JPH07191027A - Method for separately measuring glycoprotein - Google Patents

Abstract

PURPOSE:To provide new information for the diagnosis of the cancer, etc., of a patient utilizing the degree of structural change of the sugar chain of glycoproteins by providing a method by which the degree of structural changes of the sugar chain of the glycoproteins can be quickly and easily measured with high accuracy when the patient is suffering from a disease. CONSTITUTION:The method for separately measuring glycoproteins is used for glycoproteins having different sugar chain structures and substantially same glycoprotein structures as objects to be measured. In the method, a lectin which recognizes at least one specific sugar chain structure of the glycoproteins to be measured and an antibody (first antibody) having such a nature that the antibody can be bonded to all glycoproteins to be measured, but cannot be bonded to some of the glycoproteins to be measured to which lectin is bonded are used in a combined way and glycoproteins to which the first antibody is bonded and glycoproteins to which the antibody is not bonded are separately measured by utilizing such difference in nature.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for differentially measuring 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 received a great deal of attention as a substance that bears some "information" about the living body due to its structural diversity, etc., and its research is being conducted by many universities and research institutes both in Japan and abroad.

During the course of the research, it has been observed that changes in the sugar chain structure of specific glycoproteins are caused by some diseases. For example, in the case of human α-fetoprotein (AFP), addition of α-L-fucose residue or N-acetylglucosamine residue (bisecting N-acetylglucosamine) to AFP sugar chain is accompanied by the progress of hepatocellular carcinoma. It has been confirmed that is frequently observed. Moreover, since the degree of such sugar chain structure change is independent of serum AFP concentration and is confirmed from the early stage of hepatocellular carcinoma, it is considered that cancer diagnosis can be made by examining the degree of change. Has been attracting attention.

The degree of sugar chain structure change of glycoprotein can be measured by
It is mainly performed by an analysis method using a lectin, for example, a lectin column method, a lectin electrophoresis method and the like. The reason why the degree of sugar chain structure change is mainly measured by using lectins is that not only are lectins inexpensive, but the immunogenicity of sugar chains is higher than that of the protein skeleton of glycoproteins. Therefore, an effective anti-glycoprotein sugar chain antibody cannot be obtained except for a part of the non-reducing end structure, that is, it is extremely difficult to prepare an antibody against the glycoprotein sugar chain.

However, although the lectin has a high recognition specificity of sugar chain structure, the binding force (binding constant) to the sugar chain is several thousand to tens of thousands times lower than that of the antibody, so that the lectin and the sugar are bound. It is difficult to form a strong complex with the chain such as an antigen-antibody reaction. Therefore, when the lectin is used instead of the antibody in an analysis method such as an enzyme-linked immunosorbent assay (ELISA) that uses a solid-phased antibody, which requires a washing operation after complex formation on the solid phase, The phenomenon of the dissociation of the complex of the glycoprotein to be measured and the lectin again occurs during washing, which causes problems such as a marked decrease in measurement sensitivity.
Such a method cannot be said to be a practical method as a clinical diagnostic method. Further, because of the properties of the lectin as described above,
To measure glycoprotein by utilizing the reaction between lectin and glycoprotein to be measured, use a lectin-immobilized column, lectin-containing agarose gel, etc. There was a constraint that it had to be done under excessive conditions.

Therefore, under the present circumstances, it is desired to develop a rapid and simple assay method for glycoprotein sugar chain fractionation, which has both sugar recognition specificity of lectin and high sensitivity of enzyme immunoassay.

[0007]

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

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is a differential measurement method for measuring glycoproteins having different sugar chain structures but substantially the same protein structure, wherein at least one specific sugar chain of the measurement target glycoprotein is used. An antibody having the property of binding to a lectin that recognizes a structure and all of the glycoproteins to be measured, but having the property of preventing the binding to the glycoprotein of the measurement target bound by the lectin (hereinafter, abbreviated as a first antibody). ) Is used in combination, the glycoprotein bound to the first antibody and the glycoprotein not bound to the first antibody are fractionally measured by utilizing the difference in these properties, and the differential measurement method is characterized. Is the invention of.

[0009] That is, the present inventors have been earnestly researching to develop a rapid and simple fractionation assay method for glycoproteins, which has both the sugar recognition specificity of lectin and the high sensitivity of enzyme immunoassay, and is currently conducting research on glycoproteins. It was found that among antibodies, there is an antibody (primary antibody) having the property of preventing an antigen-antibody reaction in the presence of a lectin. Therefore, further research was conducted to develop a method for differentially measuring glycoproteins using this antibody. As a result, a lectin capable of discriminating a structural change of glycoprotein sugar chains due to a disease and an antibody having the above-mentioned properties (first It was found that it is possible to measure the degree of sugar chain structure change of a glycoprotein with high sensitivity by using it in combination with an antibody), and completed the present invention.

To carry out the present invention, for example, the following may be carried out. That is, first, a biological sample containing the glycoprotein to be measured and at least one of the glycoprotein to be measured
After reacting a lectin that recognizes one specific sugar chain structure with the first antibody, a complex of the measurement target glycoprotein and the first antibody, and the measurement target glycoprotein to which the first antibody is not bound And, using the difference in these properties, such as the difference in molecular weight, the difference in isoelectric point, the strength of electric charge, the strength of hydrophobicity, etc., and then measuring each by a method according to each property. Can be implemented by

When reacting the glycoprotein to be measured with the lectin and the first antibody, they may be reacted simultaneously, but after reacting the lectin first,
Reacting an antibody is preferable because the reaction between the lectin and the glycoprotein is surely caused and the measurement accuracy is improved.

