JP3070418B2 - Differential measurement of glycoproteins - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for the differential measurement of two or more glycoproteins having different sugar chain structures.

[0002]

BACKGROUND OF THE INVENTION In most animals, not only humans, most of the proteins contained in body fluids are glycoproteins having sugar chains. The sugar chain portion has recently attracted particular attention as a substance that bears some "information" about the living body due to structural diversity and the like, and its research is being carried out by many universities and research institutions both in Japan and overseas.

[0003] In the course of the research, it has been observed that a certain disease causes a change in the sugar chain structure of a specific glycoprotein. For example, in the case of human α-fetoprotein (AFP), α-L-fucose residues and N-acetylglucosamine residues (bisecting N-acetylglucosamine) are added to AFP sugar chains with the progression of hepatocellular carcinoma. Has been confirmed to be frequently observed. Moreover, the degree of such sugar chain structural change is independent of the serum AFP concentration and is confirmed from the early stage of hepatocellular carcinoma. Therefore, examining the degree of the change will enable cancer diagnosis. It is attracting attention.

[0004] The degree of change in the sugar chain structure of a glycoprotein is measured by:
It is mainly performed by an analysis method using a lectin, for example, a lectin column method, a lectin electrophoresis method, or the like. The reason that the degree of sugar chain structural change is measured mainly using lectins is not only because lectins are inexpensive, but also because the immunogenicity of sugar chains is higher than that of glycoproteins. Is low, an effective anti-glycoprotein sugar chain antibody cannot be obtained except for a part of the non-reducing terminal structure, that is, it is extremely difficult to prepare an antibody against the glycoprotein sugar chain.

[0005] However, although lectin has a high recognition specificity of the sugar chain structure, its binding force (binding constant) to the sugar chain is several thousand to several tens of thousands lower than that of an antibody. It is difficult to form a strong complex with the chain, such as an antigen-antibody reaction. Therefore, when lectin is used instead of an antibody in an analytical method that requires a washing operation after forming a complex on a solid phase, such as an enzyme-linked immunosorbent assay (ELISA) using an immobilized antibody, During washing, a phenomenon occurs in which the complex of the glycoprotein to be measured and the lectin is dissociated again, causing problems such as a significant decrease in measurement sensitivity.
Such a method is hardly a practical method as a clinical diagnostic method. Also, due to the properties of lectins as described above,
In order to measure glycoproteins using the reaction between lectin and the glycoprotein to be measured, the lectin is markedly compared to the glycoprotein to be measured using a lectin-immobilized column or lectin-containing agarose gel. There was a restriction that it had to be performed under excessive conditions.

[0006] Therefore, it is presently desired to develop a rapid and simple method for assaying glycoprotein sugar chains, which has both the lectin sugar recognition specificity and the high sensitivity of enzyme immunoassay.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and has high sensitivity using a lectin and an antibody.
It is an object of the present invention to provide a rapid and simple method for measuring glycoprotein fractionation.

[0008]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a fractionation measurement method for measuring a glycoprotein having a different sugar chain structure but substantially the same protein structure, and comprising at least one specific sugar chain of the glycoprotein to be measured. An antibody having the property of binding to a lectin recognizing the structure and all glycoproteins to be measured but having the property of preventing binding to the glycoprotein to be measured to which the lectin is bound (hereinafter abbreviated as first antibody). ), Wherein a glycoprotein bound to the first antibody and a glycoprotein not bound to the first antibody are fractionated and measured by utilizing the difference between these properties. It is the invention of.

That is, the inventors of the present invention have been working on a glycoprotein in the course of intensive research to develop a rapid and simple method for fractionating glycoproteins, which has both the specificity of lectin sugar recognition and the high sensitivity of enzyme immunoassay. It has been found that some antibodies have a property of preventing an antigen-antibody reaction in the presence of lectin (first antibody). Therefore, further studies were conducted to develop a method for differentially measuring glycoproteins using this antibody. As a result, a lectin capable of discriminating structural changes due to diseases of glycoprotein sugar chains and an antibody having the above-mentioned properties (first type) It has been found that the degree of change in the sugar chain structure of a glycoprotein can be measured with high sensitivity by using in combination with an antibody), and the present invention has been completed.

The present invention may be carried out, for example, as follows. That is, first, a biological sample containing the glycoprotein to be measured and at least one of the glycoproteins to be measured.
After reacting a lectin recognizing two specific sugar chain structures with a first antibody, a complex of the glycoprotein to be measured and the first antibody, and a glycoprotein to be measured to which the first antibody is not bound And, using the difference in these properties, for example, the difference in molecular weight, the difference in isoelectric point, the strength of charge, the strength of hydrophobicity, etc., are measured by a method corresponding to each property. Can be implemented.

When the glycoprotein to be measured is reacted with the lectin and the first antibody, these may be reacted simultaneously. However, the lectin is reacted first, followed by the first antibody.
It is preferable to react the antibody, since the reaction between the lectin and the glycoprotein occurs reliably and the measurement accuracy is improved.

In the above-described fractionation measurement according to the present invention, in order to facilitate detection of a glycoprotein to be measured,
It is desirable that a substance capable of changing the property of the complex between the glycoprotein to be measured and the antibody (hereinafter, abbreviated as a separation improving substance) or a labeling substance is bound to the first antibody.

Further, if it is desired to facilitate the detection of the glycoprotein to be measured and to measure the total amount of the glycoprotein together, all the glycoproteins to be measured including the glycoprotein to be measured to which the lectin is bound. It is desirable to further use an antibody having a binding property and to which a labeling substance is bound (hereinafter, abbreviated as a second antibody). Second
The present invention using an antibody may be carried out, for example, as follows. That is, first, a biological sample containing a glycoprotein to be measured, a second antibody, a lectin that recognizes at least one specific sugar chain structure of the glycoprotein to be measured, and a first antibody are reacted. Thereafter, a complex of the glycoprotein to be measured and the first antibody and the glycoprotein to be measured to which the first antibody is not bound are subjected to a difference in these properties, for example, a difference in molecular weight, a difference in isoelectric point, The separation can be performed by utilizing the strength of the charge, the strength of the hydrophobic property, and the like, and then measuring each of them using the properties of the labeling substance bound to the second antibody.

