken out, dialyzed, and freeze-dried. This powder is separated by Jatrobeads force chromatography to obtain sialosyl paragloboside, in which sialic acid is bound to paragloboside. IN this substance
Treat with formic acid at 80°C for 1 hour to remove sialic acid. After dialysis and freeze-drying, this powder was dissolved in CHCQ and purified by Jatrobead ram chromatography to approximately 50111? Purified paragloboside was obtained.

Preparation of glycolipid antibodies (1) Preparation of anti-Asia OGM and antiserum Asialo GM121119 and bovine serum albumin 4 mg
was suspended in 1 raQ of distilled water. 70 to this suspension
Add 1 ts (l) of India Complete Adjuvant and emulsify it.
Injected intradermally. Whole blood was collected 2 weeks after the last injection.

Antibodies against asialoGIJ2, FucoC, A1, and paragloboside could also be produced in the same manner.

(2) Purification of anti-asialo GIJ and antibodies Anti-asialo CM obtained in Example (1).

Antiserum 100+12 was salted out with ammonium sulfate at 50% saturation.

Add the precipitate to phosphate buffer (pH 7,2) 1oOI+IQ
The fibrinogen was removed by salting out with 20% saturated ammonium sulfate. The supernatant was salted out with 35% saturated ammonium sulfate, and γ
- A globulin fraction was obtained.

γ-globulin was dissolved in a small amount of phosphate buffer (pH 7.2) and subjected to Sephadex G-200 column chromatography (2.5×110 cm). A small first peak (IgM fraction) and second peak (IgG fraction) were obtained by Sephadex G-200 column chromatography. The IgG fraction was dialyzed against distilled water and then freeze-dried.

50 mg of the obtained IgG fraction was added to a phosphate buffer (pH 7,
2) A) I-Sepharose 4B 1OrnQ' dissolved in 30++++2 and bound to oligosaccharides of AsialoCM
After passing through the packed column, soak the column with phosphate buffer (
pH 7,2) 120m (2t' washed. Then 3.0
MNa5CN was eluted with 100 m (l. Elution was 10 t
The reaction was carried out at rrQ/hour, and 5II112 fractions were collected.

The specific activity of the eluted anti-asialo GM antibody is 26-2
The complement fixation titer was 1/10-20 μg protein/rxQ.

After desalting the anti-asialo GM antibody, it was transferred to a PM30 membrane (Amjc
on) is used for condensation.

The AI(-Sepharose 4B column bound with the oligosaccharide of asialoGMI) can be used repeatedly by washing with phosphate buffer (pH 7, 2) until Na5CN is removed.

*Asialo GM to AH-Sepharose 4B.

1001 g of Asialo GM1, which is a combination of sugar chain moieties (oligosaccharides), was mixed with methanol:hexane (
4: 3) After dissolving in 20IQ, aerate ozone for 30 minutes. Remove lipid-like suspended matter by centrifugation, add aqueous sodium carbonate solution, and leave at room temperature for 18 hours (p) 110.
5-11). After that, centrifuge at ooorpm for 20 minutes, add supernatant Dowe+c 50 (H” Form) to remove sodium ions, and then transfer to Sephadex G.
Approximately 40119 oligosaccharides were obtained by -25 column chromatography.

51 g of the obtained oligosaccharide and 20 mg of NaBCNHs were dissolved in 20 g of distilled water. This solution was mixed with ^H-Sepharose 4B20+ml and refluxed for 5 hours. After cooling, the AH-Sepharose 4B reacted with the oligosaccharide was centrifuged and washed several times with distilled water.

AH-Sepharose 4B bound with oligosaccharides of Asialo GM obtained above was prepared in phosphate buffer (pH 7.2).
After equilibration, it was used for antibody purification.

3. Preparation of anti-rabbit γ-globulin antibody One part of rabbit γ-globulin was dissolved in 2IIa of physiological saline and 2mQ of 70 India Complete Adjuvant was added and emulsified. This emulsion was injected into goats at 2 week intervals. Two weeks after the last injection, whole blood was collected and antiserum was obtained.

Quantification of glycolipids using competitive reaction 1. Standard Asialo GM or serum sample 10 in a test tube for quantitative measurement of Asialo GM.
After incubation, 0p2, 100 μQ of oligosaccharide tyrosine-I, and 100 μ+2 of anti-asialo GMI antibody were added, and then 500 μg of anti-rabbit γ-globulin antibody was added and incubated.

