JP2000081434A - Measuring method for thyroglobulin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method whereby various kinds of Tg(thyroglobulin) in a sample derived from a subject can be measured easily and simply and a method and reagent whereby the degree of malignancy of goiter can be determined on the basis of the measurements. SOLUTION: This measuring method uses one or more each of a protein bonding to the invariable region of thyroglobulin and a protein specifically bonding to the specific sugar chain structure of the thyroglobulin, and enables the degree of malignancy of goiter to be determined on the basis of their measurements.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring thyroglobulin (hereinafter abbreviated as "Tg"), and more particularly to a method for measuring Tg having a specific sugar chain structure. In addition, the present invention relates to a method for determining the amount of Tg having a specific sugar chain structure and discriminating the thyroid tumor malignancy based on this amount.

[0002]

BACKGROUND OF THE INVENTION Tg is secreted from thyroid tissue and has a molecular weight of
It is a glycoprotein composed of 330,000 subunit dimers and having a molecular weight of 660,000. It is known that its concentration in cells and blood increases in benign or malignant thyroid diseases. For this reason, the measurement of the amount of Tg in blood is used for follow-up after thyroid cancer surgery. However, benign or malignant thyroid tumors cannot be distinguished by the amount of Tg in the blood.

[0003] Therefore, ultrasonic diagnosis and fine needle aspiration cytology are performed to distinguish the degree of malignancy of a thyroid tumor. However, ultrasonic diagnostics require excellent diagnostic techniques, and fine needle aspiration cytology requires several days to discriminate when culturing the collected cells, or when discriminating by observing the collected cells. Requires skill,
There are problems such as. In addition, there is a problem that it is difficult to distinguish follicular adenocarcinoma from follicular adenoma in fine needle aspiration cytology.

[0004] Furthermore, a method of discriminating the difference between benign and malignant thyroid tumors by using sugar recognition proteins such as lectins on the surface of tumor cells has been attempted, and the sugar chains are changed between benign thyroid tumors and thyroid cancer. It turned out to be. However, since this method involves confirming the amount of the labeled substance under a microscope after reacting each collected cell with a labeled sugar chain-recognizing protein, it requires skill in the inspection and quantitative evaluation. Is difficult to do.

[0005] On the other hand, Tg was purified from benign tumor cells and malignant tumor cells, respectively, and the sugar chain structure was analyzed. As a result, it was found that the structures of the sugar chains bound between the benign tumor and the malignant tumor were different. It has become. But even with this method,
There is a problem that it takes a long time to purify Tg from the collected cells or to analyze the sugar chain structure.

[0006]

SUMMARY OF THE INVENTION Under the circumstances described above, the problem to be solved by the present invention is to provide a method for easily and easily measuring various Tg in a sample derived from a living body. It is an object of the present invention to provide a method and a reagent capable of discriminating the malignancy degree of the above.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made to solve such problems. (1) A protein which binds to a constant region of Tg and a Tg having a specific sugar chain structure A method of measuring Tg, characterized in that at least one protein specifically binding to the sugar chain structure is used; (2) a protein binding to a constant region of Tg, and a Tg having a specific sugar chain structure. And (3) the amount of Tg having a specific sugar chain structure or the amount of Tg having another sugar chain structure, which comprises one or more proteins that specifically bind to the sugar chain structure of the present invention. A method for differentiating the degree of malignancy of thyroid tumor,
(4) a protein specifically binding to the constant region of Tg and one or more proteins specifically binding to the sugar chain structure of Tg having a specific sugar chain structure,
The present invention relates to a reagent for differentiating thyroid tumor malignancy.

That is, the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that a protein which specifically binds to Tg and a specific sugar chain structure of Tg having a specific sugar chain structure. The total amount of Tg in a biological sample, the amount of Tg having a specific sugar chain structure, and / or the amount of Tg having another sugar chain structure can be measured by using one or more proteins that bind specifically. As a result of finding and further study, the amount of Tg having a specific sugar chain structure or the amount of Tg having another sugar chain structure, or the total amount of Tg having a specific sugar chain structure or other The present inventors have found that the ratio of Tg having a sugar chain structure is useful for discriminating the degree of malignancy of thyroid tumor, and completed the present invention.

The protein of the present invention that binds to the constant region of Tg (hereinafter abbreviated as “Tg-binding protein”) includes, for example, anti-T that binds to the constant region of Tg.
g antibody, Tg binding receptor and the like. These may be used alone or in combination as appropriate. The Tg invariable region refers to all Tg in a biological sample.
Refers to a structural region common to In addition, T according to the present invention
The g-binding protein may have a property of binding to a region other than the constant region of Tg.

[0010] The Tg-binding protein includes a Tg having a specific sugar chain structure and a protein that specifically binds to the specific sugar chain structure (hereinafter abbreviated as “specific sugar chain binding protein”). Is a Tg having the property of not binding
Binding protein (hereinafter, “competitive Tg binding protein”)
Abbreviated. ) And Tg having the property of being capable of binding to all Tg regardless of the presence or absence of the binding of a specific sugar chain binding protein
Binding proteins (hereinafter abbreviated as “non-competitive Tg binding proteins”) and the like.

[0011] Anti-Tg antibodies that bind to the constant region of Tg
If the antibody has such properties, for example, a horse, a horse, etc. may be used in accordance with a method described in a conventional method, for example, [Introduction to Immune Experiments, 2nd Printing, Nao Matsuhashi, Gakkai Shuppan Press Center, 1981].
Polyclonal antibodies produced by immunizing animals such as cows, sheep, rabbits, goats, rats, mice and the like, or by a conventional method, ie, Keller and Milstein (Nature, 256
Volume, page 495, 1975), for example, to produce a hybridoma produced by fusing cells from a mouse tumor line with mouse spleen cells previously immunized with a test substance. It may be an antibody.

The specific sugar chain-binding protein according to the present invention includes, for example, antibodies and lectins that specifically bind to the specific sugar chain structure of Tg. More specifically, the reaction for example to Lewis type sugar chain such as anti-Le ^a antibody, anti-L
e ^b antibody, anti-Le ^x antibody, anti-Le ^y antibody, antibody such as anti-S-Le ^a antibody, for example, L- fucose-binding lectins such as Lotus lectins, such as peanut lectin, soybean lectin, Himarekuchin, such as kidney bean lectin D
-Galactose or N-acetyl-D-galactosamine-binding lectins, for example, D-mannose-binding lectins such as concanavalin A, lentil lectin, pea lectin; Examples include lectins such as sialic acid binding lectins such as horseshoe crab lectin. Among them, D-galactose or N-acetyl-D-galactosamine-binding lectin, D
-Mannose-binding lectins and the like are preferred. These may be used alone or in combination as appropriate.