When the glycoprotein to be measured is reacted with the second antibody, the lectin and the first antibody, these may be reacted simultaneously, but the lectin (and the second antibody) may be used.
It is preferable to react the first antibody after the first reaction, since the reaction between the lectin and the glycoprotein surely occurs and the measurement accuracy is improved. When reacting a lectin, a first antibody or a second antibody with a glycoprotein to be measured (or before the reaction), treating the glycoprotein with glycosidase can perform the intended differential measurement more accurately. You may get. That is, the sugar chain terminal of the glycoprotein to be measured undergoes various structural changes depending on the type of disease, but depending on the type of sugar residue bonded to the non-reducing terminal side of the sugar chain, the lectin or the first antibody used Alternatively, even if the second antibody or the like is a sugar chain having a structure to be originally bound, the binding may be prevented or may not be possible. In such a case, when reacting the glycoprotein to be measured with the lectin, the first antibody or the second antibody (or in advance before reacting with the lectin, the first antibody or the second antibody), the glycoprotein is treated with an appropriate glycosidase to remove the glycoprotein sugar chain. The lectin, the first antibody or the second antibody
This is because by making it easier for the antibody to bind to the glycoprotein having the target structure, the accuracy of the target fractionation measurement can be improved. The glycosidase used for such a purpose is not particularly limited as long as it has an action as a sugar hydrolase.For example, sialidase, β-galactosidase, β-N-acetylglucosaminidase, α-mannosidase, β-mannosidase , Α-fucosidase and the like. The origin of these glycosidases is not particularly limited. In the case as described above, for example, temperature, pH, etc. in the reaction of glycosidase may be appropriately selected depending on the type of glycosidase used, but the reaction temperature is usually 0 to 50 ° C., preferably, The range is from 1 to 40 ° C, more preferably from 25 to 37 ° C, and the pH during the reaction is usually from 2 to 10, preferably from 4 to 9. The concentration of glycosidase used depends on the type of glycosidase to be used, and is not particularly limited as long as the concentration is higher than the concentration at which the target sugar hydrolysis reaction occurs. It is preferable to perform the reaction for a short time in a solution having a relatively high concentration of glycosidase than to perform the reaction for a long time, since unnecessary elimination of sugar chains can be prevented, and the total amount in the reaction solution is usually 10 to 1000 mU (unit). ), Preferably 50-500
The reaction time is selected so as to be in the range of mU, and the reaction time is usually in the range of 1 minute to 1 day and night, preferably 3 minutes to 1 hour. When the reaction with glycosidase is performed simultaneously with the reaction with lectin, the first antibody or the second antibody, the above reaction conditions are limited to some extent by the reaction conditions when lectin or the like is reacted. Not even.

The lectin used in the present invention has a property capable of recognizing a target sugar chain structure from various lectins, for example, concanavalin A, lentil lectin, kidney bean lectin, datura lectin, wheat germ lectin and the like. What is necessary is just to select and use those appropriately,
There is no particular limitation.

As the first antibody (or the antibody used for preparing the second antibody) having the above-mentioned properties used in the present invention, any antibody having the above-mentioned properties can be used by a conventional method, for example, For example, horses, cows, sheep, rabbits, goats, rats, mice, and other animals can be measured according to the method described in "Introduction to Immune Experiments, 2nd printing, Nao Matsuhashi, Gakkai Shuppan Press Center, 1981". Or a cell line from a mouse tumor line according to a conventional method, ie, the cell fusion method established by Keller and Milstein (Nature, 256, 495, 1975). And any monoclonal antibody produced by a hybridoma obtained by fusing the spleen cells of a mouse previously immunized with the measurement target with the subject, and any of these may be used alone or in an appropriate combination thereof. so That.

Specific examples of glycoproteins that can be separately measured using the present invention include biological fluids such as serum, blood, plasma, and urine, and biological samples such as lymphocytes, blood cells, and various cells. A lectin and a first antibody which recognize different at least one specific sugar chain structure of a glycoprotein to be measured, wherein the glycoproteins differ in sugar chain structure but have substantially the same protein structure. Is present without particular limitation as long as it exists, for example, amylase,
Alkaline phosphatase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactate dehydrogenase (L
DH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), renin, protein kinase, tyrosine kinase, and other enzymes such as human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), and luteinizing hormone (LH) physiologically active substances such as, for example, prostate-specific antigen (PSA), α ₂ - macroglobulin, carcinoembryonic antigen (CEA), cancer-associated antigens α- fetoprotein etc., e.g. CA19-9, sugar chain CA125 etc. An antigen is preferably exemplified.

In the fractionation measurement method of the present invention, the concentration of the lectin used varies depending on the type of the lectin used, the properties of the glycoprotein to be measured, and the like. Usually 10 times or more, preferably 10 times
It is desirable to coexist at a concentration of 0 times or more, more preferably 1000 times or more during the reaction.

The concentration of the first antibody to be used varies depending on the detection limit of the glycoprotein to be measured, but the difference in the binding constant between the lectin and the first antibody to the glycoprotein to be measured is taken into consideration. It is desirable to set after setting. For example, although it varies depending on the type and physical properties of the glycoprotein to be measured and the lectin, the physical properties of the first antibody, and the like, it is desirable to set using the following general formula or the like. First antibody concentration ≦ (Lectin binding constant) / (First antibody binding constant) × (Lectin concentration) The binding constant according to the above general formula refers to the equilibrium reaction in the following equation (1). Indicates a coupling constant and is a constant determined by the following equation (2). [A] + [B] ← → [AB] (1) Coupling constant = [AB] / ([A] × [B]) (2) [A]: Lectin or lectin in equilibrium state 1 Antibody concentration (M). [B]: Concentration of free glycoprotein to be measured in equilibrium (M). [AB]: Concentration (M) of the complex of the lectin (or the first antibody) and the glycoprotein to be measured. More specifically, for example, the binding constant of lectin to the glycoprotein to be measured is 1 × 10 ⁶ M ⁻¹ , and the binding constant of the first antibody to the glycoprotein to be measured is 1 × 10 ⁸ M ⁻¹ . In that case, the first antibody concentration will be less than one hundredth, preferably less than one thousandth of the lectin concentration. The use concentration of the first antibody is desirably equal to or higher than the concentration that can bind to all the glycoproteins to be measured at the set detection limit concentration, but may be lower (eg, about 1/10).

