JP3531372B2 - Novel polypeptide and method for measuring biological components using the same - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel polypeptide having an acid residue derived from a strong acid, and biological fluids such as serum, blood, plasma, urine, etc., lymphocytes, blood cells, and various types utilizing the polypeptide. The present invention relates to a method for measuring a substance to be measured in a biological sample such as cells.

[0002]

2. Description of the Related Art Specific substances such as antigens and antibodies, proteases and their proteinaceous protease inhibitors, sugar chains and lectins, enzymes and their corresponding substrates and coenzymes, hormones and other physiologically active substances, and their receptors and transport. It is known that a pair of polynucleotide chains such as a protein and a double-stranded DNA exert a strong interaction with each other (that is, show mutual affinity) to form a strong complex.

A method of measuring a substance to be measured in a sample utilizing such interaction is to form a complex on the solid phase by the reaction of the substances in the combination as described above, and then to form the solid phase. The so-called B / F separation is carried out by utilizing, for example, enzyme immunoassay (EIA), radioimmunoassay (RI).
A), fluorescence immunoassay (FIA) (for example, Japanese Patent Laid-Open No. 1-2270)
No. 61, JP-A-3-44399, etc.) and the like and the high-performance liquid chromatography (HPLC) developed by some of the present inventors Separation, so-called B / F separation
JP-A-2-28557, JP-A-3-206964, JP-A-3-22
1865, JP-A-6-66800, etc.) and the like.

In such a measuring method, since the accuracy of the measurement and the time required for the analysis depend to some extent on how efficiently the B / F separation is performed, various studies have been made on the B / F separating method. Has been done. For example,
B / F separation using a solid phase method such as JP-A 1-227061 and JP-A-3-44399, or B / F separation using HPLC disclosed in JP-A-6-66800. In the method of carrying out the method, for example, an anionic substance is bound in advance to an antibody or the like against the substance to be measured in a biological sample, and the so-called B /
The F separation is performed by utilizing the property (anionic property) of the anionic substance in the complex of the substance to be measured and the antibody against the substance.

Although these methods are capable of performing B / F separation more efficiently than in the past, the anionic substances used are all derived from polyamino acids, Since the B / F separation is carried out by utilizing the anionic property derived from the carboxyl group, the method developed by the inventors of the present application is a method having some problems in the B / F separation. there were.

That is, in the method using HPLC, B
/ F separation, it is necessary to separate the complex from a substance that may affect the measurement coexisting in the sample, a free antibody, and the like. Such separation is performed by using the anionic property of the carboxyl group derived from the polyamino acid. In order to fully utilize the anionic substance, it was necessary to introduce about 200 carboxyl groups (about 200 amino acid residues) into the anionic substance. Therefore, first of all, it is difficult to prepare the anionic substance, and it is difficult to prepare a large amount of anionic substance having a uniform molecular weight. When B / F separation is performed, there is a problem that the peak may be tailed or read during the separation, and the measurement accuracy may be reduced. In addition, when a polyamino acid having a carboxyl group such as polyglutamic acid is used for separation, a non-specific reaction may occur, and a phenomenon such as a blinded value (blank value) or an increased measured value may occur. In order to prevent this, there is a problem that it is necessary to add an appropriate polymer anion such as γ-polyglutamic acid. Therefore, there is a demand for further improvement.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned situation, and includes a substance to be measured in a biological sample and a substance having a binding ability (affinity) to the substance to be measured (affinity). Hereinafter, it is abbreviated as a binding ability substance.) A method of using a negative charge for a complex resulting from an interaction with a binding ability substance and a free binding ability substance or a coexisting substance that may affect the detection of the complex. , A novel polypeptide that can be used to more effectively separate the complex from a free binding substance and the like, and a substance to be measured in a biological sample using the polypeptide Its purpose is to provide a measuring method of.

[0008]

The present invention is the invention of "a polypeptide having 4 to 20 sulfated tyrosine residues and having a total of 4 to 30 amino acid residues." Further, the present invention is "the following general formula ^{[1] A- (R 4)} j-B [1] ( wherein, j is 4 to the inner of .j number of R ⁴ represents an integer of 4-20
20 are sulfated tyrosine residues, the remaining R ⁴ may be the same or different and represent an amino acid residue into which a strong acid-derived acid residue has not been introduced. A protective group may be introduced into the reactive group. A represents a protecting group for the N-terminal amino group of an amino acid into which no hydrogen atom, a sulfated tyrosine residue or an acid residue derived from a strong acid has been introduced, and B represents a hydroxyl group or a protecting group for the C-terminal carboxyl group of an amino acid. ) The polypeptide represented by. It is the invention of. Furthermore, the present invention provides "the following general formula [2] A- (R ⁵ ) k-B [2] (in the formula, R ⁵ represents an amino acid residue into which an acid residue derived from a strong acid has been introduced. May be different, k is 4 to 2
A represents an integer of 0, A represents a hydrogen atom, an acid residue derived from a strong acid or a protecting group of an N-terminal amino group of an amino acid, and B represents a hydroxyl group or a protecting group of a C-terminal carboxyl group of an amino acid.
However, 4 to 20 of k R ⁵ are sulfated tyrosine residues. ) The polypeptide represented by. It is the invention of.

That is, the present inventors have found that the complex formed as a result of the interaction between the substance to be measured and the binding substance in the biological sample and the free binding substance [or the free substance to be measured]. still,
The measurement target substance may be labeled with a substance that can be detected by some method (hereinafter, abbreviated as a detection substance). ] When the separation from coexisting substances that may affect the detection of the complex using a method utilizing a negative charge, the complex and the free binding ability substance are more clearly separated. In the course of earnest research for a possible method, a substance having appropriate properties capable of changing the properties of the complex (hereinafter, abbreviated as a separation improving substance) is bound to the complex, and the separation improving is performed. In the case of performing a separation operation of the complex and a coexisting substance that may affect the detection of the complex such as a free binding substance (or a free substance to be measured) based on the properties of the substance, It is possible to freely adjust the elution position of the complex by appropriately selecting and using the separation enhancing substance, in other words, by binding an appropriate separation enhancing substance to the complex, Clearly separate free binding substances, etc. It found that it is possible to have filed a patent application previously (JP-A-6-66800). However, in this method, when an anionic separation improving substance is bound to the complex to separate the complex, the above-mentioned problems may occur.
As a result of further studies by the present inventors, in order to find an anionic separation enhancing substance that does not cause such a problem, as a result, a polypeptide having at least three acid residues derived from a strong acid was identified as an anionic separation enhancing substance. As a result, they have found that the problems described above can be solved, and have completed the present invention.

The polypeptide of the present invention is "a polypeptide having 4 to 20 sulfated tyrosine residues and having a total number of amino acid residues of 4 to 30". In addition, the polypeptide of the present invention includes, for example, the following general formula [1] A- (R ⁴ ) j-B [1] (in the formula, j represents an integer of 4 to 20. Of j R ⁴ s) 4-
20 are sulfated tyrosine residues, the remaining R ⁴ may be the same or different and represent an amino acid residue into which a strong acid-derived acid residue has not been introduced. A protective group may be introduced into the reactive group. A represents a protecting group for the N-terminal amino group of an amino acid into which no hydrogen atom, a sulfated tyrosine residue or an acid residue derived from a strong acid has been introduced, and B represents a hydroxyl group or a protecting group for the C-terminal carboxyl group of an amino acid. ), The following general formula [2] A- (R ⁵ ) k-B [2] (In the formula, R ⁵ represents an amino acid residue into which an acid residue derived from a strong acid is introduced, and may be the same or different. K is 4 to 2
A represents an integer of 0, A represents a hydrogen atom, an acid residue derived from a strong acid or a protecting group of an N-terminal amino group of an amino acid, and B represents a hydroxyl group or a protecting group of a C-terminal carboxyl group of an amino acid.
However, 4 to 20 of k R ⁵ are sulfated tyrosine residues. ), The following general formula [3] A- (R ⁵ ) j- (R ⁶ ) I-B [3] (In the formula, R ⁵ represents a sulfated tyrosine residue, and j is 4 to 20.
Represents the integer. R ⁶ represents an amino acid residue into which an acid residue derived from a strong acid has not been introduced. Further, (j + 1) is 5 to 21. A represents a protecting group for the N-terminal amino group of an amino acid into which no hydrogen atom, a sulfated tyrosine residue or an acid residue derived from a strong acid has been introduced, and B represents a hydroxyl group or a protecting group for the C-terminal carboxyl group of an amino acid. ) And the like. "A conjugate of a polypeptide having at least three acid residues derived from a strong acid and a substance capable of binding to a measurement target substance in a biological sample." Or "at least acid residues derived from a strong acid" The compound used in “a compound having a maleimide group bound to the N-terminus of a polypeptide having three via a spacer.” May be a polypeptide having at least three acid residues derived from a strong acid, and the remaining amino acids constituting the polypeptide may be used. The type and number of groups are not particularly limited, but considering the easiness of synthesis and the like, the total number of amino acid residues is appropriately selected from the range of usually about 3 to 30, preferably about 5 to 15. Also, as a strong acid, pKa of 3 or less
Examples of the acid include inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid and phosphoric acid. The strong acid-derived acid residue in the present invention means an acid residue derived from the strong acid. In addition, the acid residue in the present invention refers to a residue obtained by removing a hydrogen atom from the strong acid. The amino acid residue means an amino acid having an N-terminal hydrogen atom and a C-terminal hydroxyl group removed.

In the polypeptide of the present invention, the above-mentioned acid residue is usually introduced into the reactive group of the polypeptide, and the reactive group may be, for example, a free hydroxyl group or amino group in the polypeptide. , Imino groups, thiol groups and the like. The amino group of the main chain of the polypeptide (the amino group at the N-terminal of the polypeptide) can also be used as the reaction-active group. However, when the polypeptide is used as a separation-improving substance, the amino group is It is preferable not to introduce the acid residue. That is, if the N-terminal amino group of a polypeptide is used when binding a polypeptide and a binding substance, the binding mode between the polypeptide and the binding substance can be made constant to some extent. This is because if the binding substance to which the obtained polypeptide (ie, the separation-improving substance) is bound is used for the measurement of the measurement target substance in the biological sample, the target measurement can be performed with higher accuracy.