[0013] In the above-mentioned classification of lectins, the term "binding property" refers to what kind of sugar the lectin once bound to an affinity column to which an appropriate sugar chain is bound is easily eluted. For example, D-galactose or N-acetyl-D-galactosamine-binding lectin means a lectin that is once bound to an affinity column and eluted with D-galactose or N-acetyl-D-galactosamine.

The antibody that specifically binds to a specific sugar chain structure may be a polyclonal antibody or a monoclonal antibody prepared according to a conventional method as described above.

In the present invention, the specific sugar chain structure is more specifically a sugar chain structure to which a lectin can bind as described above, for example, a sugar chain structure of Tg produced by a tumor cell such as a thyroid tumor cell, And more specifically, for example, Yamamoto, K., Eur. J. Biochem., Vol. 143, 133-144,
It has a sugar chain structure as described in documents such as 1984.

In the measurement method of the present invention, the Tg-binding protein and the specific sugar chain-binding protein are appropriately combined to obtain various extracts from biological samples such as plasma, serum, cerebrospinal fluid, component extracts of various biological tissues, and urine. Is measured, and the specific measurement target is, for example, the total Tg.
g, Tg having a specific sugar chain structure, Tg having another sugar chain structure, and the like. In addition, Tg is converted into various fragments by a decomposition action in a living body, and even if such a fragment can bind to a Tg-binding protein and / or a specific sugar chain-binding protein, it is a target of the present invention. It is. These measurement objects may be measured separately, or may be measured simultaneously by one measurement.

Hereinafter, embodiments of the present invention will be described. (1) Reaction of a biological sample with a Tg-binding protein or a specific sugar chain-binding protein to give [Tg-binding protein]-[Tg] or [specific sugar chain-binding protein]-[Tg]
To form a complex. Next, a specific sugar chain-binding protein (hereinafter abbreviated as “labeled specific sugar chain-binding protein”) or a Tg binding protein (hereinafter abbreviated as “labeled specific sugar chain-binding protein”) to which the appropriate complex is bound. , Abbreviated as “labeled Tg-binding protein”), and [Tg-binding protein]-[Tg]-[labeled specific sugar chain-binding protein] or [specific sugar chain-binding protein]-[Tg]-[labeled Tg [Binding protein]. Next, after removing the free labeled specific sugar chain binding protein or labeled Tg binding protein, the amount of the labeling substance in the complex obtained in is measured, and based on the value, the amount of Tg having the specific sugar chain structure is determined. Ask.

(2) The reaction between a biological sample, a labeled specific sugar chain-binding protein and a non-competitive Tg-binding protein, and the reaction is carried out to obtain [labeled specific sugar chain-binding protein]-[Tg]-[non-competitive Tg-binding protein]. [Protein] complex. Next, after separating the obtained complex from the free labeled specific sugar chain-binding protein, the amount of the labeling substance in the complex obtained in is measured, and the Tg having the specific sugar chain structure is determined based on the value. Find the quantity.

(3) The biological sample and the specific sugar chain-binding protein are reacted to form a complex of [Tg]-[specific sugar chain-binding protein]. Next, the obtained complex is separated from the Tg to which the specific sugar chain-binding protein has not bound, and the complex has a Tg amount having a specific sugar chain structure or a sugar chain structure other than the specific sugar chain structure. The Tg amount is determined.

(4) A sample derived from a living body is reacted with a Tg-binding protein to form a [Tg-binding protein]-[Tg] complex. Next, the specific sugar chain-binding protein is reacted with a competitive Tg-binding protein to which an appropriate labeling substance is bound (hereinafter, abbreviated as “label-competitive Tg-binding protein”), and [Tg-binding protein]-[ A complex of [Tg]-[specific sugar chain binding protein] and [Tg binding protein]-[Tg]-[labeled competitive Tg binding protein] is formed. Next, after removing the free labeling-competitive Tg-binding protein, the amount of the labeling substance in the complex obtained in the above was measured, and based on the value, the amount of Tg having a sugar chain structure other than the specific sugar chain structure was determined. Ask for.

(5) The complex (a) [specific sugar chain binding protein] is obtained by reacting a biological sample with a specific sugar chain binding protein and then reacting with a competitive Tg binding protein.
-[Tg] and complex (b) [competitive Tg binding protein]-[T
g] to form a complex. Next, the obtained complex (b) is separated, and the complex (b) is reacted with a labeled Tg-binding protein, whereby the complex (c) [competitive Tg-binding protein]-[Tg]-
After forming the complex of [labeled Tg-binding protein], the free labeled Tg-binding protein is separated, and the amount of the labeling substance in the complex (c) obtained in step (c) is measured. Having a sugar chain structure other than the sugar chain structure of
Determine the amount of g.

(6) The complex (a) [specific sugar chain-binding protein]-[Tg] and complex (a) are reacted by reacting a biological sample, a specific sugar chain-binding protein, and a label-competitive Tg-binding protein. b) Form a [label-competitive Tg-binding protein]-[Tg] complex. Next, the complex (b) is separated from the free labeled competitive Tg-binding protein and the complex (a), the amount of the labeling substance in the complex (b) is measured, and the specific sugar is determined based on the value. The amount of Tg having a sugar chain structure other than the chain structure is determined.

(7) reacting the biological sample, the labeled Tg binding protein, and the specific sugar chain binding protein,
A complex of the complex (a) [labeled Tg binding protein]-[Tg] and the complex (b) [labeled Tg binding protein]-[Tg]-[specific sugar chain binding protein] is formed. Next, after removing the free labeled Tg-binding protein, the amounts of the labeling substances in the complex (a) and the complex (b) obtained in the above were respectively measured, and a specific sugar chain structure was determined based on the values. Have T
g, other Tg having a sugar chain structure, and total Tg
Find the quantity.

(8) Competition between a non-competitive Tg binding protein (hereinafter abbreviated as “labeled non-competitive Tg binding protein”) to which a biological sample and an appropriate labeling substance are bound, and a specific sugar chain binding protein. (A) [labeled non-competitive Tg binding protein]-[Tg]-[competitive Tg binding protein] and complex (b)
A complex of [labeled non-competitive Tg binding protein]-[Tg]-[specific sugar chain binding protein] is formed. Next, the free labeled non-competitive Tg-binding protein, the complex (a) and the complex (b) are separated, and the amount of the labeling substance in the complex (a) or the complex (b) obtained in is measured, respectively. Based on the values, the Tg amount having a specific sugar chain structure, the Tg amount having other sugar chain structures, and the total T
Determine the amount of g.