The concentration of the second antibody used varies depending on the detection limit of the glycoprotein to be measured. However, the concentration in the reaction solution is the concentration of the glycoprotein to be measured at the set detection limit concentration. It is desirable that the concentration is not less than the concentration capable of binding to all, preferably not less than twice the concentration, more preferably not less than 5 times the concentration.

In the differential measurement method of the present invention, the glycoprotein to be measured is reacted with a lectin and a first antibody, and if necessary, a second antibody to form a complex between the lectin and the glycoprotein, Reaction conditions for forming a complex or the like of an antibody and a glycoprotein (or a complex of these complex and a second antibody) are not particularly limited as long as they do not prevent the formation reaction of these complexes. Although not performed, a reaction at the time of forming a complex or the like by a conventional method, for example, an enzyme immunoassay (EIA), a radioimmunoassay (RIA), a fluorescence immunoassay (FIA), an affinity chromatography, or the like, is known. What is necessary is just to carry out according to conditions. For example, when a buffer is used during the reaction, a buffer and other reagents to be used may be appropriately selected from those used in a method known per se. The pH during the reaction is not particularly limited as long as it does not hinder the formation reaction of these complexes, but is usually 2 to 10, preferably 5 to 9
Range. The temperature during the reaction is not particularly limited as long as it does not hinder the complex formation reaction, but is usually 0 to 50 ° C, preferably 0 to 40 ° C, more preferably 0 to 10 ° C. No. The reaction time depends on the reactivity of the glycoprotein to be measured with the lectin, the first antibody and the second antibody, and the time required for the formation of these complexes varies from several seconds to several seconds depending on each property. The reaction may be appropriately performed for several hours.

Examples of the separation improving substance used for binding to the first antibody include substances capable of changing properties such as molecular weight, hydrophobicity and isoelectric point of the complex of the glycoprotein to be measured and the first antibody. Is not particularly limited, but specifically, for example, α-chymotrypsinogen, β
Proteins such as galactosidase, lysozyme, cytochrome C and trypsin inhibitor, for example, peptides containing amino acids such as phenylalanine, proline, arginine, lysine, aspartic acid, glutamic acid, etc .; halogen atoms such as bromine, chlorine, iodine, etc. Synthetic polymers, for example, polyamino acids such as polyglutamic acid, polyaspartic acid, polylysine, polyarginine, polyphenylalanine, and polytyrosine; alkyl chains having 3 to 10 carbon atoms, for example, fatty acids such as palmitic acid, oleic acid, and stearic acid; N-
(ε-maleimidocaproyloxy) succinimide [N-
(ε-maleimidocaproyloxy) succinimide] (EMCS),
N-succinimidyl-6-maleimidohexanoate (NS
A chemical substance having a reactive group capable of binding to the first antibody and having hydrophobicity or ionicity, such as uccinimidyl-6-maleimidohexanoate, bismaleimidohexane (BMH), and octylamine, is preferably exemplified. The separation enhancing substance according to the present invention is appropriately selected in consideration of the properties (eg, pH stability, hydrophobicity, solubility in an aqueous solution, isoelectric point, etc.) of the glycoprotein and the first antibody to be measured. It is sufficient if used.

The method for binding the first antibody and the separation improving substance according to the present invention is a method for binding a specific reactive group of the first antibody to a specific reactive group of the separation improving substance, and a method for separating and improving the specific reactive group of the first antibody. Examples include a method of substituting with a substance, and a method of binding the first antibody to a separation improving substance via a substance capable of binding to the first antibody (for example, an antibody, a lectin, an antigen, an inhibitor, or DNA). More specifically, for example, 1) a method of binding an antibody to a known labeling substance and an antibody generally used in EIA, RIA, FIA or the like known per se (for example, Medical Experiment Account, Vol. 8, supervised by Yuichi Yamamura,
1st edition, Nakayama Shoten, 1971; Illustrated fluorescent antibody, Akira Kawao,
First Edition, Soft Science Co., 1983; Enzyme Immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, Second Edition, Medical Shoin,
1982, etc.), 2) Modification and binding methods of substances known per se (for example, chemical modification of proteins <top><bottom>, Ikuzo Uraya,
Kensuke Shimura, Michinori Nakamura, edited by Masaru Funatsu, 1st edition, Gakkai Shuppan Center, 1981; polyethylene glycol-modified protein, Yuji Inada et al., Biochemistry, Vol. 62, No. 11, P1351-13
62, Japan Biochemical Society, 1990; DNA PROBES, George H.
K. and Mark MM STOCKTON PRESS, 1989, etc.) without exception, and these methods may be used.

The labeling substance labeled on the first antibody (or used for preparing the second antibody) according to the present invention includes, for example, alkaline phosphatase, β-galactosidase, peroxidase, and alkaline phosphatase used in EIA.
Microperoxidase, glucose oxidase,
Enzymes such as glucose-6-phosphate dehydrogenase, acetylcholinesterase, malate dehydrogenase, and luciferase, for example, ^99m Tc, ¹³¹ I, used in RIA
Fluorescent substances such as radioisotopes such as ¹²⁵ I, ¹⁴ C, and ³ H, such as fluorescein, dansyl, fluorescamine, coumarin, naphthylamine, and derivatives thereof used in FIA, such as luciferin, isoluminol, luminol, and bis. Luminescent substances such as (2,4,6-trifluorophenyl) oxalate, for example, substances having an ultraviolet absorption such as phenol, naphthol, anthracene or derivatives thereof, for example, 4-amino-2,2,6, 6-tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 2,6-di-t-butyl-α- (3,5-di-t -Butyl-4-oxo-2,5-cyclohexadiene-1-ylidene) -p
-A substance having a property as a spin labeling agent typified by a compound having an oxyl group such as tolyloxyl, but needless to say, the present invention is not limited thereto.