According to the present invention, "the acid residue derived from a strong acid is reduced.
Also having three polypeptides and the measurement target in a biological sample
"Binding substance with substance having binding ability" or "strong acid"
N of a polypeptide having at least 3 acid residues derived from
A compound in which a maleimide group is bonded to the end via a spacer
Examples of the polypeptide used in the "product" include, for example, the following general formula [I] A- (R) m-B [I] (in the formula, m represents an integer of 3 or more, preferably 3 to 30,
It is more preferably 3 to 20, still more preferably 4 to 20, still more preferably 5 to 15, and at least 3 of m R's.
Is an amino acid residue into which an acid residue derived from a strong acid has been introduced, and the remaining R represents an amino acid residue into which an acid residue from a strong acid has been introduced or an amino acid residue into which an acid residue from the strong acid has not been introduced. Represents any of the groups. The type of the amino acid residue into which the acid residue derived from a strong acid represented by R and the amino acid residue into which the acid residue derived from the strong acid is not introduced may be the same or different, and A protective group or the like may be introduced into the reaction active group of the side chain, and the bonding order thereof is arbitrary. A represents a hydrogen atom, an acid residue derived from a strong acid, or an N-terminal amino group protecting group of an amino acid, and B represents a hydroxyl group or a C-terminal carboxyl group protecting group of an amino acid. ), General formula [II] A- (R ¹ ) m′-B [II] [In the formula, R ¹ represents an amino acid residue into which an acid residue derived from a strong acid is introduced, and m ′ is an integer of 3 or more. Represents, preferably 3
-30, more preferably 3-20, even more preferably 4-20,
Even more preferably, it is 5 to 15, and A and B are the same as above. The types of m R1s (amino acid residues into which an acid residue derived from a strong acid has been introduced) may be the same or different,
The joining order is also arbitrary. ], The general formula ^{[III] A- (R 1)} m '- (R 2) n-B [III] [ wherein, R ² represents an amino acid residue having no acid residues from a strong acid, n Represents a natural number, and R ¹ , m ′, A and B are the same as above. The types of n R ² (amino acid residues) are
They may be the same or different, and their bonding order is arbitrary. Further, the bonding order of m R ¹ and n R ² is arbitrary. Further, (m '+ n) is usually 4 to 31, preferably 4 to 2
1, more preferably 5 to 21, and even more preferably 6 to 16. ], Etc. are mentioned.

When the polypeptide of the present invention (including the one represented by the above general formula) is used as a separation-improving substance when measuring a substance to be measured in a biological sample, The acid residue derived from a strong acid is preferably an acid residue derived from a polyvalent strong acid such as sulfuric acid or phosphoric acid. Since there is a possibility that phosphatase coexists in a biological sample, a sulfuric acid residue is preferable especially when a polypeptide is used as a separation improving substance. Further, at least three acid residues may be present in the polypeptide of the present invention, but if the number of the acid residues is too large, it is difficult to use as a separation improving substance (for example, anion exchange method). It becomes difficult to separate or elute from a column or the like in the case of separation by). Therefore, the number of the acid residues when used as a separation improving substance is usually 3 to 30, preferably 3 to 20, more preferably Is 4 to 20
, More preferably 5 to 15 are appropriately selected from the range,
The total number of amino acid residues is usually about 3 to 30, preferably about 3 to 20, more preferably about 4 to 20, and even more preferably about 5 to 15 in consideration of easiness of synthesis. To be selected. If the number of the acid residues is less than 3, the separation peak based on the separation-improving substance and the elution peak derived from the serum component will overlap and the measurement (analysis) accuracy will decrease.
The problem arises. Further, in order to eliminate the overlap between the separation peak based on the separation-improving substance and the elution peak derived from the component in the biological sample such as serum and further improve the measurement accuracy, the number of acid residues is 4 or more, It is preferable that the number is 5 or more. Furthermore, when introducing a sulfuric acid residue as an acid residue derived from a strong acid, in consideration of the stability in an aqueous solution after the introduction of the sulfuric acid residue, the phenolic hydroxyl group of the tyrosine residue should be used for the introduction. Is desirable.

In the polypeptide of the present invention, an amino acid residue into which an acid residue derived from a strong acid has been introduced is an amino acid residue having a free reaction active group, and the acid derived from a strong acid is present in the reaction active group. The residue is not particularly limited as long as it has introduced a residue. More specifically, for example, amino acid residues having a free hydroxyl group such as tyrosine, serine, threonine, etc., such as amino acid residues having a free amino group such as lysine, arginine, etc., such as histidine, tryptophan, proline, oxyproline, etc. An amino acid residue having a free imino group, for example, an amino acid residue having a free thiol group such as cysteine, wherein a strong acid is derived from these free reactive groups such as a hydroxyl group, an amino group, an imino group and a thiol group. And the like in which the acid residue is introduced. Considering the ease of introduction of an acid residue derived from a strong acid, amino acid residues such as serine, threonine and tyrosine are preferred. Further, the amino acid residue having no acid residue derived from a strong acid in the polypeptide of the present invention is not particularly limited as long as it is generally recognized as an amino acid residue in the field of peptide chemistry, and examples thereof include alanine. , Amino acid residues such as glycine and β-alanine are preferred.

The polypeptide of the present invention comprises a method of introducing an acid residue derived from a strong acid into at least three free active reactive groups of a polypeptide having an appropriate number of free active reactive groups (J. Chem. Soc). .Perkin Trans I, ( 1990 ), 1739-1744 etc.),
A method for synthesizing a polypeptide having at least three strong acid-derived acid residues using an amino acid into which a strong acid-derived acid residue has been introduced (Chem.Pharm.Bull., 41 (2) , (199
3), 376-380, etc.) and the like. Considering the yield of the target polypeptide, etc., among the above methods,
The method of is preferable (especially when the number of amino acid residues is large). However, in the above method, since it is not necessary to carry out a sulfation reaction, a functional group capable of binding to a binding ability substance such as an antibody, such as a maleimidocaproic acid residue, is directly attached to the N-terminus of the obtained polypeptide. Since it can be introduced, there is an advantage that the obtained polypeptide can be immediately bound to a binding substance.

As a method for introducing an acid residue derived from a strong acid into a free active reactive group of a polypeptide or an amino acid, for example, a free reactive active group such as a hydroxyl group, an amino group, an imino group or a thiol group is derived from a strong acid. The method is not particularly limited as long as it can introduce an acid residue. More specifically, for example, amino acids having free hydroxyl groups such as tyrosine, serine and threonine (or polypeptides having residues of these amino acids), and 1 to 20 relative to the free hydroxyl groups of the amino acids (residues). Double equivalents such as HSO ₃ · Cl, HSO ₃ · Dimethylformamide, HS
A sulfating agent such as O ₃ · polyphosphorus or a phosphorylating agent such as phosphoryl chloride or phosphorus halide such as phosphorus trichloride is dissolved in a solvent (anhydrous) such as dimethylformamide (DMF) or dimethylsulfoxide (DMSO). , Such as pyridine, triethylamine, NaH, etc. as a catalyst 0-4
A method of introducing an acid residue derived from a strong acid into the hydroxyl group of the amino acid residue by reacting at 0 ° C. for 1 to 24 hours, for example, an amino acid having a free amino group such as lysine or arginine (or a residue of these amino acids Or a amino acid having a free imino group such as histidine, tryptophan, proline, oxyproline (or a polypeptide having a residue of these amino acids) and a free amino group of the amino acid (residue). or / and 1 to 20 times the imino group) equivalent of example HSO ₃ · Cl, HSO ₃ · di methyl formamidine preparative Bu, sulfating agents such as HSO ₃ · pyridine or e.g. phosphoryl chloride,
Phosphorylating agents such as phosphorus trichloride are used in, for example, pyridine, triethylamine, Na
Examples include a method of reacting with an alkaline substance such as H as a catalyst at 0 to 40 ° C. for 1 to 24 hours to introduce an acid residue derived from a strong acid into the amino group (or / and imino group) of the amino acid residue. . Incidentally, in consideration of the ease of synthesis and the stability of the introduced acid residue in an aqueous solution and the stability against enzymes such as phosphatase existing in a biological sample, etc., the acid residue derived from the strong acid according to the present invention Of these, a sulfuric acid residue is more preferable.

As the raw material of the polypeptide of the present invention, for example, a polypeptide having a free reactive group such as a hydroxyl group, an amino group, an imino group and a thiol group may be a commercially available product or peptide chemistry. Conventional methods commonly used in the field of, for example, active ester method, mixed acid anhydride method, azide method, etc. ("Basics and Experiments of Peptide Synthesis", pages 89-142, Nobuo Izumiya et al., January 20, 1985) , Maruzen Co., Ltd.) may be used. When synthesizing such a polypeptide, the protecting group for the amino group at the N-terminal is preferably one that is stable in the next sulfation step (a protecting group that is not suitable under acidic conditions is unsuitable), for example, under alkaline conditions. Preferable examples include a urethane-type protecting group such as a removable 9-fluorenylmethoxycarbonyl (Fmoc) group. Also,
The protective group for the reaction active group of the side chain of the amino acid (residue) may be appropriately selected in consideration of, for example, the synthesis conditions of the polypeptide, the conditions for introducing an acid residue derived from a strong acid, and a t-butyl group, A benzyl group and the like are preferred.

The binding substance according to the present invention is not particularly limited as long as it is a substance capable of binding to the substance to be measured in the biological sample to be measured.
Specifically, for example, antibodies and antigens having the ability to bind to a substance to be measured in a biological sample, for example, concanavalin A, lentil lectin, kidney bean lectin, datsura lectin, wheat germ lectin, and other lectins, such as amylase and creatine. Preferable examples include inhibitors for enzymes such as kinases (CK) and glutamate oxaloacetate transaminase (GOT), and polynucleotides complementary to the nucleic acid chain as the substance to be measured, for example, receptors for thyroid stimulating hormone, acetylcholine, glutamic acid and the like.

The method for preparing the binding substance of the binding ability substance according to the present invention and the polypeptide of the present invention (hereinafter, abbreviated as the binding substance of the present invention) comprises a specific reactive group of the binding ability substance and the polypeptide. And a method of substituting the specific reactive group of the binding ability substance with the polypeptide, a substance capable of binding to the binding ability substance (eg, antibody, lectin, antigen, inhibitor, DNA) Etc.) and the like and the method of binding the binding substance and the polypeptide. Specifically, for example, 1) a labeling substance and an antibody known per se which are generally used in enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA) and the like known per se. Binding method (for example, Medical Experiment Course, Volume 8, edited by Yuichi Yamamura, 1st 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)
Known methods for modifying and binding substances (for example, chemical modification of proteins <up><down>, Ikuzo Uraya, Kensuke Shimura, Michinori Nakamura, Masaru Funatsu, 1st edition, Academic Publishing Center Co., Ltd., 198
1; Polyethylene glycol modified protein, Yuji Inada et al., Biochemistry, Volume 62, No. 11, P1351-1362, Japan Biochemical Society, 1990; DNA PROBES, George HK and Mark M.
M. STOCKTON PRESS, 1989, etc.) and the like can be mentioned without exception, and the method may be carried out according to these methods.