Further, in order to facilitate the detection of the glycoprotein to be measured in the above-described fractional measurement according to the present invention,
It is desirable to bind to the first antibody a substance capable of changing the properties of the complex of the glycoprotein to be measured and the antibody (hereinafter abbreviated as separation enhancing substance) or a labeling substance.

Furthermore, if it is desired to facilitate the detection of the glycoprotein to be measured and also to measure the total amount of glycoprotein together, all glycoproteins to be measured including the glycoprotein to be measured to which the lectin has bound are measured. It is desirable to further combine and use an antibody (hereinafter, abbreviated as a second antibody) having a property of being capable of binding and having a labeling substance bound thereto. Second
To carry out the present invention using an antibody, for example, the following may be carried out. 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 the first antibody are reacted. Thereafter, the 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, It can be carried out by separating each of them by using the strength of electric charge, the strength of hydrophobicity and the like, and then measuring each by using the property of the labeling substance bound to the second antibody.

When reacting the glycoprotein to be measured with the second antibody, the lectin and the first antibody, these may be reacted simultaneously, but the lectin (and the second antibody)
It is preferable that the first antibody is first reacted with the first antibody because the reaction between the lectin and the glycoprotein is surely caused and the measurement accuracy is improved. When reacting (or before reacting) the lectin, the first antibody, or the second antibody with the glycoprotein to be measured, the glycosidase treatment of the glycoprotein enables the target differential measurement to be performed more accurately. You may get it. That is, the sugar chain end of the glycoprotein to be measured causes various structural changes depending on the type of disease, but depending on the type of sugar residue bound to the non-reducing end 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 hindered or unable to bind. In such a case, when the glycoprotein to be measured is reacted with the lectin, the first antibody or the second antibody (or before reacting with these), it is treated with an appropriate glycosidase to remove non-glycans of the glycoprotein. The lectin, the first antibody, or the second antibody by appropriately removing the sugar residue on the reducing end side
By facilitating the binding of the antibody to the glycoprotein having the target structure, the accuracy of the target differential measurement can be improved. The glycosidase used for such purpose is not particularly limited as long as it has an action as a sugar hydrolase, and for example, sialidase, β-galactosidase, β-N-acetylglucosaminidase, α-mannosidase, β-mannosidase. , Α-fucosidase and the like are preferable. The origin of these glycosidases is not particularly limited. In the case as described above, conditions such as temperature and pH when reacting glycosidase may be appropriately selected according to the type of glycosidase used, but the reaction temperature is usually 0 to 50 ° C., preferably The range of 1 to 40 ° C., more preferably 25 to 37 ° C., and the pH during the reaction are usually 2 to 10 and preferably 4 to 9. The concentration of glycosidase used varies depending on the type of glycosidase used, and is not particularly limited as long as it is at a concentration at which the desired sugar hydrolysis reaction occurs or higher, but when performing the sugar hydrolysis reaction, in a solution with a thin glycosidase concentration. It is desirable to carry out the reaction in a solution with a relatively high concentration of glycosidase for a short time rather than for a long time in order to prevent unnecessary elimination of sugar chains, and the total amount in the reaction solution is usually 10 to 1000 mU (unit ), Preferably 50-500
The reaction time is usually 1 minute to 1 day and night, preferably 3 minutes to 1 hour. When the reaction with glycosidase is carried out simultaneously with the reaction with lectin, the first antibody or the second antibody, the reaction conditions described above are to some extent restricted by the reaction conditions when reacting with lectin or the like. There is no end.

The lectin used in the present invention has a property of recognizing a target sugar chain structure from various lectins such as concanavalin A, lentil lectin, kidney bean lectin, datsura lectin, wheat germ lectin and the like. You can select and use the appropriate one,
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, an antibody having the above-mentioned properties can be prepared by a conventional method, for example, " According to the method described in "Introduction to Immunological Experiments, 2nd edition, Nao Matsuhashi et al., Academic Society Publishing Center, 1981", etc., for example, for animals such as horses, cows, sheep, rabbits, goats, rats, mice, etc. Cells produced from a tumor line of a mouse according to a conventional method, that is, a cell fusion method established by Keller and Milstein (Nature, 256, 495, 1975). The monoclonal antibody produced by the hybridoma obtained by fusing the spleen cells of a mouse previously immunized with the measurement target may be any antibody, and these may be used alone or in appropriate combination. 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 that recognizes at least one specific sugar chain structure of a glycoprotein to be measured, and a first antibody, which include glycoproteins having different sugar chain structures but substantially the same protein structure Are not particularly limited as long as they exist, for example, amylase,
Alkaline phosphatase, acid phosphatase, γ-glutamyl transferase (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), luteinizing hormone. (LH) and other physiologically active substances, such as prostate specific antigen (PSA), α ₂ -macroglobulin, carcinoembryonic antigen (CEA), α-fetoprotein and other cancer-related antigens, such as sugar chains such as CA19-9 and CA125 Antigens and the like are preferable.