(9) The complex (a) [labeled Tg-binding protein]-[Tg]-is reacted by reacting a biological sample, a labeled Tg-binding protein, a specific sugar chain-binding protein, and a competitive Tg-binding protein. A complex of [competitive Tg binding protein] and complex (b) [labeled Tg binding protein]-[Tg]-[specific sugar chain binding protein] is formed. Then, the free labeled Tg binding protein, complex (a)
And the complex (b) are separated, and the amount of the labeling substance in the complex (a) or the complex (b) obtained in is measured, and the amount of Tg having a specific sugar chain structure is determined based on the value. , Tg amount having other sugar chain structure and total T
Determine the amount of g.

A specific example of the measuring method will be described below. I. A method of measuring the objects to be measured separately. The measurement target, that is, for example, total Tg, Tg having a specific sugar chain structure, Tg having another sugar chain structure, and the like are each measured as follows, for example. I-1. Measurement of total Tg The total Tg may be measured by a method known per se using a Tg binding protein.

I-2. Measurement of Tg having specific sugar chain structure I-2-1) Method using Tg-binding protein-immobilized insoluble carrier. For example, plasma, serum, cerebrospinal fluid, component extracts of various biological tissues,
A sample derived from a living body such as urine is reacted with an insoluble carrier on which Tg-binding protein is immobilized to form the following immobilized complex. [Insoluble carrier] -Tg binding protein-Tg Then, after removing unnecessary coexisting substances by washing or the like, the immobilized complex is further reacted with a labeled specific sugar chain-binding protein to form the following immobilized complex. Let it form. [Insoluble carrier] -Tg-binding protein-Tg-labeled specific sugar chain-binding protein Then, after removing the free labeled specific sugar chain-binding protein by washing the immobilized complex or the like, the immobilized complex in the immobilized complex is removed. The amount of the labeling substance is measured by an appropriate method, and the obtained measurement value is measured by the same method using, for example, a standard solution containing Tg having a specific sugar chain structure whose concentration is known in advance. Substance quantity (measured value)
The amount of Tg having a specific sugar chain structure in the sample can be determined by applying the calibration curve or the like representing the relationship between the Tg and the Tg concentration.

I-2-2) Method using an insoluble carrier immobilized on a specific sugar chain-binding protein. For example, plasma, serum, cerebrospinal fluid, component extracts of various biological tissues,
A biological sample such as urine and a specific sugar chain-binding protein-immobilized insoluble carrier are reacted to form the following immobilized complex. [Insoluble carrier] -Specific sugar chain-binding protein-Tg Then, after removing unnecessary coexisting substances by washing or the like, the immobilized complex is further reacted with a labeled Tg-binding protein to form the following immobilized complex. Let it form. [Insoluble carrier] -specific sugar chain binding protein-Tg-labeled Tg binding protein Then, after removing the free labeled Tg binding protein such as by washing the immobilized complex,
The amount of the labeling substance in the immobilized complex is measured by an appropriate method, and the obtained measurement value is measured by the same method using, for example, a standard solution containing Tg having a specific sugar chain structure with a known concentration in advance. The labeling substance amount (measured value) and T
The amount of Tg having a specific sugar chain structure in the sample can be determined by applying to a calibration curve or the like representing the relationship with the g concentration.

I-2-3) A method using a labeled specific sugar chain-binding protein and high performance liquid chromatography (HPLC). For example, plasma, serum, cerebrospinal fluid, component extracts of various biological tissues,
A sample derived from a living body such as urine is reacted with a labeled specific sugar chain-binding protein and a non-competitive Tg-binding protein to form the following complex in the sample. Labeled specific sugar chain-binding protein-Tg-non-competitive Tg-binding protein Next, this complex and free labeled specific sugar chain-binding protein are separated by using HPLC, electrophoresis or the like filled with a suitable filler. The amount of the labeling substance in the complex is measured by an appropriate method, and the obtained measurement value is measured by the same method using, for example, a standard solution containing Tg having a specific sugar chain structure with a known concentration in advance. The Tg having a specific sugar chain structure in the sample by applying to a calibration curve or the like representing the relationship between the amount of the labeled substance (measured value) and the Tg concentration
The quantity can be determined.

I-3. Measurement of Tg having a sugar chain structure other than a specific sugar chain structure I-3-1) Method using free specific sugar chain binding protein First, for example, plasma, serum, cerebrospinal fluid, component extracts of various biological tissues, urine And the like and a specific sugar chain-binding protein are reacted with each other, and a complex of Tg having a specific sugar chain structure in the sample with the specific sugar chain-binding protein (hereinafter, referred to as “sugar chain-binding T
g ". ) Is generated. Then
The sugar chain-bound Tg and the Tg to which the specific sugar chain-binding protein did not bind, in other words, Tg having a sugar chain structure other than the specific sugar chain structure (hereinafter may be abbreviated as "non-bound Tg"). Is separated from the sample using a known separation method such as a centrifugation method, a gel filtration method, a molecular separation membrane method, an electrophoresis method, etc., to prepare a sample containing only unbound Tg. The unbound Tg thus obtained
The amount of unbound Tg can be determined by measuring the amount of Tg in a sample containing only Tg by a measurement method known per se using a Tg-binding protein. The sample containing only unbound Tg may be prepared by treating the sample by affinity chromatography using a carrier on which a specific sugar chain-binding protein is immobilized.

I-3-2) Method Using Competitive Tg-Binding Protein First, an insoluble carrier having Tg-binding protein immobilized thereon,
For example, plasma, serum, cerebrospinal fluid, component extracts of various biological tissues,
The sample is reacted with a biological sample such as urine to form the following immobilized complex. [Insoluble carrier] -Tg-binding protein-Tg Then, after removing unnecessary coexisting substances by washing or the like, the immobilized complex is reacted with a specific sugar chain-binding protein, and further reacted with a labeled competitive Tg-binding protein. To form the following immobilized complex. [Insoluble carrier] -Tg binding protein-Tg-Specific sugar chain binding protein [Insoluble carrier] -Tg binding protein-Tg-Labeling competitive Tg binding protein Then, free label competition is performed by washing the immobilized complex or the like. After removing the sex Tg-binding protein, the amount of the labeling substance in the immobilized complex is measured by an appropriate method, and the obtained measurement value is determined, for example, by having a sugar chain structure other than the specific sugar chain structure whose concentration is known in advance. Tg, that is, by applying to a calibration curve or the like representing the relationship between the amount of the labeled substance (measured value) and the Tg concentration, obtained by performing measurement in the same manner using a standard solution containing unbound Tg, and the like. And the amount of unbound Tg in the sample can be determined.