Specific examples of the method for binding the specific reactive group of the binding substance to the specific reactive group of the polypeptide include, for example, first, a maleimide group via a spacer at the N-terminus of the polypeptide of the present invention. (Hereinafter, abbreviated as “maleimide compound of the present invention”) bound to the polypeptide and the SH group of Fab ′ of the antibody prepared by a conventional method to react with the polypeptide of the present invention and a binding substance (antibody). And a method for preparing a bound product thereof. The maleimide compound of the present invention used here has, for example, the following general formulas [IV] to [V
It is a compound represented by [I]. General formula [IV] DE- (R) m-B [IV] (In the formula, D represents a maleimide group, E represents a spacer, and R, m, and B are the same as the above.) General formula [V ^{] D-E- (R 1)} m'-B [V] ( ^{wherein, D, E, R 1,} m ' and B have the same meanings as defined above.) in formula [VI] D-E- (R 1) m '-(R < ² >) n-B [VI] (In the formula, D, E, R < ¹ >, R < ² >, m', n and B are the same as above.) Here, in these general formulas, E As the spacer represented by, the N-terminal and the maleimide group of the polypeptide of the present invention can be bound to both ends thereof, and the maleimide compound of the present invention binds to the binding substance (antibody) of the present invention. is not particularly limited as long as it has a property that does not interfere with, for example, -R group (wherein, R ⁴ represents a divalent hydrocarbon group.) represented by ⁴ -CO-, more specifically For example the following general formula [VII] ~ [IX] - (CH 2) p-CO- [VII] ( wherein, p is an integer of 1 to 10.)

[Chemical 1] (In the formula, q and r represent 0 or an integer of 1 to 5.)

[Chemical 2] (In the formula, q and r are the same as above.). In addition, a substituent may be introduced into the alkylene group, the phenylene group and the cyclohexylene group in the general formulas [VII] to [IX]. Examples of such a substituent include a methyl group, an ethyl group and a propyl group. Group, lower alkyl group having 1 to 4 carbon atoms such as butyl group (which may be linear or branched), for example, a hydrophilic group such as hydroxyl group, carboxyl group, sulfo group, amino group, etc. Etc. The maleimide compound of the present invention can be easily obtained, for example, by the following method. That is, the polypeptide of the present invention and 1 to 10 relative to the N-terminal amino group of the polypeptide.
0 times mol, preferably 1 to 50 times mol, more preferably 1.2 times.
~ 10-fold molar amount of a divalent cross-linking agent (a compound having a maleimide group at one end and a group capable of reacting with an amino group at the other end), if necessary, a 2- to 5-fold molar amount relative to the amino group After reacting the reaction accelerator with an appropriate buffer solution having a pH of 6 to 9 or an appropriate organic solvent at 4 to 37 ° C for 0.2 to 10 hours, the reaction solution is purified by an appropriate column chromatography to give The maleimide compound of the invention can be obtained. As the divalent cross-linking agent used in the above reaction, the maleimide group is finally added to the N of the polypeptide of the present invention.
It is not particularly limited as long as it can be introduced into the terminal, and examples thereof include a cross-linking agent having a maleimide group and a succinimide group, and more specifically, succinimidyl-N-4-maleimidobutyrate, succinimidyl-N-6. -Maleimidocaproylate, succinimidyl-N-8-maleimidocaprylate, succiimidyl-N-11-maleimidoundecanoate, succinimidyl-N-4- (2-maleimidoethoxy)
Succinilate, sulfosuccinimidyl-N-4-maleimidobutyrate, sulfosuccinimidyl-N-6-maleimidocaproylate, sulfosuccinimidyl-N-8-maleimidocaprylate, succinimidyl-N-11 -Maleimidoundecanoate, succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, succimidyl-m-maleimidobenzoate Preferred examples thereof include sulfosuccinimidyl-m-maleimidobenzoate, succinimidyl-4- (p-maleimidophenyl) butyrate and sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate. Examples of the reaction accelerator include bases such as triethylamine and pyridine. PH6 used as solvent during reaction
Examples of the buffers of 9 to 9 include phosphate buffers having such a pH, tris (hydroxymethyl) aminomethane buffers such as N, N-bis (2-hydroxyethyl) glycine (Bicine) buffer, 2 Good buffers such as-[4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) buffer and 3-morpholinopropanesulfonic acid (MOPS) buffer are listed, and examples of the organic solvent include Dimethylformamide (DMF), dimethylsulfoxide (DMS)
O) and the like. Moreover, examples of the chromatography used for purifying the maleimide compound of the present invention after the reaction include gel column chromatography, silica gel column chromatography, octadecyl silica gel (ODS) column liquid chromatography and the like. An organic solvent is preferred as the solvent during the reaction. That is, since the maleimide group is less decomposed in the organic solvent, the maleimide compound of the present invention can be obtained in high yield as a result. Further, it is preferable to carry out the purification by ODS column liquid chromatography because the excess divalent crosslinking agent and the maleimide compound of the present invention can be separated more clearly. The maleimide compound of the present invention obtained as described above can be stably stored, for example, by treatment by concentration to dryness, freeze-drying, etc., and thus the polypeptide of the present invention can be bound to an appropriate bond according to the present invention. It is extremely useful as an intermediate for binding to an active substance (antibody). In addition, the method for binding the maleimide compound of the present invention and the binding substance according to the present invention to obtain the bound substance of the polypeptide of the present invention and the binding substance may be carried out, for example, as follows. That is, the maleimide compound of the present invention and Fa of the antibody prepared by a conventional method
reacting the SH group of b ′ with a solution that does not lose the properties of the antibody (for example, a buffer commonly used in the field of immunoassays) at 4 to 37 ° C. for 1 to 16 hours Thus, a bound product of the polypeptide of the present invention and a binding substance (antibody) can be prepared.

[0021] Examples of the method for binding the amino group of the polypeptide of the present invention to the amino group of the binding substance include the glutaraldehyde method known per se, and the like.
As a method for binding the amino group of the polypeptide of the present invention to the sugar chain of the binding substance, for example, the sugar chain is treated with periodate to form an aldehyde group, and the aldehyde group and the polypeptide of the present invention are combined. A known periodic acid method for binding an amino group can be used.

When the polypeptide of the present invention is bound to the substance capable of binding, it may be carried out by utilizing the reaction active group of the side chain of the polypeptide, but it is carried out by utilizing the amino group at the N-terminal. Is desirable.

That is, since there is usually only one N-terminal amino group, if it is used to bind to a binding substance,
The binding molar ratio between the polypeptide of the present invention and the binding substance is 1: 1. Therefore, when the substance to be measured is separated using the bound substance, it is separated as one peak, and thus the accuracy of the target measurement can be further improved. The maleimide compound of the present invention is a substance useful for preparing such a bound product.

When the conjugate of the present invention is prepared using the N-terminal amino group, it is desirable not to introduce a strong acid-derived acid residue into the N-terminal amino acid residue of the polypeptide of the present invention. . That is, the yield of the desired bound product is higher when such a polypeptide of the present invention is bound to the binding substance.

As a method for measuring a biological component using the conjugate of the present invention (hereinafter abbreviated as "the measuring method of the present invention"), a method capable of carrying out an intended measurement using the conjugate of the present invention. The method is not particularly limited as long as it is, but specific examples include the following methods. (1) Method of measuring amount of target substance in biological sample using non-competitive reaction (measurement method (1)) First, a sample of biological origin containing the target substance and a binding substance (hereinafter referred to as a binding substance labeled with a detection substance) , "Labeled substance"
Is abbreviated. ) And the binding product of the present invention (the binding site of the labeled binding substance in the measurement target substance is different from the binding site of the binding product of the present invention), if necessary, in a suitable buffer solution. After adding, mixing and reacting with each other to form a complex in which the substance to be measured in the biological sample, the label-binding substance and the binding substance of the present invention are bound to each other, the complex and the free label-binding capability are formed. A substance and a method utilizing a negative charge, for example, an HPL equipped with a column packed with a packing material for anion exchange chromatography
Separation is performed by using a device having anion exchange action (anion exchange method) such as C device, anion exchange membrane, and reaction tube having anion exchange action. Then, the amount of the detection substance contained in the separated complex is determined by a measuring method according to the property of the detection substance. Separately, measurement is performed by a similar method using a sample whose concentration of the substance to be measured is known, and a calibration curve showing the relationship between the amount of the substance to be measured in the biological sample and the amount of the substance to be detected in the complex is prepared, and this is prepared. If the amount of the substance to be measured in the biological sample corresponding to the amount of the substance to be detected in the complex is obtained by using the amount of the substance to be measured in the biological sample. In the above reaction, the concentration of the labeled binding substance and the binding substance of the present invention used in forming the complex varies depending on how much the calibration limit of the substance to be measured in the biological sample is set. However, usually, in the reaction solution, a concentration equal to or higher than the predetermined calibration limit concentration capable of binding to all the substances to be measured in the biological sample, preferably 2 times or more concentration, more preferably 5 times concentration Above, more preferably 10 times or more is preferably present in the reaction solution. When the substance to be measured itself is a substance that can be measured (detected) by some method, the above reaction is carried out without using a labeled binding substance, and the property of the substance to be measured in the obtained complex is determined. The amount of the substance to be measured in the biological sample in the sample can be similarly obtained by obtaining the amount by a corresponding measuring method.

(2) Method for measuring amount of substance to be measured in biological sample using competitive reaction (Measurement method (2)) First, a sample of biological origin containing the substance to be measured, and the substance to be measured labeled with the detection substance ( Hereinafter, abbreviated as a labeled measurement substance.), If necessary, the bound substance of the present invention is added to a suitable buffer solution, mixed and reacted to give a substance to be measured in a biological sample and the bound substance of the present invention. And a complex of the labeled measurement substance and the bound substance of the present invention (hereinafter abbreviated as a labeled complex) are formed, and then the labeled complex and the free labeled measurement substance are charged with a negative charge. , For example, HPL equipped with a column packed with a packing material for anion exchange chromatography
Separation is performed using a device having an anion exchange action such as a C device, an anion exchange membrane, and a reaction tube having an anion exchange action. Then, the amount of the detection substance contained in the separated labeled complex is determined by a measuring method according to the property of the detection substance. Separately, measurement is performed by a similar method using a sample with a known concentration of the substance to be measured, and a calibration curve showing the relationship between the amount of the substance to be measured in the biological sample and the amount of the substance to be detected in the labeled complex is prepared. If the amount of the substance to be measured in the biological sample corresponding to the amount of the substance to be detected in the labeled complex is obtained using, the amount of the substance to be measured in the biological sample in the sample can be obtained. In the above reaction, the use concentration of the binding substance of the present invention and the use concentration of the labeled measurement substance in forming the labeled complex are set to the calibration limit and the measurement sensitivity of the measurement target substance in the biological sample. It may be appropriately set depending on whether or not it is used, and is not particularly limited. However, it goes without saying that the concentration of the labeled measurement substance to be used must be set to a concentration at which it can bind to all the bound substances of the present invention present in the reaction solution.