In the differential assay method of the present invention, the concentration of the lectin used varies depending on the type of lectin used, the properties of the glycoprotein to be measured, etc., but the set detection limit concentration of the glycoprotein to be measured is set. Usually 10 times or more, preferably 10
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 used varies depending on how much the detection limit of the glycoprotein to be measured is set, but the difference in binding constant between the lectin and the first antibody to the glycoprotein to be measured is taken into consideration. It is desirable to set it after doing so. For example, although it varies depending on the glycoprotein to be measured, the type of lectin and its physical properties, the physical properties of the first antibody, etc., it is desirable to set using the general formula shown below. Concentration of first antibody ≤ (binding constant of lectin) / (binding constant of first antibody) x (concentration of lectin) The binding constant relating to the above general formula means the equilibrium reaction as shown in the following formula (1). A coupling constant is shown and is a constant calculated by the following equation (2). [A] + [B] ← → [A · B] (1) Binding constant = [A · B] / ([A] × [B]) (2) [A]: Lectin or the lectin in the equilibrium state 1 Antibody concentration (M). [B]: Free measurement target glycoprotein concentration (M) in the equilibrium state. [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 the 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 this case, the concentration of the first antibody will be 1/100 or less, preferably 1/1000 or less, of the lectin concentration. The concentration of the first antibody used is preferably a concentration at which the set detection limit concentration can bind to all of the glycoproteins to be measured, but may be lower than that (for example, about 1/10).

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

In the differential assay method of the present invention, the glycoprotein to be assayed is allowed to react with the lectin and the first antibody, and further with the second antibody to form a complex of the lectin and the glycoprotein, the first antibody. The reaction conditions for forming a complex or the like of the antibody and glycoprotein (or a complex of the complex and the second antibody) are not particularly limited as long as they do not interfere with the formation reaction of these complexes. Although not carried out, a reaction for forming a complex or the like in a conventional method such as enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), affinity chromatography, etc. It may be performed according to the conditions. For example, when a buffer solution is used in the reaction, the buffer agent and other reagents to be used may be appropriately selected from those used in the methods known per se. The pH during the reaction is not particularly limited as long as it does not interfere with the complex formation reaction, but is usually 2 to 10, preferably 5 to 9
The range of is mentioned. The temperature during the reaction is not particularly limited as long as it does not hinder the complex-forming reaction, but is usually 0 to 50 ° C, preferably 0 to 40 ° C, more preferably 0 to 10 ° C. Can be mentioned. The reaction time depends on the reactivity of the glycoprotein to be measured with the lectin, the first antibody and the second antibody, and therefore the time required for the formation of these complexes may vary from several seconds to several seconds depending on each property. The reaction may be appropriately performed for several hours.

As the separation-improving substance used for binding to the first antibody, a substance 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 There is no particular limitation so long as it is, but specifically, for example, α-chymotrypsinogen, β
Proteins such as galactosidase, lysozyme, cytochrome C, trypsin inhibitor, peptides containing amino acids such as phenylalanine, proline, arginine, lysine, aspartic acid, glutamic acid, halogen atoms such as bromine, chlorine, iodine, such as polyethylene glycol Synthetic polymers such as polyamino acids such as polyglutamic acid, polyaspartic acid, polylysine, polyarginine, polyphenylalanine and polytyrosine, alkyl chains having 3 to 10 carbon atoms, such as fatty acids such as palmitic acid, oleic acid and stearic acid, for example N-
(ε-maleimidocaproyloxy) succinimide [N-
(ε-maleimidocaproyloxy) succinimide] (EMCS),
N-Succinimidyl-6-maleimidohexanoate (NS
Preferred examples include chemical substances having a reactive group capable of binding to the first antibody, such as uccinimidyl-6-maleimidohexanoate), bismaleimidohexane (BMH), and octylamine, and having hydrophobicity or ionicity. The separation-improving substance according to the present invention is appropriately selected in consideration of the properties of the glycoprotein to be measured and the first antibody (eg pH stability, hydrophobicity, solubility in aqueous solution, isoelectric point, etc.). It is enough to use.

The method for binding the first antibody and the separation-improving substance according to the present invention includes a method for binding a specific reactive group of the first antibody and a specific reactive group of the separation-improving substance, and a separation-improving specific reactive group of the first antibody. Examples thereof include a method of substituting with a substance, a method of binding the first antibody and the separation improving substance through a substance capable of binding to the first antibody (eg, antibody, lectin, antigen, inhibitor, DNA, etc.). More specifically, for example, 1) a method for binding an antibody known per se to an antibody generally known in EIA, RIA, FIA or the like known per se (for example, Medical Experiment Account, Volume 8, supervised by Yuichi Yamamura,
First edition, Nakayama Shoten, 1971; Illustrated fluorescent antibody, Akira Kawao,
1st edition, Soft Science Co., Ltd., 1983; Enzyme Immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, 2nd edition, Medical Institute,
1982, etc.), 2) Modification of substances known per se and binding method (for example, chemical modification of protein <up><down>, Ikuzo Uraya,
Kensuke Shimura, Michinori Nakamura, Masaru Funatsu, 1st Edition, Academic Publishing 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.) can be mentioned without exception, and the method may be carried out according to these methods.