I-3-3) Method using an insoluble carrier on which a competitive Tg-binding protein is immobilized. First, a sample derived from a living body such as plasma, serum, cerebrospinal fluid, component extracts of various biological tissues, urine, etc. is reacted with a specific sugar chain-binding protein to generate sugar chain-bound Tg in the sample. Next, this sample is reacted with a competitive Tg-binding protein-immobilized insoluble carrier to generate the following immobilized complex. [Insoluble carrier] -Competitive Tg-binding protein-Non-binding Tg Next, after removing unnecessary coexisting substances by washing or the like, the immobilized complex is reacted with a labeled Tg-binding protein to form the following immobilized complex. Let it form. [Insoluble carrier]-Competitive Tg-binding protein-Non-binding Tg
-Labeled Tg-binding protein Next, after removing the free labeled Tg-binding protein by washing the immobilized complex or the like, the amount of the labeling substance in the immobilized complex was measured by an appropriate method and obtained. The measured value is converted to, for example, a calibration curve or the like that represents the relationship between the amount of the labeled substance (measured value) and the Tg concentration, obtained by performing the measurement in the same manner using a standard solution containing unbound Tg with a known concentration in advance. By applying, for example, the amount of unbound Tg in the sample can be determined.

I-3-4) Method using label-competitive Tg-binding protein and HPLC or the like. First, a specific sugar chain-binding protein is reacted with a biological sample such as plasma, serum, cerebrospinal fluid, component extracts of various biological tissues, and urine to generate sugar chain-bound Tg. Next, this sample is reacted with the label-competitive Tg-binding protein to generate the following complex. Unbound Tg-labeled competitive Tg-binding protein Next, the complex is separated from free labeled competitive Tg-binding protein using HPLC or electrophoresis, or the like, packed with a suitable filler. Is measured by an appropriate method, and the measured value obtained is measured by the same method using, for example, a standard solution containing unbound Tg with a known concentration in advance, to obtain the amount of the labeled substance ( Measured value) and Tg
The amount of unbound Tg in the sample can be determined by applying it to a calibration curve or the like representing the relationship with the concentration. still,
As a matter of course, the amount of Tg having a specific sugar chain structure (sugar chain-bound Tg) can also be determined by subtracting the amount of Tg having a sugar chain structure other than the specific sugar chain structure (unbound Tg) from the total Tg amount. Alternatively, the amount of Tg (unbound Tg) having a sugar chain structure other than the specific sugar chain structure can also be determined by subtracting the amount of Tg having a specific sugar chain structure (sugar chain bound Tg) from the total Tg amount. it can.

II. A method of measuring a measurement target with a single measurement operation. II-1. Method Using Labeled Tg-Binding Protein and Specific Sugar Chain-Binding Protein The method may be performed as follows according to the method disclosed in JP-A-7-191027. That is, first, for example, plasma,
Serum, cerebrospinal fluid, component extracts of various biological tissues, a biological sample such as urine, and a labeled Tg-binding protein and a specific sugar chain-binding protein are reacted, or a biological sample,
After reacting the labeled Tg-binding protein, a specific sugar chain-binding protein is further added to the reaction solution and reacted to form the following complex. Labeled Tg-binding protein-Tg Labeled Tg-binding protein-Tg-Specific sugar chain-binding protein Next, these complexes and free labeled Tg-binding protein are separated using HPLC or electrophoresis, etc., filled with an appropriate filler. Then, the amount of the labeling substance in each complex is measured by an appropriate method, and the obtained measured value is, for example, Tg having a specific sugar chain structure having a known concentration in advance and / or having other sugar chain structures. Identification in a sample by applying to a calibration curve or the like representing the relationship between the amount of a labeled substance (measured value) and various Tg concentrations obtained by performing measurement in the same manner using a standard solution containing Tg. The Tg having a sugar chain structure, the Tg having another sugar chain structure, and the total of these Tg, that is, the total Tg can be determined by one measurement. As a method for separating the complex and the free labeled Tg-binding protein, a method using HPLC is preferable in consideration of operability, reusability, and the like. The Tg binding protein used is preferably non-competitive.

II-2. Method using non-competitive Tg binding protein, competitive protein and specific sugar chain binding protein First, for example, a sample derived from a living body such as plasma, serum, cerebrospinal fluid, component extracts of various biological tissues, urine, etc. The following complex is formed by reacting the Tg binding protein, the competitive Tg binding protein and the specific sugar chain binding protein. Labeled non-competitive Tg-binding protein-Tg-Competitive Tg-binding protein Labeled non-competitive Tg-binding protein-Tg-Specific sugar chain-binding protein Separation using HPLC or electrophoresis, etc. filled with the reagent, the amount of the labeling substance in each complex is measured by an appropriate method, and the obtained measured value is, for example, a specific sugar chain structure having a known concentration in advance. Represents the relationship between the amount of the labeled substance (measured value) and the various Tg concentrations obtained by performing measurement in the same manner using a standard solution containing Tg having Tg or / and Tg having another sugar chain structure. By applying to a calibration curve or the like, Tg having a specific sugar chain structure and Tg having other sugar chain structures in a sample, and the total of these Tg, that is, the total Tg can be measured in one measurement. It is Mel possible. Furthermore, by simultaneously reacting a labeled non-competitive Tg-binding protein and a non-competitive Tg-binding protein having a different epitope, the difference between the properties of the complex and the properties of the serum components that affect the measurement can be increased. This is preferable because the influence of the components is reduced and the measurement accuracy is improved. As a method for separating the complex and the free labeled non-competitive Tg-binding protein, a method using HPLC is preferable in consideration of operability, repeated use, and the like.