(3) Measuring method in which the substances to be measured in the biological sample are two or more substances having the same action and the same detectable chemical property (Measuring method (3)) A biologically-derived sample containing a substance, and a substance having a property of specifically binding to at least one substance to be measured in the biological sample, but not to at least one of these substances, and bound to the polypeptide of the present invention (Less than,
It is abbreviated as "the conjugate A of the present invention". ) And, if necessary, in an appropriate buffer solution and mixed to react with each other to form a complex between the substance to be measured in a specific biological sample and the conjugate A of the present invention, and then the complex And a substance to be measured in a free biological sample by using a negative charge, for example, HPLC equipped with a column packed with a packing material for anion exchange chromatography
Separation is performed by using a device, an anion exchange membrane, a mechanical device having an anion exchange action such as a reaction tube having an anion exchange action, or the like. Next, the amount of the measurement target substance in the biological sample contained in the separated complex or / and the amount of the measurement target substance in the free biological sample is measured by a measuring method according to the property of the measurement target substance in the biological sample. Ask. Separately, for example, measurement is performed by a similar method using a sample having a known concentration of the measurement target substance, and the relationship between the amount of the measurement target substance in the biological sample and the measurement value based on the detectable property of the measurement target substance in the complex is shown. If a calibration curve is created and the amount of the substance to be measured in the biological sample corresponding to the measured value is obtained using this, any amount of the substance to be measured in the biological sample in the sample can be obtained. In the above reaction, the concentration of the conjugate A of the present invention used in forming the complex may be appropriately set depending on how much the calibration limit and the measurement sensitivity of the substance to be measured are set.
There is no particular limitation.

(4) Measuring method in which the substance to be measured in the biological sample is two or more substances having the same action or two or more substances having a similar structure but different actions (Measuring method (4)) First, a sample derived from a living body containing a substance to be measured and a substance capable of binding to all the substance to be measured in the biological sample (hereinafter, abbreviated as “binding substance A”) are labeled with a detection substance (hereinafter, Abbreviated as "labeled binding substance A") and a substance capable of binding to a specific substance to be measured in a biological sample and bound to the polypeptide of the present invention (hereinafter referred to as "the binding substance B of the present invention"). Abbreviated.), If necessary, in a suitable buffer solution, mixed and reacted, and a complex of the substance to be measured and the labeled binding substance A in the biological sample (hereinafter abbreviated as complex A). ) And a specific substance to be measured and a labeled binding substance in a biological sample After forming a complex of the substance A and the bound substance B of the present invention (hereinafter, abbreviated as the complex B), the complex A, the complex B and the free labeled binding substance A are charged with a negative charge. Method, for example, using an HPLC apparatus equipped with a column packed with a packing material for anion exchange chromatography, an anion exchange membrane, a mechanical device having an anion exchange action such as a reaction tube having an anion exchange action, etc. To separate. Next, the amount of the detection substance contained in the separated complex A or / and the amount of the detection substance contained in the separated complex B is determined by a measuring method according to the property of the detection substance. Separately, for example, measurement is performed by a similar method using a sample whose concentration of the substance to be measured is known, and the amount of the substance to be measured in the biological sample and the amount of the substance to be detected contained in the complex A or / and the amount contained in the complex B are included. Create a calibration curve that shows the relationship with the amount of the detected substance, and use this to find the amount of the substance to be measured in the biological sample that corresponds to the amount of the detected substance in the complex. The amount of any of the substances to be measured of is determined. In the case where the binding ability substance A itself is a substance that can be measured (detected) by some method, the above reaction is performed using the binding ability substance A that is not labeled with the detection substance, and The amount of the substance to be measured in the biological sample in the sample can be similarly obtained by obtaining the amount of the substance A having binding ability by a measuring method according to the property of the substance capable of binding A.

(5) Measurement method in which the substance to be measured in the biological sample is two substances having the same action or two substances having a similar structure but different actions (measurement method
(5)) First, a sample derived from a living body containing a substance to be measured, a substance capable of binding to the labeled binding substance A and the substance 1 to be measured in the biological sample, and having the polypeptide 1 of the present invention bound (hereinafter , Abbreviated as “the conjugate C of the present invention”) and a substance capable of binding to the substance 2 to be measured in a biological sample and bound to the polypeptide 2 of the present invention (hereinafter, “the conjugate D of the present invention D”). Is added to a suitable buffer solution and mixed and reacted to obtain a complex of the substance to be measured 1 in the biological sample, the bound substance C of the present invention and the labeled binding substance A. (Hereinafter, it is abbreviated as complex C.) and a complex (hereinafter, abbreviated as complex D) of the measurement target substance 2 in the biological sample, the bound substance D of the present invention and the labeled binding substance A is formed. After that, the complex C, the complex D and the free labeled binding substance A are added to the polypeptide 1 and Based on the difference between the properties of 2 and 2, a method utilizing a negative charge, for example, an HPLC apparatus equipped with a column packed with a packing material for anion exchange chromatography, an anion exchange membrane, a reaction tube having an anion exchange action, etc. Separation is performed using a machine tool having anion exchange action. Next, the amount of the detection substance contained in the separated complex C and / or the amount of the detection substance contained in the complex D is determined by a measuring method according to the property of the detection substance. Separately, for example, the measurement target substance 1 in the biological sample is measured by the same method using a sample whose concentration of the measurement target substance is known.
(Or 2) A calibration curve representing the relationship between the amount and the amount of the detection substance contained in the complex C or / and the amount of the detection substance contained in the complex D is prepared, and is used to prepare the complex If the amount of the measurement target substance in the biological sample corresponding to the amount of the detection substance in the sample is calculated, any amount of the measurement target substance in the biological sample in the sample can be calculated. In the case where the binding ability substance A itself is a substance that can be measured (detected) by some method, the above reaction is performed using the binding ability substance A that is not labeled with the detection substance, and The amount of the substance to be measured in the biological sample in the sample can be similarly obtained by obtaining the amount of the substance A having binding ability by a measuring method according to the property of the substance capable of binding A.

(6) Measurement method in which the substance to be measured in the biological sample is two or more glycoproteins having different sugar chain structures but substantially the same protein structure (measurement method (6)) A biological sample containing a glycoprotein, a lectin that recognizes at least one specific sugar chain structure of a glycoprotein to be measured, and a sugar to be measured to which all the glycoproteins to be measured have the property of binding. A binding product of an antibody having a property of preventing binding to a protein and the polypeptide of the present invention (hereinafter abbreviated as "polypeptide binding antibody"), a measurement target containing a glycoprotein to be measured to which the lectin is bound The antibody having the property of being capable of binding to all of the glycoproteins (hereinafter, abbreviated as "labeled anti-glycoprotein antibody") having the property of binding to a detection substance is reacted with the glycoprotein, lectin and labeled anti-glycoprotein antibody. Complex with And a complex of a glycoprotein, a polypeptide-binding antibody, and a labeled anti-glycoprotein antibody are formed, and then the complex and the free labeled anti-glycoprotein antibody are subjected to a method utilizing a negative charge, such as an anion. HP equipped with a column packed with packing material for exchange chromatography
Separation is performed by using an LC device, an anion exchange membrane, a mechanical device having an anion exchange action such as a reaction tube having an anion exchange action, or the like. Then, the amount of the detection substance contained in these separated complexes is determined by a measuring method according to the property of the detection substance. Separately, for example, a measurement is performed by a similar method using a sample of known glycoprotein concentration to be measured, and a calibration value indicating the relationship between the amount of glycoprotein of the measurement target in a biological sample and the amount of the detection substance contained in these complexes. If a line is created and the amount of the glycoprotein to be measured in the biological sample corresponding to the amount of the detection substance in these complexes is determined using this line, it will be determined which of the glycoproteins to be measured in the biological sample in the sample. The amount is required. Needless to say, when the glycoprotein to be measured is a substance that can be measured (detected) by some method, the above-mentioned measurement can be performed without using a labeled anti-glycoprotein antibody.

The measurement target substance in the biological sample that can be measured by the measurement methods (1) and (2) using the present invention includes i) a strong interaction with the measurement target substance in the biological sample, and A substance capable of forming a complex is present, and the substance itself can be measured (detected) by some method, or can be labeled with any detected substance, or ii) the substance to be measured itself Is a substance capable of being labeled with any detection substance, and there is a substance capable of forming a strong labeled complex by interacting strongly with the substance to be measured in the biological sample, and is not particularly limited. Without mentioning, for example, serum, blood,
Typical examples include proteins, peptides, nucleic acids, sugar chains, lipids, hormones, drugs, etc. contained in biological body fluids such as plasma and urine, lymphocytes, blood cells, and various biologically derived samples such as various cells. More specifically, for example, α-fetoprotein (AFP), CA19-9, prostate specific antigen (P
SA), carcinoembryonic antigen (CEA), cancer markers such as substances having special sugar chains produced by cancer cells, such as immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin G (IgG), β ₂ -microglobulin,
Serum proteins such as albumin, ferritin, etc., such as C-peptide, angiotensin I, etc., peptides such as amylase, alkaline phosphatase, γ-glutamyl transferase (γ-GTP), etc., such as rubella virus, herpes virus, hepatitis virus, ATL. Viruses, antiviral antibodies against clinically noticed viruses such as AIDS virus, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) of pathogens such as viruses, single-stranded polynucleotides constituting these nucleic acids, viruses, etc. Antigens derived from pathogens, such as antibodies that react with allergens such as cedar and other plant pollens and indoor dust,
For example, lipids such as lipoproteins, proteases such as trypsin, plasmin, serine proteases, such as insulin, human chorionic gonadotropin (hCG), thyroxine (T ₄ ), triiodothyronine (T ₃ ), prolactin, thyroid stimulating hormone ( Examples thereof include hormones such as TSH), drugs such as digoxin, phenytoin, morphine, and nicotine, and receptors such as thyroid stimulating hormone, acetylcholine, glutamic acid, and the like.

As the substance capable of binding to the substance to be measured in the biological sample used for preparing the labeled substance having binding ability according to the measuring method (1) utilizing the present invention, the substance to be measured in these biological samples is Substances that exert strong interactions with each other to form a strong complex, and if necessary, can be measured (detected) by some method or labeled with any detectable substance that can be measured (detected). Any substance can be used without limitation (in the case where the substance to be measured itself in the biological sample can be labeled with a substance to be detected), there is no particular limitation. For example, a substance having antigenicity ( (Including hapten), an antigen to the antibody, and an ability to bind to a sugar chain having a specific structure, such as concanavalin A, lentil lectin, kidney bean Chin, Datura lectin, lectin such as wheat germ lectin, α for example, trypsin
₁ - antitrypsin, ₂ alpha for plasmin - macroglobulin, alpha for serine protease ₂ - macroglobulin and alpha ₁ - inhibitors, for specific enzymes antichymotrypsin such as single-stranded polynucleotide is a measurement substance in a biological sample And a receptor for hormones and the like.