Examples of the labeling substance to be labeled on the first antibody (or used to prepare the second antibody) according to the present invention include alkaline phosphatase, β-galactosidase, peroxidase, 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,
Radioisotopes such as ¹²⁵ I, ¹⁴ C and ³ H, for example, fluorescent substances such as fluorescein, dansyl, fluorescamine, coumarin, naphthylamine or their derivatives used in FIA, such as luciferin, isoluminol, luminol, bis. Luminescent substances such as (2,4,6-trifluorophenyl) oxalate, such as phenol, naphthol, anthracene or derivatives thereof having absorption in the ultraviolet, such as 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
Examples thereof include substances having a property as a spin labeling agent represented by a compound having an oxyl group such as -tolyloxyl, but it goes without saying that the substance is not limited thereto.

As a method for labeling the first antibody or the like with the above-mentioned labeling substance on the first antibody (or for preparing the second antibody), generally known EIA, RIA or FIA is known. Known labeling methods that are used (for example, Medical Chemistry Laboratory Course, Volume 8, edited by Yuichi Yamamura, 1st edition,
Nakayama Shoten, 1971; Illustrated fluorescent antibody, Akira Kawao, first edition,
Soft Science Co., Ltd., 1983; Enzyme-linked immunosorbent assay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, 2nd edition, Medical School, 1982, etc.)
Can be mentioned without exception, and it may be carried out according to these. Further, it goes without saying that a conventional method utilizing the reaction of avidin (or streptavidin) and biotin may be used as the labeling method.

When the glycoprotein to be measured according to the present invention is one that can be measured (detected) by some method itself, for example, an enzyme, such a property of the glycoprotein itself is used to measure the object. It goes without saying that it is possible to measure (detect) the glycoprotein to be measured in which the complex of the glycoprotein of 1) and the first antibody (or / and the second antibody) and the first antibody are not bound. Needless to say, the measurement at that time may be carried out according to a method for measuring enzyme activity known per se.

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 are utilized by utilizing the difference in these properties. The fractionation and measurement may be carried out by using an ordinary column chromatography method or electrophoresis method, but if using a high performance liquid chromatography (HPLC) method, a capillary electrophoresis method or the like, a shorter time is obtained. In addition, it is desirable because it enables accurate and precise measurement.

When the separation and measurement of these complexes are performed by HPLC, the complex of the glycoprotein to be measured and the first antibody (or / and the second antibody) and the measurement target in which the first antibody is not bound Separation by means of HPLC equipped with a column packed with a packing material selected depending on the difference in properties from the glycoprotein, or when the separation-improving substance is bound to the first antibody. . Then, an appropriate measuring method for the separated complexes or the like depending on their properties, or, if a second antibody is used, a measuring method according to the property of the labeling substance in the second antibody The amount of any of the glycoproteins to be measured in the sample can be determined by measuring with.

Further, the type of the packing material of the column used in HPLC is also determined by the complex of the glycoprotein to be measured and the first antibody (or / and the second antibody) and the measurement object in which the first antibody is not bound. It may be appropriately selected depending on the difference in properties from the glycoprotein and the properties of the separation enhancing substance. The differences in the 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 below. The method of selecting the filler in accordance with the method will be described in more detail.

When a gel filtration filler is used Since the filler has a property of separating the target substance from other coexisting substances by utilizing the difference in molecular weight, the glycoprotein to be measured and the first antibody (or And / or the second antibody) has a molecular weight of 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more the molecular weight of the glycoprotein to be measured to which the first antibody is not bound, or Is preferably a polymer, a synthetic polymer such as polyethylene glycol, or a polymeric substance such as polyamino acid as the separation-improving substance, because high separation ability can be obtained when the molecular weight ratio of these is as described above. Used. The separation method using the separation-enhancing substance based on the above principle is particularly effective when the sample containing the substance to be measured contains various substances such as serum that affect the analysis and the molecular weights thereof are wide. It is valid. That is, in order to avoid the influence of coexisting substances in such a sample, the molecular weight of the complex should be made larger than the maximum value of the molecular weight of the substance that affects the analysis. It is sufficient to select and use those having a molecular weight that satisfies Further, when utilizing a separation method using a separation improving substance, when a separation improving substance having a molecular weight larger than the cut-off molecular weight of the gel filtration filler is bound, the complex containing the substance to be measured is Since it elutes in the void portion, the analysis can be performed in the shortest time. In this case, it goes without saying that the separation improving substance does not have to be a substance having a single molecular weight, that is, a substance having a molecular weight higher than the molecular weight cut-off of the filler for gel filtration. Further, as the gel filtration filler, for example, YMC-pack Diol-200
(YMC Co., Ltd. product name), YMC-Pack Diol-300
(YMC product name), TSK gel (Toso Co., Ltd. product name) and the like.