As a method of labeling the first antibody or the like with the labeling substance as described above for the first antibody (or for preparing the second antibody), a method generally used in EIA, RIA or FIA known per se is used. Labeling methods known per se (for example, Medical Chemistry Laboratory Course, Volume 8, supervised by Yuichi Yamamura, 1st edition,
Nakayama Shoten, 1971; Illustrated fluorescent antibody, Akira Kawao, 1st edition,
Soft Science Inc., 1983; Enzyme-linked immunosorbent assay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, 2nd edition, Medical Shoin, 1982, etc.)
Can be cited without exception, and may be performed according to these. In addition, it goes without saying that a conventional method using a reaction between avidin (or streptavidin) and biotin may be used as the labeling method.

When the glycoprotein to be measured according to the present invention is itself measurable (detectable) by any method, for example, when it is an enzyme, such a property of the glycoprotein itself is used to make the measurement. Needless to say, the measurement (detection) of a glycoprotein to be measured in which the complex of the glycoprotein and the first antibody (or / and the second antibody) is not bound to the first antibody can be performed. Needless to say, the measurement at that time may be performed according to a method for measuring enzyme activity known per se.

The complex between the glycoprotein to be measured and the first antibody (or / and the second antibody) and the glycoprotein to be measured to which the first antibody is not bound are determined by utilizing the difference between these properties. For separation and measurement, ordinary column chromatography or electrophoresis may be used, but if high-performance liquid chromatography (HPLC) or capillary electrophoresis is used, a shorter time will be required. This is desirable because it allows for separate and accurate measurement.

When the separation and measurement of these complexes are carried out by HPLC, the complex between the glycoprotein to be measured and the first antibody (or / and the second antibody) is not bound to the first antibody. If a separation improving substance is bound to the first antibody depending on the difference in properties with the glycoprotein of the above, separation is performed by HPLC equipped with a column packed with a packing material selected in accordance with the property. . Next, an appropriate measuring method for the separated complexes and the like according to these properties, or a measuring method according to the properties of the labeling substance in the second antibody when the second antibody is used. , The amount of any of the glycoproteins to be measured in the sample is determined.

In addition, the kind of the packing material of the column used in the HPLC is also different from the measurement object in which the complex of the glycoprotein to be measured and the first antibody (or / and the second antibody) is not bound to the first antibody. It suffices to select appropriately according to the difference in properties from the glycoprotein and the properties of the separation improving substance. In the following, differences in properties between the complex of the glycoprotein to be measured and the first antibody (or / and the second antibody) and the glycoprotein to be measured to which the first antibody is not bound and the properties of the separation improving substance are described. A method of selecting a filler according to the method will be described in more detail.

When a filler for gel filtration is used Since it is a filler having a property of separating a target substance from other coexisting substances by utilizing a difference in molecular weight, the glycoprotein to be measured and the first antibody (or) are used. / And the molecular weight of the complex with the second antibody) is at least 1.2 times, preferably at least 1.5 times, more preferably at least 2 times, the molecular weight of the glycoprotein to be measured to which the first antibody is not bound. As a separation improving substance, a polymer, a synthetic polymer such as polyethylene glycol, or a polymer substance such as a polyamino acid is preferably used, as long as the molecular weight ratio is as described above, a high separation ability can be obtained. Used. The separation method using a separation-improving substance based on the above principle is particularly effective when the sample containing the substance to be measured contains various substances that affect the analysis, such as serum, and their molecular weights have a wide range. It is valid. That is, in order to avoid the influence of the coexisting substance in such a sample, the molecular weight of the complex should be larger than the maximum value of the molecular weight of the substance affecting the analysis. It is sufficient to select and use a compound having a molecular weight that satisfies the above. When a separation method using a separation-improving substance is used, and when a separation-improving substance having a molecular weight larger than the molecular weight cut off of the packing material for gel filtration is bound, the complex containing the substance to be measured is not applied to the column. Since it elutes in the void part, it can be analyzed in the shortest time. In this case, it goes without saying that the separation improving substance may not be a substance having a single molecular weight, that is, a substance having a molecular weight larger than the molecular weight cut off of the packing material for gel filtration. As a filler for gel filtration, for example, YMC-Pack Diol-200
(YMC sea product name), YMC-Pack Diol-300
(Trade name of YMC Corporation), TSK Gel (trade name of Tosoh Corporation) and the like.

When a packing material for hydrophobic chromatography is used Since the packing material has a property of separating a target substance and other coexisting substances by utilizing a difference in hydrophobicity, the glycoprotein to be measured and the first antibody ( And / or the second antibody) has a difference in hydrophobicity from that of the glycoprotein to be measured to which the first antibody is not bound, or as a separation improving substance such as α-
Highly hydrophobic proteins such as chymotrypsinogen and β-galactosidase, for example, peptides containing highly hydrophobic amino acids such as phenylalanine and proline, for example, homopolymers of hydrophobic amino acids such as polyphenylalanine and polytyrosine, and those having 3 to 10 carbon atoms Hydrophobicity of alkyl chains, for example, halogen atoms such as bromine, chlorine, iodine, etc., and highly hydrophobic chemical substances, for example, octylamine, EMCS, BMH, etc., for example, complexes of fatty acids, for example, palmitic acid, oleic acid, stearic acid, etc. Can be used when a substance that can be set as appropriate is used. When a peptide is used as the separation improving substance, a peptide containing a highly hydrophobic amino acid is preferable, and the hydrophobicity may be adjusted by selecting the chain length. In the case of peptides and polyamino acids composed of only hydrophobic amino acids, when the number of amino acids is 15 or more, the solubility in water is reduced. When the separation improving substance is a halogen atom, it can be easily obtained by directly halogenating the first antibody, and the hydrophobicity can be appropriately adjusted by changing the amount of introduced halogen.
Examples of the chemical substance having high hydrophobicity include substances having a long alkyl chain other than those described above. The hydrophobicity can be adjusted by appropriately selecting the alkyl chain length. Further, since the separation improving substance having too high hydrophobicity has low solubility in water, it becomes necessary to use an organic solvent during the binding reaction between the first antibody and the separation improving substance, and the resulting modified first antibody is denatured. In some cases, such a problem occurs that the modified primary antibody becomes insoluble in water, or the like, or that the obtained modified first antibody becomes insoluble in water. Further, as a packing material for hydrophobic chromatography, for example, butyl-NPR (trade name of Tosoh Corporation), butyl
MCI gel (trade name of Mitsubishi Kasei Co., Ltd.), phenyl MCI gel (trade name of Mitsubishi Kasei Co., Ltd.) and the like.