As the substance capable of binding to the substance to be measured in the biological sample used for preparing the bound product of the present invention according to the measuring methods (1) and (2) using the present invention, the binding substance in these biological samples is Target substance (or labeled target substance,
Or a complex of a substance to be measured and a label-binding substance) that strongly interacts with each other to form a strong complex, and examples thereof include, but are not limited to, substances having antigenicity. Antibodies to (including haptens), antigens to antibodies, and abilities to bind to sugar chains having a specific structure, such as concanavalin A, lentil lectin, kidney bean lectin, datsura lectin, wheat germ lectin, and the like, α for trypsin, for example.
₁ - antitrypsin, alpha for plasmin ₂ - macroglobulin, ₂ alpha for serine protease - inhibitors, for specific enzymes macroglobulin etc., single-stranded polynucleotide complementary to the polynucleotide as the measurement substance in a biological sample A chain and the like (however, the binding site is different from the binding ability substance used for preparing the labeled binding ability substance).

The measurement target substance in the biological sample that can be measured by the measurement method (3) utilizing the present invention is a substance that can be measured (detected) by itself and is a measurement target substance in the biological sample. And at least one of them strongly interact with each other to form a strong complex,
There is no particular limitation as long as there is a substance having a property of not binding to at least one of them, and examples thereof include biological fluids such as serum, blood, plasma and urine,
Representative examples include enzymes contained in biological samples such as lymphocytes, blood cells, and various cells. More specifically, for example, amylase, alkaline phosphatase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate. Pyruvate transaminase (GPT), renin,
Enzymes such as protein kinase (PK) and tyrosine kinase are included.

As a substance capable of binding to a specific substance to be measured in a biological sample used for preparing the bound substance A of the present invention according to the measuring method (3) of the present invention, the measurement in the biological sample is performed. At least one of the target substances interacts strongly with each other to form a strong complex,
There is no particular limitation as long as it is a substance that does not bind to at least one of the measurement target substances in the biological sample,
For example, an antibody to a specific partial structure or antigenic determinant site of a substance having an antigenicity (including hapten), an antibody having a binding ability to a sugar chain of a specific structure such as concanavalin A,
Lectins such as lentil lectin, kidney bean lectin, honeysuckle lectin, hilochawantake lectin, chickpea lectin, peanut lectin, wheat germ lectin, such as amylase, creatine kinase (C
K), inhibitors of enzymes such as glutamate oxaloacetate transaminase (GOT), and the like.

The measurement target substance in the biological sample that can be measured by the measurement method (4) using the present invention has the property of binding to all of the measurement target substance and being detectable by some method itself. Or a substance that can be labeled with a detection substance and at least one of the substances to be measured in the biological sample have a strong interaction with each other (affinity or affinity) to form a strong complex. As long as there is a substance that does not bind to at least one of them, there is no particular limitation, and examples thereof include biological fluids such as serum, blood, plasma, urine, lymphocytes, blood cells, and various cells. Representative examples include enzymes, physiologically active substances, cancer-related antigens, sugar chain-containing substances, etc. contained in the biological sample. More specifically, for example, amylase, alkaline phosphatase, acid phosphatase, γ-glutamyl transferase (γ-G
TP), lipase, creatine kinase (CK), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), renin, protein kinase, tyrosine kinase and other enzymes such as steroid hormones. , Human chorionic gonadotropin (hCG), prolactin, thyroid stimulating hormone (TSH), luteinizing hormone (LH), and other physiologically active substances such as prostate specific antigen (PSA), α ₂ -macroglobulin, carcinoembryonic antigen (CEA) ), Cancer-related antigens such as α-fetoprotein, and the like.

As the binding ability substance for the measurement target substance in the biological sample used for preparing the labeled binding ability substance A according to the measurement method (4) utilizing the present invention, the measurement target substance in the biological sample is There is no particular limitation as long as it is a substance capable of binding to all and having a property of being detectable by some method itself or being labelable with a detection substance. Specific examples thereof include, for example, concanavalin A, lentil lectin, and kidney bean having the ability to bind to an antibody against a specific partial structure or an antigen-determining site of a substance having an antigenicity (including a hapten) or a sugar chain of a specific structure. Lectins such as lectins, datura lectins, hiirochawantake lectins, chickpea lectins, peanut lectins, wheat germ lectins, and inhibitors for enzymes such as amylase, creatine kinase (CK), and glutamate oxaloacetate transaminase (GOT). .

As a binding substance having a binding ability to a specific substance to be measured in a biological sample, which is used for preparing the bound substance B of the present invention according to the measuring method (4) utilizing the present invention Does not inhibit the complex formation reaction between a specific substance to be measured in the biological sample and the free labeled binding substance A and the reaction of detecting the detection substance (or binding substance A) in the complex. A substance capable of binding to a specific substance to be measured in a biological sample, or a substance capable of binding to the substance to be detected when the substance A to be labeled is bound to the substance to be detected. Examples thereof are not particularly limited. Specifically, for example, an antibody having an ability to bind to a specific substance to be measured in a biological sample, for example, lectins such as concanavalin A, lentil lectin, kidney bean lectin, datura lectin, wheat germ lectin, etc. (The binding site is different from substance A.)
Etc., or, for example, an antibody having the binding ability to the binding substance A or the detection substance, such as concanavalin A, lentil lectin, kidney bean lectin, datura lectin,
Lectins such as wheat germ lectins are preferred.

The measurement target substance in the biological sample that can be measured by the measurement method (5) using the present invention has the property of binding to all of the measurement target substance and being detectable by some method itself. Substance or a substance that can be labeled with a detection substance and the measurement target substance 1 in the biological sample have a strong interaction with each other (affinity; affinity or affinity)
Substances that form a strong complex but do not bind to the measurement target substance 2 and exert strong interaction (affinity; affinity or affinity) with the measurement target substance 2 in the biological sample. If there is a substance that forms a strong complex but does not bind to the measurement target substance 1,
Examples thereof include without limitation, biological fluids such as serum, blood, plasma, urine, etc., enzymes, physiologically active substances, cancer-related antigens contained in biological samples such as lymphocytes, blood cells, and various cells. Representative examples include substances having sugar chains. More specifically, 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 steroid hormones, human chorionic gonadotropin (hCG), prolactin, thyroid stimulating hormone (TSH). ), Luteinizing hormone (LH)
Bioactive substances such as prostate specific antigen (PSA),
α ₂ -macroglobulin, carcinoembryonic antigen (CEA), α-
Preferable examples include cancer-related antigens such as fetoprotein.

As the binding substance for the measurement target substance in the biological sample used for preparing the labeled binding substance A according to the measurement method (5) of the present invention, the measurement target substance in the biological sample is There is no particular limitation as long as it is a substance capable of binding to all and having the property of being detectable by some method itself or being labeled with a detection substance. Specific examples thereof include, for example, concanavalin A, lentil lectin, and kidney bean having the ability to bind to an antibody against a specific partial structure or an antigen-determining site of a substance having an antigenicity (including a hapten) or a sugar chain of a specific structure. Lectins such as lectins, datura lectins, hiirochawantake lectins, chickpea lectins, peanut lectins, wheat germ lectins, and inhibitors for enzymes such as amylase, creatine kinase (CK), and glutamate oxaloacetate transaminase (GOT). .

The binding ability to a specific target substance 1 or 2 in a biological sample, which is used for preparing the bound substance C or D of the present invention according to the measuring method (5) utilizing the present invention, is shown. The binding substance to be possessed includes a complex formation reaction between the measurement target substance 1 or 2 in the biological sample and the free labeled binding substance A and a reaction for detecting the detection substance (or the binding substance A) in the complex. Any substance that does not inhibit and has a binding ability to the specific substance 1 or 2 to be measured in the biological sample can be mentioned without particular limitation. Specifically, for example, an antibody, such as concanavalin A, which has a binding ability to the measurement target substance 1 or 2 in a biological sample,
Lectins such as lentil lectin, kidney bean lectin, datura lectin, wheat germ lectin (however, the binding site is different from the binding ability substance A), or the like, or the binding ability to the binding ability substance A or the detection substance. Preferable examples include antibodies such as concanavalin A, lentil lectin, kidney bean lectin, datura lectin, wheat germ lectin and the like.

As the lectin used in the assay method (6) using the present invention, various lectins such as concanavalin A, lentil lectin, kidney bean lectin, datsura lectin, wheat germ lectin, etc. A substance having a property capable of recognizing the structure may be appropriately selected and used without any particular limitation.

The antibody used for preparing the polypeptide-binding antibody or the labeled anti-glycoprotein antibody used in the assay method (6) of the present invention is an antibody having the above-mentioned properties. , A conventional method, for example, “Introduction to Immunological Experiments, 2nd edition, Nao Matsuhashi et al., Academic Publishing Center, 198.
1) etc. according to the method described in, for example, horse, cow, sheep, rabbit,
Polyclonal antibodies prepared by immunizing animals such as goats, rats, and mice with a measurement target, or by a conventional method, that is, Keller and Milstein (Nature, 256, 495, 1975)
According to the cell fusion method established by, the monoclonal antibody produced by the hybridoma obtained by fusing the cells from the tumor line of the mouse and the splenocytes of the mouse previously immunized with the substance to be measured may be any antibody. It is optional to use these alone or in combination thereof.

Specific examples of glycoproteins that can be fractionally measured by the measuring method (6) using the present invention include biological fluids such as serum, blood, plasma and urine, and biological fluids such as lymphocytes, blood cells and various cells. A lectin which is present in a sample derived from a glycoprotein having different sugar chain structures but substantially the same protein structure, and which recognizes at least one specific sugar chain structure of the glycoprotein to be measured. And an antibody capable of preparing a polypeptide-binding antibody are not particularly limited, and examples thereof include amylase, alkaline phosphatase, acid phosphatase, γ-glutamyl transferase (γ-GTP), lipase. ，
Creatine kinase (CK), lactate dehydrogenase (LD
H), 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.

The concentrations of reagents used in the measuring method (6) of the present invention will be described below. That is, first, the concentration of lectin used varies depending on the type of lectin used, the properties of the glycoprotein to be measured, etc., but is usually 10 times or more the detection limit concentration of the set glycoprotein to be measured, preferably 100.
It is desirable to coexist at a concentration of 2 times or more, more preferably 1000 times or more during the reaction.

The concentration of the polypeptide-binding antibody used varies depending on the detection limit of the glycoprotein to be measured, but the difference in the binding constant between the lectin and the polypeptide-binding antibody to the glycoprotein to be measured. It is desirable to set it after considering. For example, although it varies depending on the glycoprotein to be measured, the type and physical properties of the lectin, the physical properties of the polypeptide-bound antibody, etc., it is desirable to set using the general formula shown below. Polypeptide-binding antibody concentration ≤ (binding constant of lectin) ÷ (binding constant of polypeptide-binding antibody) x (concentration of lectin) In addition, the binding constant according to the above general formula means an equilibrium reaction as shown in the following formula (1). Is a constant obtained by the following equation (2). [A] + [B] ← → [AB] (1) Binding constant = [AB] / ([A] × [B]) (2) [A]: Lectin or poly in equilibrium state Concentration of peptide-bound antibody (M). [B]: Free measurement target glycoprotein concentration (M) in the equilibrium state. [AB]: Concentration (M) of the complex of lectin (or polypeptide-binding antibody) and the glycoprotein to be measured.