When a packing material for hydrophobic chromatography is used Since the packing material has a property of separating the target substance and other coexisting substances by utilizing the difference in hydrophobicity, the glycoprotein to be measured and the first antibody ( Or / and the hydrophobicity 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 as a separation enhancing substance, for example, α-
Proteins with high hydrophobicity such as chymotrypsinogen and β-galactosidase, for example, peptides containing highly hydrophobic amino acids such as phenylalanine and proline, homopolymers of hydrophobic amino acids such as polyphenylalanine and polytyrosine, 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 such as octylamine, EMCS, BMH, etc., such as complex of fatty acids such as palmitic acid, oleic acid, stearic acid, etc. It is available when a substance that can be set appropriately is used. When a peptide is used as the separation-improving substance, a peptide containing highly hydrophobic amino acids is preferable, and the hydrophobicity may be adjusted by selecting its chain length. Further, 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 decreases, so 2 to 15 are preferable. 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 highly hydrophobic chemical substances include substances having a long alkyl chain in addition to those mentioned above, and the hydrophobicity can be adjusted by appropriately selecting the alkyl chain length. In addition, since the separation enhancing substance having too high hydrophobicity has low solubility in water, it is necessary to use an organic solvent during the binding reaction between the first antibody and the separation enhancing substance, which results in denaturation of the resulting modified first antibody. It may be difficult to say that it is preferable as a separation improving substance because it may cause a problem such as a decrease in the activity, or the obtained modified first antibody becomes insoluble in water. Further, as the packing material for hydrophobic chromatography, for example, butyl-NPR (Toso Co., Ltd. trade name), butyl
Examples include MCI gel (trade name of Mitsubishi Kasei Co., Ltd.) and phenyl MCI gel (trade name of Mitsubishi Kasei Co., Ltd.).

When using a packing material for ion exchange chromatography: Since the target substance and other coexisting substances are separated by utilizing the difference in ionicity, the glycoprotein to be measured and the first antibody (or //
And a second antibody) has an ionicity 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 enhancing substance, For example, a peptide containing an acidic protein such as trypsin inhibitor, a residue of a basic amino acid such as arginine and lysine, or a residue of an acidic amino acid such as aspartic acid and glutamic acid, a polyamino acid containing 50 or more amino acid residues as described above. , For example, when fatty acids such as palmitic acid, oleic acid and stearic acid are used. In ion exchange chromatography, in general, it is better to adsorb the measurement target once on the column and then to elute, so that high resolution and high specificity can be obtained. Is a packing material for cation exchange chromatography,
When using an anionic separation enhancing substance, it is preferable to use a packing material for anion exchange chromatography. When using a peptide or polyamino acid composed of only basic amino acid residues (or acidic amino acid residues) as a separation improving substance, the elution time of the complex can be freely adjusted by adjusting the number of amino acid residues. However, if a peptide or polyamino acid consisting of 5 or more, preferably 50 or more, and more preferably 100 or more amino acid residues is used, the elution position of the complex is the same as that of biological components in serum or urine. It is desirable because it can be completely separated. Further, when the peptide or polyamino acid is a synthetic peptide or a synthetic polyamino acid, the length of the peptide (or polyamino acid) and the ionicity are in a proportional relationship. By adjusting and using it as a separation improving substance, the elution position of the complex or the like can be easily adjusted. Even when there are multiple serum components that affect the measurement, these effects can be avoided by using a separation-enhancing 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 measurement, it is preferable to use the stepwise gradient method rather than the linear gradient method. When 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 are separated 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 bound 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 and a sialic acid residue is bound to the end thereof, the glycoprotein to be measured and the first antibody are affected by the carboxyl group of the sialic acid residue. This is to prevent this because the separation between the complex with (or / and the second antibody) and the glycoprotein to be measured to which the first antibody is not bound may be insufficient. Since the packing material for ion exchange chromatography generally has a high exchange capacity (absolute adsorption capacity for ionic substances), it can be used when analyzing samples with a large absolute amount of ionic coexisting substances such as biological samples such as serum. Even if there is any, it is possible to adsorb all the complex or the like to which the separation improving substance is bound, so that the complex can be eluted at a position where the influence of the coexisting substance can be almost avoided. In addition, since the separation-enhancing substance that can be used in this method has high solubility in water, the water solubility of the complex to which it is bound is higher than that before the binding. There is almost no possibility of denaturation or deactivation of the measurement object during the reaction for forming the complex. Examples of the packing material 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.), Wako beads DEAE gel (Wako Pure Chemical Industries, Ltd.) Product name), Poros Q
(Perseptive), Poros PI (Perseptive), and other anion-exchange chromatographic fillers, or for example SP M
CI gel (trade name of Mitsubishi Kasei), CM MCI gel (Mitsubishi Kasei)
(Trade name), Wako beads CM gel (Wako Pure Chemical Industries, Ltd.)
Product name), Poros S (manufactured by Perseptive), Poros CM (Per
and a packing material for cation exchange chromatography such as septive).