In the case of using a filler for ion exchange chromatography The target substance and other coexisting substances are separated by utilizing the difference in ionicity, so that the glycoprotein to be measured and the first antibody (or /
And the ionicity of the complex with the second antibody) is different from that of the glycoprotein to be measured to which the first antibody is not bound, or a basic protein such as lysozyme or cytochrome C as a separation improving substance; For example, acidic proteins such as trypsin inhibitor, for example, residues containing basic amino acid residues such as arginine and lysine, and peptides containing acidic amino acid residues such as aspartic acid and glutamic acid, and polyamino acids containing 50 or more amino acid residues as described above For example, it can be used when utilizing fatty acids such as palmitic acid, oleic acid and stearic acid. In addition, in the case of ion exchange chromatography, in general, it is better to elute the target substance once it is adsorbed on the column, because high resolution and high specificity can be obtained. Contains a packing material for cation exchange chromatography,
When an anionic separation improving substance is used, it is preferable to use a filler for anion exchange chromatography. When a peptide or polyamino acid composed of only basic amino acid residues (or acidic amino acid residues) is used as the separation improving substance, the elution time of the complex can be freely adjusted by adjusting the number of amino acid residues. Although it is possible to use a peptide or polyamino acid consisting of usually 5 or more, preferably 50 or more, and more preferably 100 or more as amino acid residues, the elution position of the complex is different from that of biological components in serum or urine. It is desirable because it can be completely separated. When the peptide or polyamino acid is a synthetic peptide or synthetic polyamino acid, the length of the peptide (or polyamino acid) is proportional to the ionicity. By adjusting and using it as a separation improving substance, the elution position of the complex or the like can be easily adjusted. In addition, even when a plurality of serum components affect the measurement, these effects can be avoided by using a separation improving substance to make the ionicity of the complex or the like larger than the ionicity of these serum components. . In this case, if it is desired to reduce the time required for the measurement, it is preferable to use the stepwise gradient method rather than the linear gradient method. In the case where the complex of the measurement target glycoprotein and the first antibody (or / and the second antibody) is separated from the measurement target glycoprotein to which the first antibody is not bound by ion exchange chromatography, In some cases, it may be better to treat the glycoprotein to be measured with sialidase in advance. That is, when a sialic acid residue is bonded to the end of the sugar chain of the glycoprotein to be measured,
In particular, when the sugar chain is branched into 3 to 4 chains and a sialic acid residue is bonded to the terminal, the glycoprotein to be measured and the first antibody are affected by the carboxyl group of the sialic acid residue. This is in order to prevent the separation of the complex with (or / and the second antibody) from the glycoprotein to be measured to which the first antibody is not bound, which may be insufficient. Packing materials for ion-exchange chromatography generally have a high exchange capacity (absolute adsorption capacity for ionic substances), so when analyzing samples with a large absolute amount of ionic coexisting substances such as biological samples such as serum. Even so, all the complexes and the like to which the separation improving substance is bonded can be adsorbed, so that the complexes can be eluted at a position where the influence of the coexisting substances can be almost avoided. In addition, since the separation improving substance that can be used in the present method has high solubility in water, the water solubility of the complex to which the separation improving substance is bound is higher than that before the binding. There is almost no possibility that the object to be measured will be denatured or deactivated during the formation reaction of the complex. Examples of fillers for ion exchange chromatography include DEAE-MCI gel (trade name of Mitsubishi Kasei Co., Ltd.), QAE MCI gel (trade name of Mitsubishi Kasei Co., Ltd.), and Wako Beads DEAE gel (Wako Pure Chemical Industries, Ltd.) Product name), Poros Q
(Perseptive), a filler for anion exchange chromatography such as Poros PI (Perseptive), or SPM
CI gel (trade name of Mitsubishi Kasei Corporation), CM MCI gel (Mitsubishi Chemical Corporation)
(Trade name), Wako Beads CM Gel (Wako Pure Chemical Industries, Ltd.)
Product name), Poros S (Perseptive), Poros CM (Per
and a filler for cation exchange chromatography such as Septive.

In the differential measurement method of the present invention, the complex between the glycoprotein to be measured and the first antibody (or / and the second antibody) and the glycoprotein to be measured to which the first antibody is not bound The fractionation and measurement can be carried out by HPLC using any of the above-mentioned chromatography, and among them, the use of ion-exchange chromatography is most preferable for the present invention. The reasons are as follows, for example. For example, to perform separation and measurement using gel filtration chromatography, it is necessary to use a column of an appropriate length, so when gel filtration chromatography is used, ion exchange chromatography is used. There is a drawback that the separation time is longer than in the case where it is used. Therefore, it is preferable to use ion exchange chromatography when a reduction in the separation time is required. In addition, gel filtration chromatography has the disadvantage that it is not suitable for separating substances having very high molecular weights (molecular size greater than about 1000 angstroms). In addition, in the case of hydrophobic chromatography, there may be a problem that the organic solvent used in the separation operation destroys the high-dimensional structure of the glycoprotein and loses the activity of the glycoprotein to be measured in the complex. For these reasons, it is not preferable to use a substance having high hydrophobicity as the separation improving substance. Further, there is another problem that the complex to which the first antibody to which the hydrophobic substance is bound as the separation improving substance is reduced in water solubility and is easily precipitated, so that separation becomes difficult. On the other hand, ion-exchange chromatography can perform more effective separation and measurement based on subtle differences in ionicity. Furthermore, according to ion exchange chromatography, a separation improving substance having various ionic properties can be arbitrarily selected, so that the glycoprotein to be measured can be separated and measured under the optimum pH condition. Furthermore, since the separation enhancer used in ion exchange chromatography has high water solubility itself, even if the separation enhancer is bound to the target substance, there is almost no possibility that the target substance will precipitate. Thus, the separation operation can be performed in a stable state.