More specifically, for example, the binding constant of lectin to the glycoprotein to be measured is 1 × 10 ⁶ M ^-1 , and the binding constant of the polypeptide-binding antibody to the glycoprotein to be measured is 1.
In the case of × 10 ⁸ M ^-1 , the concentration of the polypeptide-binding antibody is 1/100 or less, preferably 1/1000 or less of the lectin concentration. The concentration of the polypeptide-conjugated antibody used is preferably a concentration at which the set detection limit concentration is capable of binding to all of the glycoproteins to be measured, but may be lower than that (for example, about 1/10). .

The concentration of the labeled anti-glycoprotein antibody used is
Although it varies depending on how much the detection limit of the glycoprotein to be measured is set, the concentration in the reaction solution is not less than the concentration that can bind to all of the glycoprotein to be measured at the set detection limit concentration, preferably twice that. It is desirable that the concentration is not less than the concentration, more preferably not less than 5 times the concentration, still more preferably not less than 10 times the concentration. In carrying out the above-described measurement methods (1) to (6) using the present invention, the complex is bound to a free binding substance (including a labeled substance) and a substance to be measured (not labeled). The method of using negative charges instead of HPLC as described above, and the electric charges based on the difference in the negative charges of substances usually used in this field are used. Separation method, for example, an electrokinetic separation method that does not use a separation carrier (Japanese Patent Publication No. 7-111398, etc.), an electrophoresis method that uses a separation carrier, a capillary electrophoresis method, such as a nucleic acid hub reconstitution method. , A method of electrically separating free nucleic acid chains or antibodies after promoting the antigen-antibody reaction by utilizing the difference in charge (International Publication No. WO 95/12808 etc.), etc. may be used. . In addition, of these, carriers used in electrophoresis etc. using a carrier for separation are also carriers usually used in this field (for example, filter paper, such as agar gel, agar gel, agarose gel, polyacrylamide gel, starch gel, etc. , Cellulose acetate membrane, etc.) can be used without particular limitation.

Examples of the detection substance used in the measuring method of the present invention include alkaline phosphatase, β-galactosidase, peroxidase, microperoxidase, glucose oxidase, glucose-used in enzyme immunoassay (EIA). Enzymes such as 6-phosphate dehydrogenase, acetylcholinesterase, malate dehydrogenase, and luciferase, for example, ^99m Tc, ¹³¹ I, ¹²⁵ used in radioimmunoassay (RIA)
Radioisotopes such as I, ¹⁴ C and ³ H, for example, fluorescent substances such as fluorescein, dansyl, fluorescamine, coumarin, naphthylamine or their derivatives used in fluorescence immunoassay (FIA), such as luciferin,
Luminous substances such as isolminol, luminol, bis (2,4,6-trifluorophenyl) oxalate, etc., such as phenol, naphthol, anthracene or derivatives thereof having ultraviolet absorption, eg 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) Examples thereof include substances having a property as a spin labeling agent represented by a compound having an oxyl group such as -p-tolyloxyl, but it goes without saying that the substance is not limited thereto.

As a method of labeling the binding substance with the above-mentioned detection substance, EIA, RIA or F known per se can be used.
Known labeling methods generally used in IA and the like (for example, Medical Chemistry Laboratory Course, Volume 8, edited by Yuichi Yamamura, 1st edition, Nakayama Shoten, 1971; Illustrated fluorescent antibody, written by Kawao Akira, 1
Edition, Soft Science Co., Ltd., 1983; enzyme immunoassay,
Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, 2nd Edition, Medical School, 1982
Etc.) can be mentioned without exception and may be performed 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.

Examples of the binding substance according to the present invention which can be measured (detected) by itself by any method include, for example, an enzyme, a fluorescent substance, a luminescent substance or a substance having absorption in the ultraviolet region. , Which themselves have the properties as a detection substance as described above.

In the measuring method of the present invention, the elution time of the complex is determined by the type of the polypeptide of the present invention (the type and number of acid residues derived from the strong acid to be introduced, the type of constituent amino acid residues, etc.). It can be adjusted freely, so if you use this property,
Separate measurement is also possible.

That is, by using two or more kinds of polypeptides of the present invention having different number of acid residues derived from strong acid and appropriately selecting the kind of the binding substance that binds each polypeptide, It becomes possible to perform simultaneous measurement (separate measurement).

Further, even when there are a plurality of serum components that affect the measurement, if the ionicity of the complex is made larger than that of these serum components by using the polypeptide of the present invention, the serum components will be The effect that the influence on the measurement can be avoided occurs. In this case, by using the stepwise gradient, there is an effect that the time required for the analysis can be shortened.

Furthermore, the chromatography carrier (gel carrier, membrane carrier, glass carrier, etc.) used in the anion exchange method generally has a high exchange capacity (absolute adsorption capacity for ionic substances), so that serum Even when a sample having a large absolute amount of an ionic coexisting substance such as a biological sample is analyzed, the complex bound with the bound substance of the present invention can be completely adsorbed. Can be eluted at a position where the influence of the coexisting substance can be almost avoided. Further, since the polypeptide of the present invention used in the measuring method of the present invention has high solubility in water, the water solubility of the complex to which it is bound is higher than that before binding, so that the measuring method of the present invention In that case, there is almost no possibility that the substance to be measured is denatured or deactivated during the reaction of forming a complex to which the separation improving substance (polypeptide) is bound.

The carrier used in the anion exchange method for separating the target substance and other coexisting substances by utilizing the anionic property of the polypeptide of the present invention has anion exchange ability. The carrier is not particularly limited, and examples thereof include diethylaminoethyl (DEAE).
Group, a carrier having an anion exchange group such as a quaternary ammonium group (eg, Q group, QAE group, etc.), more specifically, for example,
Packing for DEAE-MCI gel (trade name of Mitsubishi Kasei Co., Ltd.), QAE MCI gel (trade name of Mitsubishi Kasei Co., Ltd.), Wako beads DEAE gel (trade name of Wako Pure Chemical Industries Co., Ltd.) for anion exchange chromatography Examples include Memsep DEAE (trade name of Japan Millipo Limited Co., Ltd.) using agents and membranes. It is needless to say that a commercially available carrier for chromatography or a container made of resin or glass and having the above-mentioned anion-exchange group bound to the surface thereof by a conventional method may be used by itself. ..

The method for measuring a biological component using the conjugate of the present invention is an excellent method because it uses a method utilizing negative charges. That is, for example, since it is necessary to use a column having an appropriate length in order to carry out the method for measuring a biological component using the gel filtration method, when the gel filtration method is used, a negative charge is generated. There is a drawback in that the separation time becomes longer than when the method used is used. Therefore, when shortening the separation time is required, a method using a negative charge, for example, an HPLC apparatus equipped with a column packed with a packing material for anion exchange chromatography, an anion exchange membrane, an anion exchange action It is preferable to use the anion exchange method for a machine or the like having an anion exchange action such as a reaction tube having a. Further, the gel filtration method has a drawback that it is not suitable for separating a substance to be measured in a biological sample having a very high molecular weight (molecular size is about 1000 angstrom or more).

Further, in the case of the hydrophobic separation method, when the substance to be measured in the biological sample is a physiologically active substance having a higher-order structure such as protein, the higher-order structure is destroyed by the organic solvent used in the separation operation. As a result, there may occur a problem that the activity of the substance to be measured in the biological sample in the complex is lost.

On the other hand, the anion exchange method can more effectively separate the substance to be measured in the biological sample based on the subtle difference in ionicity. Furthermore, according to the anion exchange method, the polypeptide of the present invention having various ionic properties can be arbitrarily selected, so that the substance to be measured in the biological sample can be separated under the optimum pH condition. . Furthermore, since the polypeptide of the present invention itself has high water solubility, even if it is bound to a substance to be measured in a biological sample, there is almost no risk of precipitation of the substance to be measured in the biological sample, The separation operation can be executed in a stable state.

When the measurement target substance in a biological sample is measured using the measurement method of the present invention, the peak of the measurement target substance in the target biological sample is not affected by components such as serum and urine. Can be moved to a position. Not only that,
By selecting and using the conjugate of the present invention having appropriate properties depending on the substance to be measured in the biological sample, it is possible to match the elution positions of the complexes containing the substance to be measured in various biological samples. Therefore, there is an effect that the substance to be measured in various biological samples can be measured by using the anion exchange method (for example, HPLC) under the same analysis condition.

In the measuring method of the present invention, a detection substance (binding substance, binding substance A or measurement in a biological sample) contained in the complex (labeled complex) separated by the method utilizing negative charge The amount of the target substance) is measured by the detection substance (the binding substance, the binding substance A or the measurement target substance in the biological sample).
Are carried out according to the respective predetermined methods according to the properties that can be detected by some method. For example, when the property is enzymatic activity, a standard method of EIA, for example, “enzyme immunoassay, protein / nucleic acid / enzyme separate volume No. 31, edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, 51-63, Kyoritsu Shuppan Co., Ltd., issued September 10, 1987 ”, etc., and the radiation emitted by the radioactive substance can be measured according to the usual method of RIA when the substance to be detected is a radioactive substance. Depending on the type and strength of the liquid, a liquid immersion type GM counter, a liquid scintillation counter, a well type scintillation counter, a counter for HPLC, etc. can be appropriately selected and used for the measurement (for example, a medical chemistry experiment course,
See Volume 8, edited by Yuichi Yamamura, first edition, Nakayama Shoten, 1971. ). Further, when the property is fluorescent, a conventional method of FIA using a measuring instrument such as a fluorometer, for example, “illustrated fluorescent antibody, written by Kawao Akira, 1st edition, Soft Science Co., Ltd.,
1983 ”and the like according to the method described,
When the property is luminescent, a conventional method using a measuring device such as a photon counter, for example, “enzyme-linked immunosorbent assay, protein / nucleic acid / enzyme separate volume No.31, Tsunehiro Kitagawa / Toshio Minamihara / Akio Tsuji / Eiji Ishikawa, 252 ~ 263 pages, Kyoritsu Shuppan Co., Ltd., September 1987
The measurement may be performed in accordance with the method described in "Issued 10th of a month". Further, when the property is that which 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 substance to be detected has 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, 264-271.
Page, Kyoritsu Shuppan Co., Ltd., published on September 10, 1987 ”and the like.