II-3. Method Using Labeled Tg-Binding Protein and Competitive Tg-Binding Protein First, after reacting a biologically derived sample such as plasma, serum, cerebrospinal fluid, component extracts of various biological tissues, urine, etc. with the labeled Tg-binding protein, The reaction solution is further reacted with a specific sugar chain binding protein and a competitive Tg binding protein,
The following complex is formed: Labeled Tg-binding protein-Tg-Competitive Tg-binding protein Labeled Tg-binding protein-Tg-Specific sugar chain-binding protein Next, these complexes and free labeled Tg-binding protein were subjected to HPLC or electrophoresis using a suitable filler. And the like, and the amount of the labeling substance in each complex is measured by an appropriate method. The obtained measured value is, for example, Tg having a specific sugar chain structure with a known concentration in advance, and / or Tg having a specific sugar chain structure. By applying to a calibration curve or the like representing the relationship between the measured value of the labeling substance and various Tg concentrations obtained by performing measurement in the same manner using a standard solution containing Tg having the sugar chain structure of Tg having a specific sugar chain structure and Tg having another sugar chain structure, and the sum of these Tg, that is, the total Tg can be determined by one measurement. Further, by reacting the labeled Tg-binding protein with the Tg-binding protein having a different epitope after the reaction of the labeled Tg-binding protein, the difference between the properties of the complex and the properties of the serum component which affects the measurement can be increased. This is preferable because the influence of serum components is reduced and measurement accuracy is improved. As a method for separating the complex and the free labeled Tg-binding protein, a method using HPLC is preferable in consideration of operability, reusability, and the like.

Known T using a Tg binding protein
The g measurement method uses, for example, an anti-Tg antibody as a Tg-binding protein, so-called enzyme immunoassay (EIA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),
What is necessary is just to carry out according to the immunological measuring method, such as a fluorescence immunoassay (FIA) and the measuring method using HPLC (Japanese Patent Application Laid-Open No. 9-301995). The measurement principle may be any of the sandwich method, the competition method, the two-antibody method and the like.

As the insoluble carrier used for immobilizing various Tg-binding proteins and specific sugar chain-binding proteins, those usually used in the field of immunoassay as described above may be used. ceramic,
A special tray made of silicone rubber, for example, polystyrene, polyvinyl chloride, polypropylene, acrylic resin, synthetic polymer such as polymethyl methacrylate, beads, tubes, and many tubes are integrally molded.
Examples of the immobilization method include a microtiter plate and the like. Examples of the immobilization method include a physical adsorption method and a chemical bonding method which are usually used in the field of immunoassay as described above.

The Tg-binding protein and specific sugar according to the present invention
Labeling substances that bind to chain-binding proteins include, for example,
Alkaline phosphatase used in EIA,
β-galactosidase, peroxidase, micrope
Oxidase, glucose oxidase, glucose
-6-phosphate dehydrogenase, acetylcholinesterase,
Enzymes such as malate dehydrogenase, luciferase, etc.
Used in RIA ^99mTc,¹³¹I,¹²⁵I,¹⁴C,^Three
H and other radioisotopes, such as the full
Olecein, dansyl, fluorescamine, coumarin,
Fluorescent substances such as naphthylamine or their derivatives, eg
For example, luciferin, isoluminol, luminol, bis
Luminescent substances such as (2,4,6-trifluorophenyl) oxalate
Quality, such as phenol, naphthol, anthracene or
Substances having an ultraviolet absorption such as these derivatives, for example,
4-amino-2,2,6,6-tetramethylpiperidine-1-oxy
1,3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxo
Sil, 2,6-di-t-butyl-α- (3,5-di-t-butyl-4-oxo
-2,5-cyclohexadiene-1-ylidene) -p-tolyloxy
Spirra represented by compounds having an oxyl group such as
Substances having properties as a belling agent may be mentioned.

A method for binding (labeling) the above-mentioned labeling substance to a Tg binding protein or a specific sugar chain binding protein is generally performed in EIA, RIA, FIA or the like known per se. What is necessary is just to carry out according to the labeling method known per se. Further, as a labeling method, a conventional method using a reaction between avidin (or streptavidin) and biotin may be used.

The apparatus for HPLC used in the measurement method using HPLC of the present invention may be any apparatus usually used in this field.

In the measurement method using HPLC of the present invention, in order to more clearly separate the complex from the free labeled Tg-binding protein (or labeled specific sugar chain-binding protein), for example, the method disclosed in Tg-binding proteins and specific sugars to which substances for improving their separation (hereinafter abbreviated as “separation improving substances”) disclosed in JP 191027, JP-A-9-301995 and the like are bound. A chain binding protein or the like may be used.

Examples of the separation improving substance used for such a purpose include proteins such as α-chymotrypsinogen, β-galactosidase, lysozyme, cytochrome C, trypsin inhibitor, such as phenylalanine, proline, arginine, lysine, aspartic acid, and the like.
Peptides containing amino acids such as glutamic acid, for example, halogen atoms such as bromine, chlorine, and iodine, for example, synthetic polymers such as polyethylene glycol, for example, polyglutamic acid,
Polyamino acids such as polyaspartic acid, polylysine, polyarginine, polyphenylalanine and polytyrosine;
An alkyl chain having 3 to 10 carbon atoms, for example, a fatty acid such as palmitic acid, oleic acid, and stearic acid, for example, N- (ε-maleimidocaproyloxy) succinimide [N- (ε-maleimi
docaproyloxy) succinimide] (EMCS), N-succinimidyl-6-maleimidohexanoate (N-Succinimidy
l-6-maleimidohexanoate), bismaleimidohexane (BMH), octylamine and other Tg-binding proteins and chemical substances having a reactive group capable of binding to a specific sugar chain-binding protein and having hydrophobicity or ionicity; For example, as described in JP-A-9-301995
4- (p-maleimidophenyl) butyryl Ala- (Tyr (SO
_{3 H)) 5, 4- (} p- maleimidophenyl) butyryl Ala- (Tyr (S
O ₃ H)) Preferable examples include peptides containing a strong acid residue such as ₈ . The substance for improving the separation is appropriately selected in consideration of the properties (eg, pH stability, hydrophobicity, solubility in an aqueous solution, isoelectric point, etc.) of Tg to be measured, Tg binding protein, and specific sugar chain binding protein. It may be used.

The method of binding the separation improving substance to the Tg binding protein and / or the specific sugar chain binding protein is also
(1) A method of binding an antibody to a known label substance generally used in a known EIA, RIA, FIA, or the like (for example, Medical Experimental Account, Vol. 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 Shoin, 1982, etc. ), (2) Modification and binding methods of substances known per se (for example, chemical modification of proteins <upper><lower>, Ikuzo Uraya, Kensuke Shimura, Michinori Nakamura, edited by Masaru Funatsu, First Edition, Inc.)
Gakkai Shuppan Center, 1981; Polyethylene glycol-modified protein, Yuji Inada et al., Biochemistry, Vol. 62, No. 11, P1
351-1362, Japan Biochemical Society, 1990; DNA PROBES, Ge
orge HK and Mark MM STOCKTON PRESS, 1989, etc.)
Etc. may be performed.

The total T obtained by the measuring method of the present invention
By appropriately combining values such as g, Tg having a specific sugar chain structure, and Tg having other sugar chain structures, the degree of malignancy of a thyroid tumor can be identified.