After centrifugation at 3,000 rpm for 20 minutes, the supernatant was removed and the radioactivity of the precipitate was measured.

Preparation of sugar chain moiety of l-1 asialo GM1
1001g methanol:hexane (4:3) 2
After dissolving in 0IQ, ozone is bubbled through for 30 minutes. Lipid-like suspended matter is removed by centrifugation, and an aqueous sodium carbonate solution is added, and the mixture is left at room temperature for 18 hours (pH 1O, 5-11). After that, centrifuge for 10, OOOrpm for 20 minutes and put it in the upper groove.
Wex 50 (H'' form) was added to remove sodium ions, and approximately 40Il1g of oligosaccharide was obtained by Sephadex G-25 column chromatography.

1-2 Preparation of tyrosine derivative of oligosaccharide of asialo GM, oligosaccharide 5 of asialo GM obtained in step 11 and NaB
320 μg of CNH was dissolved in 220 μg of distilled water (JtaQ), 5 rRg of diaminohexane was added thereto, and the mixture was refluxed for 5 hours.

After cooling, the reaction solution was concentrated under reduced pressure, and hexylamine oligosaccharide was purified by Sephadex G-25 column chromatography.

On the other hand, tyrosine of IOIllg is formed into N-hydroxysuccinimide ester in the presence of N-hydroxysucciimide and dicyclohexylcarbodiimide. The previously obtained hexylamine oligosaccharide 5119 is added to this and reacted at room temperature for 24 to 72 hours. Approximately 6-door asialo CM, a tyrosine derivative of oligosaccharide, was obtained by Sephadex G-25 column chromatography.

1 mCl Na12'I 20tLQ, 0
．． 5M phosphate buffer (pH 7,4) 25 μg Q1 tyrosine-introduced asialo GM oligosaccharide and chloramine T (l rrry/mQ) 25 μa
After adding 100 μQ of sodium pyrosulfite (l rag/m(2)), the reaction was stopped by shaking for about 30 seconds.

Free 1!51 was removed by Sephadex G-25 column chromatography, and oligosaccharide-tyrosine-11 was
I got 61.

Quantification of glycolipids using the sandwich method 1. Standard Asialo GM or serum sample 1 on the plate for quantitative measurement of Asialo GM.
00 μa and O, IM borate buffer (pH 8,6) 100
Add μg. After adding one anti-Asialo GM antibody bead and incubating, remove the reaction solution and wash twice with physiological saline. Furthermore, anti-Asialo GM, Antibody-1■ Engineering 200
Add μa and incubate. After removing the reaction solution, the beads were washed three times with physiological saline, and the beads were transferred to a counting test tube to measure radioactivity.

1-1 Anti-Asialo GM, 500 polystyrene beads homologated with antibody are placed in a 100+mff lidded bottle.

Anti-asialo GM, 2 μg antibody per bead, 10.
16m! Add 0.1M phosphate buffer (pH 7, 8), cap tightly, and rotate overnight. After washing 5 times with 0.9% NaCQ solution, it was dried in a desiccator to obtain the immobilized antibody.
child .

12 Anti-Asialo GM, 1 I labeling of the antibody 1 + nCi of Na''I 20110 in a siliconized tube.
Add 25 μa of 0.5 M phosphate buffer (pH 7.5). Add 50μQ of anti-asialo GM and antibody solution, and then add chloramine T (3rtrg/mQ>25μQ). Shake well for 20-30 seconds to react, then add 100μQ of sodium metabisulfite (3mg/mQ) and react. This is followed by potassium iodide (50+n/IN(2
) 25μQ1 Bovine serum albumin solution (5%) 1o
Ou2 was added and free 1281 was removed by gel filtration with Sephadex G-25 to obtain a 1!5■ labeled anti-asialo GM antibody.