In the measuring method of the present invention, the substance to be measured in the biological sample and the bound substance of the present invention (bound substances A, B, C, D)
Etc.), if necessary, a reaction condition for forming a complex by reacting with a binding ability substance (including binding ability substance A) that is labeled or not, or a measurement target in a biological sample. The reaction conditions for forming the labeled complex by reacting the substance, the labeled measurement substance, and the bound substance of the present invention are not particularly limited as long as they do not interfere with the formation reaction of the complex (or the labeled complex). Without limitation, conventional methods such as EIA, RIA, FI
A, a method known per se such as affinity chromatography may be used according to the reaction conditions for forming the complex or the like. 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 hinder the formation reaction of the complex (or the labeled complex), but it is usually 2 to 10, preferably 5 to 9. To be The temperature during the reaction is not particularly limited as long as it does not hinder the complex (or labeled complex) forming reaction, but is usually 0 to 50 ° C., preferably 20 to 40.
The range of ° C is preferably mentioned. The reaction time is the time required for the complex (or the labeled complex) to form, the reactivity between the substance to be measured in the biological sample and the bound substance of the present invention, the labeled binding substance, or the like. Since it depends on the reactivity with the bound substance of the invention and the labeled measurement substance, it may be appropriately reacted for several seconds to several hours depending on each property.

As the HPLC used for carrying out the anion exchange method in the measuring method of the present invention, the apparatus itself may be one which is usually used in the field of analysis and which can obtain a constant flow rate. It can be used without any particular problem. When the measuring method of the present invention is carried out using HPLC,
The measurement of the substance to be measured may be performed using the peak area or the peak height.

Anion exchange method, more specifically HPLC
As the solvent (eluent) used when separating the complex (or the labeled complex) or the free labeled binding substance by the method, the formed complex (or the labeled complex) or the like is again used as the biological sample. A complex (or labeled complex) that does not decompose into the substance to be measured or the substance capable of binding to the label inside
There is no particular limitation as long as it does not lose the property of the binding ability substance or the like contained in the substance and the substance to be detected, which can be detected by some method. Usually, for example, EIA, RIA, FIA Those used as buffers in methods known per se such as affinity chromatography are preferably used. Specific examples include, for example, phosphates, acetates, citrates, Good's buffers, buffers such as tris (hydroxymethyl) aminomethane, salts such as sodium chloride, potassium chloride, ammonium sulfate, etc. methanol,
Polar organic solvents such as ethanol, isopropyl alcohol, acetonitrile, tetrahydrofuran, and surfactants are appropriately selected according to the properties of the complex (or labeled complex) or the free labeled binding substance, and then added and mixed. A buffer solution having a pH of 2 to 10 prepared by the above method is preferably used.

By appropriately adding a suitable surfactant to the eluent, tailing of the peak of the complex (or labeled complex) can be prevented and the complex (or labeled complex) or free label can be labeled. The elution position of the binding substance and the like can be adjusted more freely. The surfactant that can be used for such a purpose may be appropriately selected according to the properties of the complex (or the labeled complex), the free label-binding substance, and the like, and the type thereof is not particularly limited. Cationic surfactants such as n-dodecyltrimethylammonium bromide and 1-laurinpyrimidinium chloride, such as lauryl betaine, lauryl amidopropyl betaine, coconut oil fatty acid amidopropyl betaine, 2-alkyl-N-carboxymethyl-N Preferred examples include amphoteric surfactants such as -hydroxyethylimidazolium betaine, 2-undecyl-N-carboxymethyl-N-hydroxyethylimidazolium betaine, and N-lauroyl-N-methyl-β-alanine sodium. Further, the concentration of the surfactant added to the eluent for such a purpose is not particularly limited as long as it is a concentration at which the intended effect is produced, but it may be slightly changed depending on the kind of the surfactant. 0.01-2%, preferably
It is appropriately selected from the range of 0.05 to 1%.

Further, in the measuring method of the present invention, HPLC
As a measurement method after separation by, for example, "Latest Liquid Chromatography, Shoji Hara and Akio Tsuji, First Edition, 92-104"
Page, Nanzandou, published February 1, 1978 ”, etc., and the effluent from the HPLC column is directly guided to the detection section, and is transferred to the complex (or labeled complex) in the effluent. Detected substances included (or substances with binding ability and substances to be measured)
The method of directly measuring the amount of is more preferable because the measurement can be performed quickly. In this case, the binding substance (or the measurement target substance) or the detection substance held by the labeled binding substance or the like (or the labeled measurement substance) has a property that can be detected by some method, For example, if it is enzymatic activity, HP
It goes without saying that it is necessary to provide a so-called post-column method reaction section for adding a reagent for measuring enzyme activity and reacting with the effluent between the LC column and the detection section. When the property of the substance to be detected (or the substance having binding ability or the substance to be measured) is enzymatic activity, the reagent for measuring the enzyme activity used in the reaction part is a conventional method, for example, "enzyme immunoassay,
Protein Nucleic Acid Enzyme Separate Volume No.31, Tsunehiro Kitagawa, Toshio Minamihara
Edited by Akio Tsuji and Eiji Ishikawa, pages 51-63, Kyoritsu Shuppan Co., Ltd., 198
It may be prepared according to the method described in "Published September 10, 1977" or the like, or may be appropriately selected and used from reagents of commercially available clinical test kits. In addition, even when the property of the substance to be detected (or the substance having binding ability or the substance to be measured) is other than enzyme activity, in order to increase the detection sensitivity, a predetermined reagent may be added and reacted with HPL.
Providing a suitable reaction part between the column of C and the detection part is optional.

HP used in the measuring method of the present invention
When a plurality of LC eluents having different components are used, the operation method may be either a concentration gradient method (linear gradient method) or a stepwise method. Is more desirable because it has the advantages of simple operation, shortening the actual analysis time, and sharpening the target peak. Further, the reagent for measuring a substance to be measured in the biological sample of the present invention is a reagent containing a bound substance of the polypeptide of the present invention and a substance having a binding ability to the substance to be measured in the biological sample. In addition, if necessary, for example, a substance to be measured as a label, a substance capable of binding with a label,
It appropriately contains a buffering agent, a surfactant and the like. still,
The preferable modes, specific examples, etc. of the respective constituent elements are as described above. Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

[0068]

EXAMPLES The abbreviations used in the examples are as follows. Ala; alanine, βAla; β-alanine, Ser; serine, Ty
r; tyrosine, Asp; aspartic acid, Fmoc; 9-fluorenylmethoxycarbonyl group, Alko; p-alkoxybenzyl alcohol, tBu; t-butyl group, TFA; trifluoroacetic acid, BOP; benzothiazol-1-yl- Oxy-tris (dimethylamino) phosphonium hexafluorophosphate, HOBt; N-hydroxy-1H-benzotriazole, DM
F; N, N-dimethylformamide, DIEA; N, N-diisopropylethylamine.

Example 1. Synthesis of Ala- (Ser (SO ₃ H)) ₅ -βAla (Polypeptide 3) (1) Synthesis of Fmoc-Ala- (Ser) ₅ -βAla Fmoc-βAla-Alko resin (100-200mesh, Watanabe Chemical Industry
Co., Ltd.) 2.3 g (1.5 mmol as βAla) as a starting material,
According to the method (BOP / HOBt method) described in the literature (J. Org. Chem., 53 , 617-624 (1988)), it was synthesized by the solid phase method. That is, first, Fmoc-βAla-Alko resin was treated with piperidine to remove the Fmoc group. To this, a DMF solution containing 3 times equivalent of a given amino acid with respect to βAla on resin, 3 times equivalent BOP, 3 times equivalent HOBt and 5.3 times equivalent DIEA were added,
Amino acids were sequentially introduced by an operation of performing a coupling reaction (twice) at room temperature for 2 hours. The order of the introduced amino acids is Fmoc-Ser (tBu) (manufactured by Wako Pure Chemical Industries, Ltd.), Fmoc-
Ser (tBu), Fmoc-Ser (tBu), Fmoc-Ser (tBu), Fmoc-Ser (t
Bu) and Fmoc-Ala (manufactured by Wako Pure Chemical Industries, Ltd.). After introducing all amino acids, the resin was washed with MeOH, and TFA: anisole (9
5: 5) Add 20 ml of the mixed solution and stir at room temperature for 1 hour to react.
At the same time that the desired polypeptide was cleaved from the resin, the tBu group (a protecting group for the Ser hydroxyl group) was cleaved. After the reaction,
The resin was filtered off, the filtrate was concentrated under reduced pressure and ether was added to precipitate the reaction product. The precipitate was collected and dried with a desiccator to obtain 0.84 g of Fmoc-Ala- (Ser) ₅ -βAla. (2) Synthesis of Ala- (Ser (SO ₃ H)) ₅ -βAla (Polypeptide 3) Fmoc-Ala- (Ser) ₅ -βAla 547 mg (0.67 mm) synthesized in (1)
ol) is dissolved in 40 ml of DMF, and further 30 ml of DMF ・ SO ₃ solution [DMF ・ SO ₃
2.57 g (Fluka) DMF: pyridine (4: 1) mixed solution 30 ml
Dissolved in.] Was added and reacted at 4 ° C. overnight. The same amount of DMF · SO ₃ solution was added to the reaction solution, and the mixture was further reacted at 4 ° C. overnight. After the reaction was completed, the reaction solution was added to 400 ml of acetone, the formed precipitate was collected by filtration, the precipitate was dissolved in 40 ml of DMF, 8 ml of piperidine was added, and the mixture was stirred at room temperature for 40 minutes for reaction.
The c group was cleaved. The reaction solution was added to 500 ml of acetone, and the formed precipitate was collected by filtration, washed with acetone and ether, and dried with a desiccator. The obtained precipitate was subjected to anion exchange chromatography and then gel filtration to obtain Ala- (Ser (SO ₃ H)) _5-.
210 mg of βAla (polypeptide 3) was obtained. The results of amino acid analysis and ion chromatography of the reaction product are shown in Table 1. The amino acid analysis was carried out by the Wako PTC-amino acid analysis system (manufactured by Wako Pure Chemical Industries, Ltd.) (hereinafter the same).

Example 2. Synthesis of Ala-Tyr (SO ₃ H) ₃ -βAla (Polypeptide 11) Using 0.77 g (0.5 mmol) of Fmoc-βAla-Alko resin as a starting material, the same reagent as in Example 1 (1) was used, and the same procedure was performed. Was carried out to introduce a predetermined amino acid. The order of the introduced amino acids was Fmoc-Tyr (SO ₃ Na) (Bachem), Fmoc-Tyr (SO
₃ Na), Fmoc-Tyr (SO ₃ Na), and Fmoc-Ala. After the reaction was completed, the resin was washed with MeOH, and a DMF: piperidine (4: 1) mixed solution 50 was added.
ml was added and the reaction was stirred at room temperature for 1 hour to cleave the Fmoc group. After removing the solvent by filtration and washing the resin with MeOH, TF
A: H ₂ O: m-cresol (45: 5: 2) was added, and after substitution with nitrogen gas, the mixture was reacted with stirring at 4 ° C. for 16 hours to cleave the desired polypeptide from the resin. The resin was filtered off from the reaction solution, the filtrate was concentrated under reduced pressure, and the reaction product was precipitated with ether. Precipitation
It was subjected to anion exchange chromatography and then gel filtration to obtain 180 mg of Ala-Tyr (SO ₃ H) ₃ -βAla (polypeptide 11). The results of amino acid analysis and ion chromatography of the reaction product are also shown in Table 1.