That is, for example, by determining the percentage of Tg having a specific sugar chain structure or Tg having another sugar chain structure in the total Tg, the malignancy of the thyroid tumor, in other words, whether the tumor is benign or malignant, Can be identified.

More specifically, for example, specific sugar chain-binding proteins include, for example, D-galactose or N-acetyl-D-galactosamine-binding lectin such as peanut lectin, soybean lectin, castor lectin, and kidney bean lectin, such as concanavalin A, Using a D-mannose-binding lectin such as lentil lectin or pea lectin or the like, the ratio of Tg having a specific sugar chain structure or Tg having another sugar chain structure to the total Tg is calculated. Based on the above, it becomes possible to distinguish papillary thyroid cancer from, for example, benign thyroid tumor, Graves' disease, etc., and distinguish follicular thyroid cancer from follicular thyroid adenoma.

That is, the method of the present invention for discriminating the degree of malignancy of thyroid tumors is based on these facts first discovered by the present inventors.

The reagent for measuring Tg of the present invention comprises a protein that binds to the constant region of Tg and a Tg having a specific sugar chain structure.
g contains at least one protein that specifically binds to the sugar chain structure, and the preferred embodiments and specific examples of the components are as described above.

In addition, the reagent for differentiating thyroid tumor malignancy according to the present invention specifically binds to a protein that specifically binds to a constant region of Tg and a specific sugar chain structure of Tg having a specific sugar chain structure. It contains one or more proteins, and the preferred embodiments and specific examples of the components are as described above.

In these reagents, there are usually used reagents in this field, such as buffers, reaction accelerators, stabilizers such as saccharides, proteins, salts, and surfactants, and preservatives, and coexist. Inhibits the stability of reagents, etc.
Those that do not inhibit the reaction with the g-binding protein and / or the specific sugar chain-binding protein may be included. Also,
The concentration may be appropriately selected from the concentration range usually used in this field.

It is well known that metal ions such as magnesium affect lectin activity and stability, and these may be included.

The buffer which can be used in the reagent of the present invention includes, for example, Tris buffer, phosphate buffer,
All buffers used in normal immunoturbidimetry, immunoturbidimetry, RIA, and EIA, such as veronal buffer, borate buffer, and good buffer, are listed. It is not particularly limited as long as the reaction between Tg and lectin or the like is not suppressed, but is usually 6 to 10.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0055]

EXAMPLES Example 1 Method for Differentiating Papillary Carcinoma Using Concanavalin A (ConA) (Peroxidase-Labeled Anti-Tg Antibody) An anti-Tg antibody (hereinafter abbreviated as “anti-Tg-1”) and peroxidase were used. Peroxidase-labeled anti-Tg antibody (hereinafter abbreviated as "anti-Tg-1.POD") was prepared by binding by a conventional method. (Sample) 0.1 g (wet weight) of human thyroid tissue fragments was homogenized in 1 ml of 0.1 M phosphate buffer (pH 7.5, containing 0.9% NaCl, hereinafter abbreviated as “PBS”) using a glass homogenizer, and then 4 ° C. , 30000 g for 5 minutes, and the obtained supernatant was used as a sample. The human thyroid tissue used was papillary carcinoma 11
There were 5 cases of benign goiter, 5 cases of Basedow's disease, and 5 normal cases. (Reagent 1) ConA (Honen Corporation) dissolved in PBS to a concentration of 15 mg / mL was used as Reagent 1.
And (Pretreatment of sample) Mix 50 μL each of sample and Reagent 1 and 4 ℃
Incubate overnight at 4 ° C, 3000g, 20min
Then, the precipitate was removed, and the obtained supernatant was used as pretreatment liquid 1. The Tg bound to ConA is removed by centrifugation. In addition, a supernatant obtained in the same manner as above using PBS instead of Test Solution 1 was used as Pretreatment Solution 2. (Measurement of Tg concentration) 1 ml of PBS containing 5 μg of anti-Tg antibody having an epitope different from that of anti-Tg-1 (hereinafter abbreviated as “anti-Tg-2”)
Into each well of a 96-well microplate,
For 1 hour and then washed with PBS to immobilize anti-Tg-2. Next, 0.2 ml of PBS containing 1% fetal calf serum was injected into each well of the microplate, left at 20 ° C. for 1 hour, washed with PBS, and subjected to a blocking treatment. 100 μL of PBS containing 1% fetal bovine serum and 50 μL of Pretreatment Solution 1 or Pretreatment Solution 2 were injected into the wells of the microplate and reacted at 20 ° C. for 1 hour. After the completion of the reaction, each well of the microplate was washed with PBS, and then 100 μL of anti-Tg-1 · POD diluted 10000-fold was injected, and reacted at 20 ° C. for 1 hour. After the reaction is completed, wash with PBS and then 3.3'5.5'-tetramethy
lbenzidine (Kirkegaad and Perry Labs Inc) solution
Add 100 μL, react at 20 ° C for 30 minutes, and then add 1M phosphoric acid 50
The reaction was stopped by adding μL. The absorbance at 450 nm of each well was measured with a microplate reader Spectra 1 (manufactured by Wako Pure Chemical Industries, Ltd.), and the obtained absorbance was obtained by performing the same operation using a Tg solution of which concentration was known in advance. T
The Tg concentrations of the pretreatment solutions 1 and 2 were calculated by fitting the calibration curve representing the relationship between the g concentration and the absorbance. The Tg concentration in the pretreatment liquid 1 indicates the amount of non-ConA-binding Tg, and the Tg concentration in the pretreatment liquid 2 indicates the total Tg amount. (Calculation of ConA non-binding Tg ratio) The ConA non-binding Tg ratio (%) was calculated by the following equation. ConA non-binding Tg ratio (%) = [(ConA non-binding Tg amount) / (total Tg amount)] × 100 (Results) The measured ConA non-binding Tg ratio (%) is shown in FIG. As is clear from FIG. 1, ConA
It can be seen that the unbound Tg ratio (%) is significantly higher than that of the benign goiter tissue extract, Graves' disease extract or normal tissue extract. In addition, benign goiter tissue extract,
ConA between the Graves disease tissue extract and the normal tissue extract
It can also be seen that there is no difference in the non-binding Tg ratio (%). That is, in the papillary carcinoma tissue, the Tg having an altered sugar chain, ie, Cog
The increase in non-A binding Tg, in other words, the percentage of non-ConA binding Tg (%) was found to be very useful in differentiating benign thyroid tumors from papillary carcinomas.