■-3 anti-asialo GM, fluorescein-labeled antibody anti-asialo GM, and antibody 5IIIg were dissolved in 0.1 M) lithium-hydrochloric acid buffer (pH 3,0) 1 wlQ. Add fluorescein inthiocyanate (FITC) to this solution.
) After adding 50μ9 saline solution with stirring I
N NaOH was slowly added with stirring until the pH reached 9.5, and the reaction was further allowed to proceed at room temperature for 1 hour with stirring. After completion of the reaction, dialysis was performed overnight at 4°C against 0.01M phosphate buffer (pH 7,6), followed by Sephadex G-25.
The labeled antibody was obtained by gel filtration.

■-4 Anti-Asialo GM, peroxidase-labeled antibody Peroxidase 5+ag was dissolved in 0.3M sodium bicarbonate buffer (pH 8-1) l raQ, then 1% l
-Fluoro-2,4-dinitrobenzene (FDNB) ethanol solution 0.1m (2) was added and reacted at room temperature for 1 hour.Additionally, 0.06M sodium periodate 1IIQ
After adding and reacting at room temperature for 30 minutes, 0.16M ethylene glycol lad was added and reacting at room temperature for 1 hour. Then, after overnight dialysis at 4°C against 0.01 M sodium carbonate buffer (pH 9,5), anti-asialo GM, antibody 5
9 was added, and the mixture was allowed to react at room temperature for 2 hours. More sodium borohydride 5I! 9 was added and left overnight at 4°C.
After dialysis against 01M phosphate buffered sodium chloride solution at 4°C overnight, the labeled antibody was obtained by gel filtration with Sephadex G-200.

[Brief explanation of drawings]

No. 111ffl shows the test results determined by the method of the present invention using three types of specimens at the dilution ratios shown in the figure. FIG. 2 is a diagram showing a plot of the values of asialoGM in the serum of cancer patients measured by the method of the present invention, and is shown in comparison with the values obtained from patients with benign disease and normal subjects. The horizontal axis is asialo GM concentration (ng/mff). FIG. 3 shows a plot of asialoGM values in the serum of cancer patients measured by the method of the present invention for each cancer site. FIG. 4 shows the results of tracking over time the values of asialoGM in the serum of lung cancer patients undergoing chemotherapy, measured by the method of the present invention.

Claims (1)

[Claims] 1. A method for quantifying glycolipids asialo GM_1, asialo CM_2, fucoGA_1 or paragloboside in body fluids, which comprises:
A labeled glycolipid obtained by labeling a glycolipid corresponding to the glycolipid with a labeling agent is subjected to a competitive reaction with an anti-glycolipid antibody, and then the labeled glycolipid to which the antibody is bound and the labeled glycolipid to which the antibody is not bound. 1. A method for immunologically measuring glycolipids in body fluids, which comprises separating glycolipids and quantifying glycolipids by measuring the activity of a labeling agent in one or both of these fractions. 2. The immunoassay method according to claim 1, wherein the labeling agent is a radioactive substance, an enzyme, or a fluorescent substance. 3. A method for quantifying glycolipids asialo GM_1, asialo GM_2, fucoGA_1 or paragloboside in body fluids, the glycolipids in body fluids to be quantified;
A labeled oligosaccharide obtained by labeling an oligosaccharide that is a sugar chain portion of a glycolipid corresponding to the glycolipid with a labeling agent is subjected to a competitive reaction with an anti-glycolipid antibody, and then the labeled oligosaccharide to which the antibody is bound and the antibody is separated from unbound labeled oligosaccharides,
1. A method for immunologically measuring glycolipids in body fluids, which comprises quantifying glycolipids by measuring the activity of a labeling agent in one or both of the fractions. 4. The immunoassay method according to claim 3, wherein the labeling agent is a radioactive substance, an enzyme, or a fluorescent substance. 5. A method for quantifying glycolipids asialo GM_1, asialo GM_2, furoGA_1 or paragloboside in body fluids, in which an insoluble substance is used as a carrier, and an anti-glycolipid antibody of the glycolipid to be quantified is bound to it. The insolubilized antibody is reacted with the glycolipid in the body fluid to be quantified, and then the labeled anti-glycolipid antibody labeled with a labeling agent is reacted with the labeled anti-glycolipid bound to the glycolipid on the insolubilized antibody. 1. An immunological measurement method for glycolipids in body fluids, the method comprising quantifying glycolipids by measuring the activity of a lipid antibody labeling agent. 6. The immunoassay method according to claim 5, wherein the labeling agent is a radioactive substance, an enzyme, or a fluorescent substance.