When the fractionation measurement method of the present invention is carried out using a separation improving substance, the peak of the target glycoprotein to be measured can be moved to a position not affected by components such as serum and urine. . In addition, by appropriately selecting and using the separation improving substance according to the measurement target, the elution positions of the complexes relating to various measurement targets can be matched, so using HPLC under specific analysis conditions, There is also an effect that various kinds of measurement target substances can be measured.

In the differential measurement method of the present invention using the first antibody (or / and the second antibody) to which a labeling substance is bound,
The glycoprotein to be measured and the first protein separated by HPLC
A labeling substance contained in a glycoprotein to be measured (including a substance to which the second antibody is bound) to which the complex with the antibody (or / and the second antibody) is not bound to the first antibody (including a substance to which the second antibody is bound); When the glycoprotein itself is measurable by any method, the amount of itself can be measured (detected) by any method possessed by the labeling substance (or the glycoprotein itself to be measured). It is carried out according to a predetermined method depending on the nature. For example, if the property is enzyme activity, E
IA standard method, for example, "Enzyme Immunoassay, Protein Nucleic Acid Enzyme Supplement No.31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, pp. 51-63, Kyoritsu Shuppan Co., Ltd., September 10, 1987 The measurement may be carried out according to the method described in “Issue of Japan” etc., and when the labeling substance is a radioactive substance, the immersion type is used according to the type and intensity of the radiation emitted by the radioactive substance according to the RIA standard method. The measurement may be performed by appropriately selecting and using a measuring device such as a GM counter, a liquid scintillation counter, a well-type scintillation counter, and a HPLC counter (for example, Medical Chemistry Experiment Course, Vol. 8, supervised by Yuichi Yamamura, First Edition) , Nakayama Shoten, 1971, etc.). If the property is fluorescent, FIA using a measuring instrument such as a fluorometer
For example, "illustration fluorescent antibody, Akira Kawao, 1st edition,
The measurement may be performed according to the method described in Soft Science Co., Ltd., 1983, etc.If the property is luminescent, a conventional method using a measuring instrument such as a photon counter, for example, an enzyme immunoassay , Proteins, Nucleic Acids and Enzymes Separate Volume No. 31, Edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 252-263
Page, Kyoritsu Shuppan Co., Ltd., published on September 10, 1987 ”. Further, when the property is a property having absorption in the ultraviolet, measurement may be performed by a conventional method using a measuring instrument such as a spectrophotometer, and when the labeling substance is a substance having a spin property, electron spin resonance is used. A conventional method using an apparatus, for example, "Enzyme immunoassay, protein nucleic acid enzyme, Supplement No.31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 264-271, Kyoritsu Shuppan Co., Ltd., September 1987 The measurement may be performed according to the method described in “10th issue”.

In the differential measurement method of the present invention, the complex of the glycoprotein to be measured and the first antibody (or / and the second antibody) is combined with the glycoprotein to be measured to which the first antibody is not bound. As the HPLC used for fractionation and measurement, any device can be used without any particular problem as long as it is a device which is generally used in the field of analysis and which can obtain a constant flow rate.

The glycoprotein to be measured by HPLC and the first
As a solvent (eluent) used for performing separation and measurement of a complex with an antibody (or / and a second antibody) and a glycoprotein to be measured to which the first antibody is not bound, the complex formed The body and the like again measure the glycoprotein and lectin (or
Antibody), and a marker substance contained in the glycoprotein itself or contained in a labeling substance held by the first antibody (or / and the second antibody). The method is not particularly limited as long as the property that can be detected by the method is not lost, but is usually used as a buffer in a method known per se such as, for example, EIA, RIA, FIA, and affinity chromatography. What is used is preferably used. Specific examples include phosphate, acetate, citrate, and Good (G
ood), a buffer such as tris (hydroxymethyl) aminomethane, for example, sodium chloride, potassium chloride,
Salts such as ammonium sulfate, for example, polar organic solvents such as methanol, ethanol, isopropyl alcohol, acetonitrile, and tetrahydrofuran, and surfactants are appropriately selected, added, and mixed according to the properties of the complex, etc. A buffer having a pH of 2 to 10 is preferably used.

[0038] In the differential measurement method of the present invention, the complex of the glycoprotein to be measured and the first antibody (or / and the second antibody) is not bound to the glycoprotein to be measured. As a measurement method when the measurement is carried out after separation by HPLC, for example, "Latest Liquid Chromatography, Shoji Hara
Akio Tsuji, 1st edition, pp. 92-104, Nanzando, February 1, 1978
Effluent from the HPLC column, as described in “Issue Publication” and the like, directly to the detection unit, and the glycoprotein itself or the first antibody (or / and / or the second antibody) contained in the complex or the like in the effluent is used. The method of directly measuring the amount of the labeling substance held by the (antibody) is more preferable because the measurement can be performed quickly. In this case, the property of the glycoprotein itself contained in the complex or the like or the labeling substance held by the first antibody (or / and the second antibody) has a property that can be detected by any method, such as an enzyme. If it is active, it goes without saying that it is necessary to provide a so-called post-column method reaction section between the HPLC column and the detection section, in which a reagent for enzyme activity measurement is added and reacted with the effluent. When the property is enzyme activity, the reagent for measuring the enzyme activity used in the reaction section is prepared by a conventional method, for example, "Enzyme-linked immunosorbent assay, protein, nucleic acid, enzyme, extra volume No. 3
1.Edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 51-6
3 pages, published by Kyoritsu Shuppan Co., Ltd., September 10, 1987, etc., or a reagent of a commercially available clinical test kit may be appropriately selected. You may use it. In addition, even when the property is other than enzyme activity, an appropriate reaction section should be provided between the HPLC column and the detection section to add and react with a predetermined reagent for the purpose of increasing the detection sensitivity. Is optional.

When a plurality of eluents having different components are used as eluents when HPLC is used in the fractionation measurement method of the present invention, the operation may be performed by a concentration gradient method (linear gradient method). The stepwise gradient method may be used for any purpose, but the stepwise gradient method is advantageous in that the operation is simple, the actual analysis time can be shortened, and the target peak becomes sharp. There is more desirable.