Example 2 Castor lectin-120 (RCA120)
Method for Differentiating Papillary Carcinoma by Using (Peroxidase-Labeled Anti-Tg Antibody) (Sample) It was prepared in the same manner as the sample of Example 1. The human thyroid tissue used was papillary carcinoma 7 cases, Graves' disease 5 cases,
And 4 normal cases. (Reagent 1) Reagent RCA120 (manufactured by HONEN CORPORATION) dissolved in PBS to a concentration of 2.5 mg / mL was used as Reagent 1. (Pretreatment of Sample) Pretreatment liquids 1 and 2 were prepared in the same manner as in the sample pretreatment method of Example 1. (Measurement of Tg Concentration) The measurement was performed in the same manner as in the method of measuring the Tg concentration in Example 1. That is, the Tg concentration in the pretreatment liquid 1 is RCA120
It represents the amount of non-binding Tg, and the Tg concentration in the pretreatment liquid 2 is the total Tg
Express the amount. (Calculation of RCA120 non-binding Tg ratio) RCA120 by the following formula
The non-binding Tg ratio (%) was calculated. RCA120 non-binding Tg ratio (%) = [(RCA120 non-binding Tg amount) /
(Total Tg amount)] × 100 (Results) The measured RCA120 non-binding Tg ratio (%) is shown in FIG. As can be seen from FIG. 2, the papillary carcinoma tissue extract contained RC
It can be seen that the A120 non-binding Tg ratio (%) is significantly higher than that of Graves' disease extract or normal tissue extract.
That is, in the papillary carcinoma tissue, the Tg having an altered sugar chain, ie, RCA120
The increase in non-binding Tg, in other words, the percentage of RCA120 non-binding Tg was found to be useful in differentiating papillary carcinoma.

Example 3 Differentiation between follicular carcinoma and follicular adenoma using concanavalin A (ConA) (Preparation of peroxidase-labeled anti-Tg antibody) Same as in Example 1. (Sample) It was prepared in the same manner as the sample of Example 1. The human thyroid tissues used were 4 cases of follicular carcinoma, 7 cases of follicular adenoma, and 5 normal cases. (Reagent 1) Same as in Example 1. (Pretreatment of Sample) Pretreatment liquids 1 and 2 were prepared in the same manner as in the sample pretreatment method of Example 1. (Measurement of Tg Concentration) The measurement was performed in the same manner as in the method of measuring the Tg concentration in Example 1. That is, the Tg concentration in the pretreatment liquid 1 indicates the amount of non-ConA-binding Tg, and the Tg concentration in the pretreatment liquid 2 indicates the total Tg amount. (Calculation of ConA non-binding Tg ratio)
The nA non-binding Tg ratio (%) was calculated. (Results) FIG. 3 shows the measured ConA non-binding Tg ratio (%). As is clear from FIG. 3, the ratio (%) of ConA non-binding Tg
Is higher in the order of normal tissue extract, follicular carcinoma tissue extract, and follicular adenoma tissue extract, indicating that there is a significant difference between that in the follicular carcinoma tissue extract and that in the follicular adenoma tissue extract. That is, from the results, the ConA non-binding Tg ratio (%)
As a result, it was found that follicular carcinoma and follicular adenoma, which are difficult to diagnose even by cytodiagnosis, can be easily distinguished.

Example 4 Differential Measurement of Tg with Different Sugar Chain Structure Using Lentil Lectin (LCA) (Peroxidase-Labeled Anti-Tg Antibody Fab ′ Fragment) LC
Two types of anti-Tg antibodies (hereinafter referred to as “anti-Tg-86” and “anti-Tg
-78 ". ) Was processed in a conventional manner to obtain a Fab ′ fragment, to which peroxidase (manufactured by Toyobo) was bound by a conventional method to obtain a peroxidase-labeled anti-Tg antibody Fa.
b ′ fragments (hereinafter abbreviated as “anti-Tg-86 • Fab′-POD” and “anti-Tg-78 • Fab′-POD”, respectively) were prepared. (Antibody solution 1) A 50 mM phosphate buffer solution (pH 7.5, 0.15 M NaCl, containing 0.5 (w / v)% bovine serum albumin) containing 2 nM of anti-Tg-86 • Fab-POD was prepared, and antibody solution 1 was prepared. And (Antibody solution 2) A 50 mM phosphate buffer solution (pH 7.5, 0.15 M NaCl, containing 0.5 (w / v)% bovine serum albumin) containing 3 nM of anti-Tg-78 • Fab-POD was prepared, and antibody solution 2 was prepared. And (Reagent 1) 50 mM phosphate buffer pH 7.5, 0.15 M containing 15 μM LCA (Honen Corporation)
NaCl was prepared and used as sample solution 1. (Sample) 0.1 g (wet weight) of human thyroid tissue fragments was homogenized in 1 ml of 0.1 M phosphate buffer (pH 7.2, containing 0.9% NaCl) using a glass homogenizer, and then 4 ° C., 100000 g, 6 g.
The supernatant was obtained by centrifugation for 0 minutes, and the supernatant was further diluted 200- to 1100-fold with 50 mM phosphate buffer (pH 7.5, 0.15 M NaCl, containing 0.5 (w / v)% bovine serum albumin), and used as a sample. did. still,
The human thyroid tissue used was 4 benign diseases (follicular adenoma,
Adenomatous goiter (2 cases, Basedow's disease) and papillary carcinoma (4 cases). (Measurement method) Mix 25 μL of sample and 15 μL of test solution, and mix at 8 ℃
For 1 hour. Add 15 μL of antibody solution to 90 μL of this reaction solution.
L was further mixed and reacted at 8 ° C. for 30 minutes. 50 μL of the obtained reaction solution was subjected to high performance liquid chromatography (HPL
Analysis was performed using C) to determine the amount of non-LCA-bound Tg. The same sample was subjected to the same measurement using a 50 mM phosphate buffer (pH 7.5, 0.15 M NaCl, containing 0.5 (w / v)% bovine serum albumin) instead of LCA-containing test solution 1, and the total Tg was measured. The amount was measured. The results of these two measurements were substituted into the following formula to obtain the anti-Tg-8
6. The ratio (%) of LCA non-binding Tg when using Fab-POD was calculated. LCA non-binding Tg ratio (%) = [(LCA non-binding Tg amount) / (total Tg
g)) × 100 In addition, the same operation was performed except that the antibody solution 2 was used instead of the antibody solution 1, and the amount of L when the anti-Tg-78 • Fab'-POD was used was determined.
It was calculated together with the non-CA-binding Tg ratio (%). (HPLC conditions) Column: Wakopack Wakosil-5Diol-200 8.0 mm x 300 mm (w) Eluent: 50 mM phosphate buffer pH 7.5, 0.15 M NaCl Substrate solution: 15 mM citrate buffer (pH 5.5,313 mM acetaminophenol) (Manufactured by Dojindo Laboratories Inc.), containing 0.12% H ₂ O ₂ Flow rate: 1.0 mL / min of eluent, 0.1 mL / min of substrate solution Detection: Ex 328 nm, Em 432 nm (Result) Anti-Tg-86 FIGS. 4 and 5 show the percentage (%) of non-LCA-binding Tg when Fab-POD and anti-Tg-78 Fab-POD were used. 4 and 5 show that LCA in benign disease tissue extract
It can be seen that it is significantly higher in the papillary carcinoma tissue extract compared to the unbound Tg percentage (%). That is, it is understood that the differential diagnosis between benign disease and papillary cancer can be made by using LCA.