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) and the glycoprotein to be measured to which the first antibody is not bound Fractionation and measurement can be performed by HPLC using any of the above-mentioned chromatographies, but of these, the use of ion-exchange chromatography is most preferable for the present invention. The reason is as follows, for example. For example, in order to carry out fractionation and measurement using gel filtration chromatography, it is necessary to use a column of an appropriate length. Therefore, when gel filtration chromatography is used, ion exchange chromatography should be used. There is a drawback in that the separation time is longer than when using it. Therefore, it is preferable to utilize ion exchange chromatography when shortening the separation time is required. Further, gel filtration chromatography has a drawback that it is not suitable for separating substances having a very high molecular weight (molecular size is about 1000 angstrom or more). Further, 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 the activity of the glycoprotein to be measured in the complex is lost. For this reason, it is not preferable to use a substance having high hydrophobicity as the separation improving substance. Further, there is a problem that the complex becomes difficult to separate because the water solubility of the complex bound with the first antibody bound with the hydrophobic substance as the separation-improving substance is lowered and the complex is easily precipitated. On the other hand, ion-exchange chromatography can more effectively perform separation and measurement based on the subtle difference 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 fractionated and measured under optimum pH conditions. Furthermore, since the separation-improving substance used in ion exchange chromatography has a high water solubility itself, even if the separation-improving substance is bound to the measurement target substance, there is almost no risk of precipitation of the measurement target substance. , It becomes possible to execute the separation operation in a stable state.

When the fractionation-measuring method of the present invention is carried out using a separation-enhancing substance, the peak of the target glycoprotein to be measured can be moved to a position that is not affected by components such as serum and urine. . Not only that, by appropriately selecting and using a separation-enhancing substance according to the measurement target, it is possible to match the elution position of the complex relating to various measurement targets, so using HPLC under specific analysis conditions, The effect that various kinds of substances to be measured can be measured is also produced.

In the differential assay method of the present invention using the first antibody (or / and the second antibody) to which the labeling substance is bound,
Glycoprotein to be measured and first separated by HPLC
The labeling substance (measurement target) contained in the glycoprotein (including the second antibody binding) of the measurement target in which the complex with the antibody (or / and the second antibody) is not bound to the first antibody When the glycoprotein itself is measurable by any method, the amount of the glycoprotein 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 enzymatic activity, E
Conventional methods of IA, for example, “Enzyme-linked immunosorbent assay, protein / nucleic acid / enzyme separate volume No. 31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, pages 51-63, Kyoritsu Publishing Co., Ltd., September 10, 1987. According to the standard method of RIA when the labeled substance is a radioactive substance, the immersion type is used according to the type and intensity of the radiation emitted by the radioactive substance. A measurement device such as a GM counter, a liquid scintillation counter, a well-type scintillation counter, or an HPLC counter may be appropriately selected and used for measurement (eg, medical chemistry experiment course, Volume 8, supervised by Yuichi Yamamura, 1st edition). , Nakayama Bookstore, 1971 etc.). When the property is fluorescent, FIA using a measuring device such as a fluorometer
Common method, for example, "Illustrated fluorescent antibody, Akira Kawao, 1st edition,
The measurement may be performed according to the method described in "Soft Science Co., Ltd., 1983" and the like. When the property is luminescent, a conventional method using a measuring device such as a photon counter, for example, "enzyme immunoassay method" , Protein Nucleic Acid 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 ”and the like. Further, when the property has absorption in the ultraviolet, the measurement may be carried out by a conventional method using a measuring device such as a spectrophotometer, and when the labeled substance is a substance having a spin property, electron spin resonance A conventional method using an apparatus, for example, “enzyme immunoassay, protein / nucleic acid / enzyme separate volume No. 31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, pages 262-171, Kyoritsu Publishing Co., Ltd., September 1987. The measurement may be performed according to the method described in "Issued on the 10th".

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) and the glycoprotein to be measured to which the first antibody is not bound As the HPLC used for the fractionation and measurement, the apparatus itself can be used without any particular problem as long as it is one that is usually used in the field of analysis and a constant flow rate can be obtained.

Glycoprotein to be measured by HPLC and the first
As the solvent (eluent) used for the separation and measurement of the complex with the antibody (or / and the second antibody) and the glycoprotein to be measured to which the first antibody is not bound, the complex formed is used. The body etc. again measure the glycoprotein and lectin (or the first
Antibody) and is not possessed by the glycoprotein itself, or is possessed by the labeling substance retained by the first antibody (or / and the second antibody), There is no particular limitation as long as it does not lose the property detectable by the method, but it is usually used as a buffer in a method known per se such as EIA, RIA, FIA, affinity chromatography and the like. Those described above are preferably used. Specific examples include, for example, phosphate, acetate, citrate, Good (G
ood) buffers, buffers such as tris (hydroxymethyl) aminomethane, such as sodium chloride, potassium chloride,
Salts such as ammonium sulfate, for example, polar organic solvents such as methanol, ethanol, isopropyl alcohol, acetonitrile, tetrahydrofuran and surfactants, etc. are appropriately selected according to the properties of the complex, etc., added, prepared by mixing. A buffer having a pH of 2 to 10 is preferably used.