Further, the differential measurement method of the present invention can naturally be applied to a known immunological measurement method such as EIA, RIA, or FIA. Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

[0041]

【Example】

Embodiment 1 FIG. Fractionation measurement by AFP sugar chain using lentil lectin (LCA) -A (lectin solution) LCA-A (Honen Co., Ltd.)
A lectin solution was prepared by dissolving 1 mg / ml in 50 mM 3- (N-morpholino) propanesulfonic acid (MOPS) buffer (pH 7.5). (First antibody solution) An anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.), which has the property of binding to all AFPs but has been confirmed to prevent binding to AFP bound to LCA-A, is usually used. And Fab- ', to which N- (8-maleimidocapryloxy), a bivalent crosslinking agent, is added.
Polyaspartic acid (average molecular weight 2880) by a conventional method using sulfosuccinimide (manufactured by Dojindo Chemical Co., Ltd.)
0, manufactured by Sigma) to obtain a polyaspartic acid-conjugated anti-AFP-Fab '(Fab'-pAsp), which was then treated with 50 mM MO.
A solution added to a PS buffer (pH 7.5) so as to have a concentration of 13.5 μM was used as a first antibody solution. (Second antibody solution) The antibody used to prepare the first antibody solution has a different antigen-recognition site, has the property of binding to all AFPs, and can also bind to AFP bound to LCA-A. An anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) confirmed to have Fab 'was processed into Fab' by a conventional method, and N- (8-maleimidocapriloxy), a divalent crosslinking agent, was added thereto. Horseradish peroxidase (POD, manufactured by Toyobo Co., Ltd.) was bound by a conventional method using sulfosuccinimide (manufactured by Dojindo Co., Ltd.),
A D-labeled anti-AFP-Fab ′ was obtained, and this was added to a 50 mM MOPS buffer (pH 7.5) so as to have a concentration of 100 nM, which was used as a second antibody solution. (Sample solution) LCA-A binding type AFP (hepatocellular carcinoma type AF
P) or LCA-A non-binding AFP (normal AFP)
Was added to a 50 mM MOPS buffer (pH 7.5, containing 0.2 W / V% bovine serum albumin) at a concentration of 100 ng / ml to prepare a hepatocellular carcinoma type AFP solution and a normal type AFP solution, respectively. Both AFP solutions were mixed at a ratio as shown in Table 1 to prepare a sample solution.

[Table 1] * The numbers in the table represent the mixing ratio (%) of each solution. (HPLC conditions) FIG. 3 shows a HPLC configuration diagram. -Column: WAKOPAK MCI CQA-31S (4.6mmIDx10mm). Eluent A: 50 mM MOPS buffer (pH 7.5, containing 50 mM sodium chloride). Eluent B: 50 mM MOPS buffer (pH 7.5, containing 1 M sodium chloride). -Substrate solution: 50 mM MOPS buffer [pH 7.5, containing 100 mM 3- (p-hydroxyphenyl) propionic acid and 20 mM hydrogen peroxide].・ Gradient: 0 to 5 minutes Eluent B 0% 5 to 8 minutes Eluent B 20% 8 to 12 minutes Eluent B 80% ・ Flow rate: Eluent A + B 1 ml / min Substrate solution 0.1 ml / min ・ Coloring coil temperature : 55 ° C ・ Fluorescence detection: excitation wavelength 320nm / fluorescence wavelength 404nm. (Measurement method) Lectin solution 200μl and second antibody solution 10
Add 10 μl of the specified sample solution to 10 μl of the mixed solution, and add
The reaction was performed for 5 minutes. 10 μl of the first antibody solution was added to this reaction mixture.
Was added and the mixture was further reacted for 2 minutes, and then 100 μl of the reaction mixture was analyzed by HPLC. (Results) The separation pattern by HPLC is shown in FIG.
In FIG. 1, peak 1 indicates the peak of AFP to which the first antibody is not bound, and peak 2 indicates the peak of AFP to which the first antibody is bound. FIG. 2 shows the respective peak area values and the sum of the two peak area values for the various sample solutions based on the mixing ratio of the hepatocellular carcinoma-type AFP solution and the normal AFP solution in the various sample solutions. still,
In FIG. 2, □ indicates the area value of peak 1, and + indicates peak 2
Indicates the total area value of peak 1 and peak 2, respectively. As is clear from the results of FIG. 2, the area value of peak 1 increases in proportion to the increase in the amount of hepatocellular carcinoma-type AFP solution in the sample solution, and the area value of peak 2 indicates the amount of normal AFP solution in the sample solution. However, the sum of the area values of the peak 1 and the peak 2 shows a substantially constant value for various sample solutions. In other words, by using these two peak area values, the sample solution is reduced. It turns out that it is possible to measure simultaneously the amount of hepatocellular carcinoma-type AFP and the total AFP concentration contained therein. Embodiment 2. FIG. Change in measured value of fucose addition rate of AFP by sialidase treatment (Sialidase solution) Sialidase (Toyobo Co., Ltd.)
Was dissolved in 50 mM MOPS buffer (pH 7.5) at a concentration of 200 Unit / ml to obtain a sialidase solution. (Lectin solution) Lentil lectin (LCA) -A (Honen Co., Ltd.) was added to 50 mM MOPS buffer (pH 7.5) at 1 mg / ml.
The lectin solution was dissolved in such a manner that (Antibody solution 1) An anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) that competes with LCA-A was treated by a conventional method to obtain Fab ′, and a divalent crosslinking agent [N- (8- (Maleimidocapryloxy) sulfosuccinimide, manufactured by Dojindo Chemical Co., Ltd.] and polyaspartic acid (average molecular weight: 28800, manufactured by Sigma) to bind to polyaspartic acid-conjugated anti-AFP-Fab ′ (Fab′-pAsp). And
This was added to a 50 mM MOPS buffer solution (pH 7.5) so as to have a concentration of 13.5 μM, and was used as an antibody solution 1. (Antibody solution 2) An anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.), which does not compete with the antibody used in the antibody solution 1, was treated by a conventional method to give Fab ′, which was added with a bivalent crosslinking agent [N -(8-maleimidocapryloxy) sulfosuccinimide, manufactured by Dojin Chemical Co., Ltd.] to label horseradish peroxidase (POD, manufactured by Sigma).
OD-labeled anti-AFP-Fab '. This is 50mM MOPS
The antibody solution 2 was added to a buffer solution (pH 7.5) so as to have a concentration of 100 nM. (Sample solution) Fucose was added in advance by lectin gel electrophoresis using AFP-lectin fractionation kit L (manufactured by Wako Pure Chemical Industries, Ltd.) (operation is performed according to the standard operation method described in the instruction manual attached to the kit). Three hepatocellular carcinoma patient sera whose rates were measured were mixed with 50 mM MOPS buffer (pH 7.5, 0.2 W / V%
Contains bovine serum albumin. Sample solutions 1, 2, and 3 were diluted so that the AFP value became 100 ng / ml in the above). (HPLC conditions) FIG. 3 shows a schematic diagram of HPLC. -Column: Wakopak MCI CQA-31S (4.6 mm ID x 10 mm, manufactured by Wako Pure Chemical Industries, Ltd.). Eluent A: 50 mM MOPS buffer (pH 7.5, containing 50 mM sodium chloride). Eluent B: 50 mM MOPS buffer (pH 7.5, containing 1 M sodium chloride). Substrate solution: 50 mM MOPS buffer (pH 7.5, containing 100 mM 3- (p-hydroxyphenyl) propionic acid and 20 mM hydrogen peroxide). Gradient conditions: 0-5 minutes Eluent B 0% 5-8 minutes Eluent B 20% 8-12 minutes Eluent B 80% Flow rate: Eluent A + B 1 ml / min. Substrate solution 0.1 ml / min.・ Coloring coil temperature: 55 ° C. -Fluorescence detection: excitation wavelength 320nm / fluorescence wavelength 404nm. (Measurement method) Sialidase solution 10μl, lectin solution
100 µl, antibody solution 25 µl and predetermined sample solution 10 µ
were mixed and reacted at 10 ° C. for 3 minutes. 110 μl of the antibody solution was added to the reaction mixture, and the mixture was further reacted at 37 ° C. for 4 minutes. After completion of the reaction, 100 μl of the reaction mixture was subjected to HPL under the above conditions.
C, the area of peak 1 (the peak of AFP to which the first antibody was not bound) and the area of peak 2 (the peak of AFP to which the first antibody was bound) were determined for each sample solution. The peak 1 ratio (%) was determined by fitting. Peak 1 ratio (%) = (area of peak 1) / (sum of areas of peaks 1 and 2) × 100 Further, instead of the sample solution, AF having a fucose addition rate of 0% was used.
P or AFP with fucose addition rate of 100% [Fucose addition rate was determined by AFP-lectin fractionation kit L (manufactured by Wako Pure Chemical Industries, Ltd.). 50mM MOPS containing 100ng / ml
Except for using a buffer solution (pH 7.5, containing 0.2% bovine serum albumin), a reaction solution obtained by performing the same reaction using the same solutions as described above was also subjected to HPLC by the same operation as above. The area of peak 1 and the area of peak 2 were determined for each sample solution, and the peak 1 ratio (%) was determined by applying the above equation. A calibration curve representing the relationship between the peak 1 ratio and the fucose addition rate was created from the obtained data, and the peak 1 ratio obtained for the sample solutions 1, 2, and 3 was applied to the calibration curve, and the The fucose addition rate of AFP was determined. Furthermore, sialidase solution
The same reaction was performed using the same solutions as above except that 10 μl of 50 mM MOPS buffer (pH 7.5) was used instead of 10 μl. The fucose addition rate of AFP in the solution was determined. (Results) Table 2 shows the values obtained by lectin gel electrophoresis and the values obtained by the method of the present invention as to the fucose addition rates of the sample solutions 1 to 3.