Example 5 Different sugar chain structures using ConA
Differential measurement of Tg (Tg) (Peroxidase-labeled anti-Tg antibody Fab 'fragment) The anti-Tg-86 • Fab'-POD prepared in Example 4 was used. (Antibody solution 1) A 50 mM phosphate buffer solution (pH 7.5, 0.15 M NaCl, containing 0.5 (w / v)% bovine serum albumin) containing 4 nM of anti-Tg-86 / Fab-POD was prepared, and antibody solution 1 was prepared. And (Reagent 1) 50 mM phosphate buffer pH 7.5, 0.15 M containing 15 μM ConA (Honen Corporation)
NaCl was prepared and used as sample solution 1. (Sample) The same one as in Example 4 was used. (Measurement method) After mixing 10 μL of sample and 145 μL of test solution,
The reaction was performed at 8 ° C for 1 hour. Add 45 μL of antibody solution 1 to this reaction solution.
Was added and the mixture was further reacted at 8 ° C. for 1 hour. Obtained reaction liquid 50
High-performance liquid chromatography (HPLC) with the following conditions
And the amount of ConA non-binding Tg was determined. For the same sample, 50 mM phosphate buffer (pH 7.
5, containing 0.15M NaCl, 0.5 (w / v)% bovine serum albumin)
The total Tg amount was measured in the same manner as described above. The results of these two measurements were substituted into the following formula to calculate the ConA non-binding Tg ratio (%). ConA non-binding Tg ratio (%) = [(ConA non-binding Tg amount) / (total Tg amount)] × 100 (HPLC conditions) Same as in Example 4. (Results) The obtained ConA non-binding Tg ratio (%) is shown in FIG. FIG. 6 shows that ConA non-binding Tg in benign disease tissue extract
It can be seen that it is significantly higher in the papillary carcinoma tissue extract compared to the percentage (%). That is, by using ConA,
It can be seen that differential diagnosis between benign disease and papillary cancer is possible.

[0060]

The present invention provides a method and a reagent for easily and accurately measuring various Tg in various biological samples, and various reagents obtained by the method of the present invention. By appropriately using the measured values of Tg, it is possible to distinguish papillary thyroid cancer from benign thyroid tumor and distinguish follicular thyroid cancer from follicular thyroid adenoma.

[0061]

[Brief description of the drawings]

FIG. 1 shows the ratio (%) of concanavalin A (ConA) non-binding thyroglobulin (Tg) in various thyroid tissue extracts obtained in Example 1.

FIG. 2 shows the percentage (%) of Tg non-binding Tg-120 (RCA-120) in various thyroid tissue extracts obtained in Example 2.

FIG. 3 shows concanavalin (ConA) non-binding thyroglobulin (T) in various thyroid tissue extracts obtained in Example 3.
g) is shown.

FIG. 4 shows that anti-Tg-8 was obtained as an anti-Tg antibody in Example 4.
6 shows the ratio (%) of lentil lectin (LCA) non-binding thyroglobulin (Tg) in various thyroid tissue extracts when No. 6 was used.

FIG. 5 shows that anti-Tg-7 was obtained as an anti-Tg antibody in Example 4.
8 shows the ratio (%) of lentil lectin (LCA) non-binding thyroglobulin (Tg) in various thyroid tissue extracts when No. 8 was used.

FIG. 6: Concanavalin (ConA) non-binding thyroglobulin (T) in various thyroid tissue extracts obtained in Example 5.
g) is shown.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Shinji Satomura 3-1-2, Doshomachi, Chuo-ku, Osaka-shi, Osaka Inside Wako Pure Chemical Industries, Ltd.

Claims (11)

[Claims] 1. A method for measuring thyroglobulin, comprising using at least one protein that binds to a constant region of a thyroglobulin and one or more proteins that specifically bind to the sugar chain structure of a thyroglobulin having a specific sugar chain structure. Method. 2. The method according to claim 1, wherein the thyroglobulin to be measured is total thyroglobulin, thyroglobulin having a specific sugar chain structure, and / or thyroglobulin having another sugar chain structure. 3. The method according to claim 1, wherein the specific sugar chain structure is a lectin-binding sugar chain structure. 4. The lectin is D-galactose- or N-acetyl-D-galactosamine-binding lectin, or D-galactose-binding lectin.
The method according to claim 3, which is a mannose-binding lectin. 5. The lectin is concanavalin A, lentil lectin or castor lectin-120.
Measurement method described in 1. 6. The method according to claim 1, wherein the specific sugar chain structure is a sugar chain structure of thyroglobulin produced by a tumor cell. 7. The method according to claim 6, wherein the tumor cells are derived from a thyroid tumor. 8. A reagent for measuring thyroglobulin, comprising at least one protein that binds to the constant region of thyroglobulin and one or more proteins that specifically bind to the sugar chain structure of thyroglobulin having a specific sugar chain structure. 9. A method for differentiating thyroid tumor malignancy, wherein the method is performed based on the amount of thyroglobulin having a specific sugar chain structure or the amount of thyroglobulin having another sugar chain structure. 10. The ratio of a thyroglobulin having a specific sugar chain structure or a thyroglobulin having another sugar chain structure to a thyroglobulin having a specific sugar chain structure is determined by comparing the ratio of a protein that binds to a constant region of the thyroglobulin with a protein that binds to a thyroglobulin constant region. The identification method according to claim 9, wherein the protein specifically binding to the sugar chain structure is determined using one or more proteins. 11. The malignancy of a thyroid tumor, which comprises at least one protein that binds to a constant region of thyroglobulin and one or more proteins that specifically binds to the sugar chain structure of a thyroglobulin having a specific sugar chain structure. Differentiation reagent.