In the differential assay method of the present invention, the complex of the glycoprotein to be assayed and the first antibody (or / and the second antibody) and the glycoprotein to be assayed in which the first antibody is not bound As a measurement method in the case of performing measurement with and after separation by HPLC, for example, “Latest Liquid Chromatography, Shoji Hara
Atsuo Tsuji, 1st edition, pages 92-104, Nanzandou, February 1978 1
The effluent from the column of HPLC as described in "Issuance by the Sun" etc. is directly introduced to the detection unit, and the glycoprotein itself or the first antibody (or / and the second antibody) contained in the complex etc. in the effluent is 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 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, for example, an enzyme. Needless to say, if it is active, it is necessary to provide a reaction part of a so-called post-column method between the HPLC column and the detection part, in which a reagent for measuring enzyme activity is added to react with the effluent. When the property is enzymatic activity, the reagent for measuring the enzymatic activity used in the reaction part is a conventional method, for example, “enzyme immunoassay, protein nucleic acid enzyme separate volume No. 3”.
1, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 51-6
3 page, Kyoritsu Shuppan Co., Ltd., published September 10, 1987 "may be used, or the reagents of commercially available clinical test kits may be appropriately selected. You may use it. Even when the property is other than enzyme activity, an appropriate reaction part should be provided between the HPLC column and the detection part in order to add and react a predetermined reagent for the purpose of increasing the detection sensitivity. Is optional.

When a plurality of eluents having different components are used when HPLC is used in the fractional measurement method of the present invention, the concentration gradient method (linear gradient method) may be used as the operating method. Although it may be carried out by the wise gradient method or any method, the stepwise gradient method has advantages such as easy operation, shortening the actual analysis time, and sharpening the target peak. Is more desirable.

The fractional measurement method of the present invention can be naturally applied to immunological measurement methods such as EIA, RIA and FIA known per se. Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

[0041]

【Example】

Example 1. Fractional measurement by AFP sugar chain using lentil lectin (LCA) -A (lectin solution) LCA-A (Hornen Co., Ltd.)
A lectin solution was prepared by dissolving 1 mM / 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 was confirmed to prevent binding to AFPs bound to LCA-A, is always used. Fab 'by treatment with a divalent cross-linking agent N- (8-maleimidocapryloxy)
Polyaspartic acid (average molecular weight 2880) was prepared by a conventional method using sulfosuccinimide (manufactured by Dojindo Co., Ltd.).
0, manufactured by Sigma) was bound to obtain polyaspartic acid-conjugated anti-AFP-Fab '(Fab'-pAsp), which was 50 mM MO.
The first antibody solution was prepared by adding 13.5 μM to PS buffer (pH 7.5). (Second antibody solution) The antibody used for preparing the first antibody solution has a different antigen recognition site from the antigen recognition site, has a property of binding to all AFPs, and also has a property of binding to AFPs bound to LCA-A. An anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) confirmed to have Fab was treated with a conventional method to give Fab ′, to which a divalent cross-linking agent N- (8-maleimidocapryloxy) was added. Horseradish peroxidase (POD, manufactured by Toyobo Co., Ltd.) was bound by a conventional method using sulfosuccinimide (manufactured by Dojindo Co., Ltd.) to obtain PO.
D-labeled anti-AFP-Fab ′ was obtained, and this was added to 50 mM MOPS buffer (pH 7.5) at 100 nM to give 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 50 mM MOPS buffer (pH 7.5, containing 0.2 W / V% bovine serum albumin) at 100 ng / ml to prepare a hepatocellular carcinoma type AFP solution and a normal type AFP solution, respectively. Both AFP solutions were mixed at the ratios 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) An HPLC configuration diagram is shown in FIG. -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 1M 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 ・ Color coil temperature : 55 ° C ・ Fluorescence detection: excitation wavelength 320 nm / fluorescence wavelength 404 nm. (Measurement method) 200 μl of lectin solution and second antibody solution 10
Add 10 μl of the specified sample solution to the mixed solution of μl, and add 1 at 10 ℃.
Allowed to react for 5 minutes. 10 μl of the first antibody solution was added to this reaction mixture.
Was added and reacted for another 2 minutes, then 100 μl of the reaction mixture was analyzed by HPLC. (Result) The separation pattern by HPLC is shown in FIG.
In addition, in FIG. 1, peak 1 shows the peak of AFP to which the first antibody is not bound, and peak 2 shows the peak of AFP to which the first antibody is bound. In addition, each peak area value and the sum of the two peak area values for each sample solution are shown in FIG. 2 based on the mixing ratio of the hepatocellular carcinoma type AFP solution and the normal type AFP solution in each sample solution. still,
In Fig. 2, □ is the area value of peak 1, and + is the peak 2.
Indicates the area value of, and ⋄ indicates the sum of the area values of peak 1 and peak 2. 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 shows the amount of normal type AFP solution in the sample solution. However, the sum of the area values of peak 1 and peak 2 shows a substantially constant value for each sample solution, in other words, by using these two peak area values, the sample solution It is understood that it is possible to simultaneously measure the amount of hepatocellular carcinoma type AFP contained therein and the total AFP concentration. Example 2. Change in measured value of fucose addition rate of AFP by sialidase treatment (sialidase solution) sialidase (Toyobo Co., Ltd.)
The product was dissolved in 50 mM MOPS buffer (pH 7.5) at 200 Unit / ml to give a sialidase solution. (Lectin solution) Lentil lectin (LCA) -A (Hornen Co., Ltd.) was added to 50 mM MOPS buffer (pH 7.5) at 1 mg / ml.
What was melt | dissolved so that it was set as lectin solution. (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 give Fab ', to which a divalent crosslinking agent [N- (8- Maleimidocapryloxy) sulfosuccinimide, manufactured by Dojindo Co., Ltd.] to bind polyaspartic acid (average molecular weight 28800, manufactured by Sigma) by a conventional method, and polyaspartic acid-bonded anti-AFP-Fab '(Fab'-pAsp). And
This was added to 50 mM MOPS buffer (pH 7.5) so as to have a concentration of 13.5 μM, which was designated as antibody solution 1. (Antibody solution 2) An anti-AFP monoclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) that does not compete with the antibody used in antibody solution 1 was treated by a conventional method to give Fab ', and a divalent crosslinking agent [N -(8-maleimidocapryloxy) sulfosuccinimide, manufactured by Dojindo Chemical Co., Ltd.] was used to label horseradish peroxidase (POD, manufactured by Sigma) by a conventional method.
It was designated as OD-labeled anti-AFP-Fab '. This is 50mM MOPS
Antibody solution 2 was prepared by adding 100 nM to a buffer solution (pH 7.5). (Sample solution) Add fucose in advance by lectin gel electrophoresis using the AFP-lectin fractionation kit L (manufactured by Wako Pure Chemical Industries, Ltd.) (the operation is according to the standard operation method in the instruction manual attached to the kit). The sera of 3 patients with hepatocellular carcinoma whose rates were measured were treated with 50 mM MOPS buffer (pH 7.5, 0.2 W / V%
Contains bovine serum albumin. The sample solutions 1, 2 and 3 were diluted so that the AFP value was 100 ng / ml. (HPLC conditions) A schematic HPLC diagram is shown in FIG. -Column: Wakopak MCI CQA-31S (4.6mmIDx10mm, 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 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.・ Color coil temperature: 55 ℃. -Fluorescence detection: excitation wavelength 320 nm / fluorescence wavelength 404 nm. (Measurement method) Sialidase solution 10 μl, lectin solution
100 μl, antibody solution 25 μl and predetermined sample solution 10 μl
1 was mixed and reacted at 10 ° C. for 3 minutes. To this reaction mixture, 10 μl of antibody solution 1 was added and further reacted at 37 ° C. for 4 minutes. After the reaction was completed, 100 μl of the reaction mixture was added to HPL under the above conditions.
The area of peak 1 (peak of AFP to which the first antibody is not bound) and peak 2 (peak of AFP to which the first antibody is bound) are obtained for each sample solution by C, and the following formula is used. The peak 1 ratio (%) was determined by fitting. Peak 1 ratio (%) = (area of peak 1) / (sum of areas of peaks 1 and 2) × 100 In place of the sample solution, AF with a fucose addition rate of 0%
P or AFP with a fucose addition rate of 100% [Fucose addition rate was determined by AFP-lectin fractionation kit L (manufactured by Wako Pure Chemical Industries, Ltd.). ] 50 mM MOPS including 100 ng / ml
The reaction solution obtained by performing the same reaction using the same solutions as above, except that the buffer solution (pH 7.5, containing 0.2% bovine serum albumin) was used, was analyzed by the same procedure 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 ratios obtained for sample solutions 1, 2 and 3 were applied to this calibration curve to determine the concentration in each sample solution. The fucose addition rate of AFP was calculated. Furthermore, sialidase solution
The same reaction was carried out 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) With respect to the fucose addition rates of the sample solutions 1 to 3, Table 2 shows the values obtained by the lectin gel electrophoresis method and the values obtained by the method of the present invention.