[Table 2] As is clear from the results in Table 2, it is found that by removing the sugar residue on the non-reducing terminal side of the AFP sugar chain by sialidase treatment, the amount of fucose-added AFP can be measured quickly and accurately.

[0042]

As is apparent from the above description, the present invention provides a method capable of measuring the degree of structural change of a glycoprotein sugar chain in a disease with high accuracy, quickly and easily. By using the present invention, it is possible to provide new information for diagnosis of cancer or the like using the degree of structural change of a glycoprotein sugar chain, which has an effect of contributing to the industry. This is a great invention.

[Brief description of the drawings]

FIG. 1 shows a separation pattern obtained by high performance liquid chromatography (HPLC) obtained in Example 1.

FIG. 2 shows the sum of each peak area value and two peak area values of various sample solutions obtained in Example 1 as hepatocellular carcinoma α-fetoprotein (A) in various sample solutions.
FIG. 2 is a diagram based on a mixing ratio between an FP) solution and a normal AFP solution.

FIG. 3 is a schematic view of the HPLC apparatus used in Example 1.

[Explanation of symbols]

In FIG. 2, □ indicates the area value of peak 1, and + indicates peak 2
Indicates the total area value of peak 1 and peak 2, respectively.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/53 C07K 1/14 G01N 33/538 BIOSIS (DIALOG)

Claims (4)

(57) [Claims] The present invention relates to a fractionation measurement method for measuring glycoproteins having different sugar chain structures but substantially the same protein structure,
A lectin that recognizes at least one specific sugar chain structure of the glycoprotein to be measured, and a property that binds to all the glycoproteins to be measured but prevents the binding of the lectin to the glycoprotein to be measured Antibody (hereinafter, referred to as
Abbreviated as first antibody. ), Wherein a glycoprotein bound to the first antibody and a glycoprotein not bound to the first antibody are fractionated and measured by utilizing the difference between these properties. . 2. The method according to claim 1, wherein a substance or a labeling substance capable of changing the property of a complex between the glycoprotein to be measured and the first antibody is bound to the first antibody. 3. An antibody having a property capable of binding to all glycoproteins to be measured including the glycoprotein to be measured, to which the lectin is bound, and having a labeling substance bound thereto (hereinafter abbreviated as a second antibody). 3. The method according to claim 1, wherein
Fractionation measurement method described in 1. 4. The glycoprotein to be measured has been treated with glycosidase in advance, or the glycosidase coexists when reacting the glycoprotein to be measured with a lectin and a first antibody and, if necessary, a second antibody. The method for fractionation measurement according to any one of claims 1 to 3.