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

[0042]

EFFECTS OF THE INVENTION As is clear from the above, the present invention provides a method capable of measuring the degree of structural change of a glycoprotein sugar chain in a disease with high precision, quickly and simply. Therefore, by utilizing the present invention, it is possible to provide new information in the diagnosis of cancer and the like using the degree of structural change of the glycoprotein sugar chain, it has an effect of contributing to the industry This is a great invention.

[Brief description of drawings]

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

FIG. 2 shows the peak area values and the total of two peak area values of various sample solutions obtained in Example 1 and shows the hepatocellular carcinoma type α-fetoprotein (A) in various sample solutions.
It is the figure shown based on the mixing ratio of a (FP) solution and a normal type AFP solution.

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

[Explanation of symbols]

In Fig. 2, □ is the area value of peak 1, and + is the peak 2.
Indicates the area value of, and ⋄ indicates the sum of the area values of peak 1 and peak 2.

Claims (4)

[Claims] 1. A differential measurement method for measuring glycoproteins having different sugar chain structures but substantially the same protein structures, the method comprising:
A lectin that recognizes at least one specific sugar chain structure of a glycoprotein to be measured, and a property of binding to all of the glycoproteins to be measured, but a property of hindering the binding to the glycoprotein to be measured bound by the lectin An antibody having
Abbreviated as the first antibody. ) Is used in combination, the glycoprotein bound to the first antibody and the glycoprotein not bound to the first antibody are fractionally measured by utilizing the difference in these properties, and the differential measurement method is characterized. . 2. The differential measuring method according to claim 1, wherein a substance or a labeling substance capable of changing the properties of the complex of the glycoprotein to be measured and the first antibody is bound to the first antibody. 3. An antibody having the property of being 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 second antibody). The method according to claim 1 or 2, wherein
Separated measurement method described in. 4. The glycoprotein to be measured is glycosidase-treated in advance, or coexistence of glycosidase when reacting the glycoprotein to be measured with a lectin and a first antibody, and optionally a second antibody. The method of fractionation measurement according to claim 1, wherein