Example 3. Synthesis of Ala- (Tyr (SO ₃ H)) ₅ -βAla (Polypeptide 14) (1) Synthesis of Fmoc-Ala- (Tyr) ₅ -βAla Fmoc-βAla-Alko Resin 2.3 g (1.5 mmol) as a starting material Then, the same reagent as in Example 1 (1) was used and the same operation was performed to introduce a predetermined amino acid. The order of the introduced amino acids is Fmoc-Tyr (tBu) (Watanabe Chemical Co., Ltd.), Fmoc-Tyr (tB
u), Fmoc-Tyr (tBu), Fmoc-Tyr (tBu), Fmoc-Tyr (tBu), F
It was moc-Ala. After the reaction is completed, the resin is washed with MeOH and TF
A: A mixture of thioanisole: 1,2-ethanedithiol (95: 5: 1) was added, and the mixture was stirred and reacted at room temperature for 1 hour to cleave the polypeptide from the resin and simultaneously cleave the tBu group (protecting group for the hydroxyl group of Tyr). Let After the reaction was completed, the resin was filtered off, the obtained filtrate was concentrated under reduced pressure, and ether was added to precipitate the reaction product. The precipitate was collected by filtration, dried in a desiccator, and Fmoc-Ala- (Tyr) ₅ -βAl.
a 1.71 g was obtained. (2) Synthesis of Ala- (Tyr (SO ₃ H)) ₅ -βAla (Polypeptide 14) 0.5 g of Fmoc-Ala- (Tyr) ₅ -βAla obtained in (1) was added to ₃ ml of DMF.
15 ml of DMF ・ SO ₃ solution (DMF ・ SO ₃ 3.2 g
MF: pyridine (dissolved in 15 ml of 4: 1)) was added and 4
After reacting at 0 ° C. overnight, ether was added to precipitate the reaction product. The precipitate was dissolved in 10 ml of DMF, 2.5 ml of piperidine was added, and the mixture was reacted with stirring at room temperature for 1 hour. Ether in the reaction solution
After adding 150 ml, the precipitate formed was filtered off. The obtained precipitate was dissolved in 4 ml of water, and the reaction product was fractionated by ODS column liquid chromatography [column: Wakosil ₁₀ C
₁₈ (2.0φx25cm) (manufactured by Wako Pure Chemical Industries, Ltd.), elution conditions:
10mMAcONa (pH6.0), 2 → 60% acetonitrile]. The obtained fraction containing the desired product was further processed by gel filtration to obtain Ala- (Tyr
(SO ₃ H)) ₅ -βAla (polypeptide 14) 203 mg was obtained. The results of amino acid analysis and ion chromatography of the reaction product are also shown in Table 1.

Example 4. Synthesis of Sulfated Polypeptides (Polypeptides 1, 2, 4-9, 18 and 20-22) Using the same reagents as in Example 1, the same operation was carried out and Table 1
Polypeptides 1,2,4-9,18 and 20-22 described in 1. were synthesized. In addition, the same reagents as in Example 2 were used and the same operation was performed to synthesize the polypeptide 10 shown in Table 1. Fmoc-Ser was used as the starting material for the synthesis of polypeptide 6.
Fmoc-Tyr was used as a starting material when synthesizing polypeptides 21 and 22 using (tBu) -O-polymer (manufactured by Kokusan Kagaku Co., Ltd.).
(tBu) -Alko Resin (100-200 mesh, Watanabe Chemical Co., Ltd.
Manufactured) was used. The results of amino acid analysis and ion chromatography of the obtained various polypeptides are also shown in Table 1.

Example 5. Synthesis of Sulfated Polypeptides (Polypeptides 12, 13, 15 to 17 and 19) Using the same reagents as in Example 3, the same procedure was carried out and Table 1
Polypeptides 12, 13, 15-17 and 19 described in 1. were synthesized.
Fmoc-βAla-Alko resin (100-200mesh, Watanabe Chemical Co., Ltd.) was used as a starting material for synthesizing the polypeptides 12 and 16.
Fmoc-Tyr (tBu) -Alko Resin (1
00-200 mesh, manufactured by Watanabe Chemical Industry Co., Ltd. was used. The results of amino acid analysis and ion chromatography of the reaction product are also shown in Table 1.

Example 6. Synthesis of Ala- (Tyr (PO ₃ H ₂ )) ₅ -βAla (Polypeptide 23) Fmoc-βAla-Alko Resin 780 mg (0.5 mmol) was used as a starting material, and the same reagents as in Example 1 (1) were used. Then, a predetermined amino acid was introduced. The order of the introduced amino acid _{Fmoc-Tyr (PO 3 H 2} ) (Nova biochem Inc.), Fmoc-
Tyr (PO ₃ H ₂ ), Fmoc-Tyr (PO ₃ H ₂ ), Fmoc-Tyr (PO ₃ H ₂ ), Fmoc
-Tyr (PO ₃ H _2), was Fmoc-Ala. After the reaction is completed, the resin is MeO
After washing with H, 50 ml of DMF: piperidine (4: 1) mixture was added, and the mixture was reacted with stirring at room temperature for 1 hour to cleave the Fmoc group.
After the reaction was completed, the resin was collected by filtration, washed with MeOH, and then mixed with TFA: phenol: H ₂ O: thioanisole: ethane (33: 2: 2: 2: 1).
The polypeptide was cleaved from the resin by adding 20 ml and stirring the mixture at room temperature for 1 hour. After the reaction was completed, the resin was filtered off and ether was added to the filtrate to precipitate the reaction product. The obtained precipitate was subjected to anion column chromatography and gel filtration to obtain Ala- (Tyr (PO ₃ H ₂ )) ₅ -βAla (polypeptide 2
3) Obtained 760 mg. The results of amino acid analysis and ion chromatography of the reaction product are also shown in Table 1.

Example 7.4 4-Maleimidobutyryl-Ala- (Tyr (PO
Synthesis of ₃ H ₂ )) ₅ -βAla (Polypeptide 24) Starting from 780 mg (0.5 mmol) of Fmoc-βAla-Alko Resin as a starting material, the same reagent as in Example 1 (1) was used and the same operation was performed to give a predetermined product. Was introduced. The order of the introduced amino acid _{Fmoc-Tyr (PO 3 H 2} ) (Nova biochem Inc.), Fmoc-
Tyr (PO ₃ H ₂ ), Fmoc-Tyr (PO ₃ H ₂ ), Fmoc-Tyr (PO ₃ H ₂ ), Fmoc
_{-Tyr (PO 3 H 2),} Fmoc-Ala, was maleimidobutyryl acid. After the reaction was completed, the resin was washed with MeOH and treated with a TFA: anisole (95: 5) mixture to cleave the polypeptide from the resin. The resin was filtered off, and ether was added to the obtained filtrate to cause precipitation. The obtained precipitate was subjected to ODS column [column: Wakosil ₅ C ₁₈ (2.0φx25 cm) (manufactured by Wako Pure Chemical Industries, Ltd.), elution conditions: 0.1% TFA, 0 → 10% acetonitrile], and then subjected to gel filtration. , 4-Maleimidvutilyl-Ala- (Tyr
(PO ₃ H ₂ )) _5- βAla (polypeptide 24) 820 mg was obtained. The results of amino acid analysis and ion chromatography of the reaction product are also shown in Table 1.

[0076]

[Table 1]

Example 8. Examination of Elution Position of Anionic Polypeptide on Anion Exchange Column (Sample) An aqueous solution containing 5 mg / ml of each of the polypeptides 1 to 24 obtained in Examples 1 to 7 was used as a sample. As a control, (Asp) ₉ -βAla (hereinafter abbreviated as “Asp9”. BOP / HOBt
Made by law. ), Polyaspartic acid having an average molecular weight of 6000 (hereinafter abbreviated as “Asp6K”, manufactured by Sigma) or polyaspartic acid having an average molecular weight of 50,000 (hereinafter “pAs”).
Abbreviated as "p". An aqueous solution containing 5 mg / ml of Sigma) was also prepared as a sample. (Analysis conditions) Column: POROS-DEAE (4.6φ x 10 mm, manufactured by Perceptive). Eluent A: 50 mM phosphate buffer (pH 7.6). Eluent B: 50 mM phosphate buffer (pH 7.6, containing 5 M NaCl). Flow rate: 1 ml / min. Detection: For polypeptides containing sulfated serine residues; UV 220 nm. Polypeptide containing sulfated tyrosine residue; UV260n
m. Polypeptide containing phosphorylated tyrosine residue; UV26
0 nm. Gradient conditions: 0 → 5 min A = 100% 5 → 30 min B = 0 → 100% 30 → 35 min B = 100% (Measurement procedure) A 20 μl sample was analyzed by HPLC under the above conditions. (Result) Salt concentration required for elution of each polypeptide (hereinafter,
It is abbreviated as “eluting salt concentration”. ) Is shown in FIG. Incidentally, FIG.
In the figure, ● indicates the salt concentration at the top of the elution peak, and I indicates the range of the salt concentration at the beginning and the end of elution, that is, the peak width. Further, each symbol and number on the horizontal axis of the figure indicates the type of polypeptide (polypeptide No. and abbreviation in Table 1). The following can be seen from the results shown in FIG. It can be seen that the concentration of eluting salt is increased by increasing the number of acid residues (sulfate group, phosphate group) derived from strong acid. Polypeptides 3 (sulfate residue number: 5) and 11 (sulfate residue number: 3) showed the same eluted salt concentration as Asp9, and polypeptide 6 (sulfate residue number: 10), 8 (sulfate residue number) : 9), 9 (sulfate residue number: 10), 12, 13 (sulfate residue number: 4) and 14,15
(Sulfate residue number: 5) exhibits an elution salt concentration equivalent to that of Asp6K or pAsp, in other words, the polypeptide of the present invention
Shorter chain length (smaller number of amino acid residues) than a conventional polypeptide having a carboxylic acid residue, and equivalent effect (effect that the degree of retention on anion exchange column is equivalent) ) Is obtained. The degree of strength retained in the anion exchange column varies depending on the type and number of acid residues derived from a strong acid, the type of amino acid residue into which the acid residue is introduced, the properties of the column packing material, and the like. For example, comparing the results for polypeptides 2 to 9 having a sulfated serine residue with the results for polypeptides 10 to 20 having a sulfated tyrosine residue, when the number of acid residues derived from a strong acid is the same, It can be seen that the polypeptide having a sulfated tyrosine residue has a higher concentration of eluted salt.
It should be noted that this result indicates that since the base material of the column packing material used this time is slightly hydrophobic, the polypeptide having a tyrosine residue having a benzene ring is more likely to be retained on the column, and the result is apparent. It is probable that the dissolved salt concentration of was increased. In addition, comparing the results for the polypeptides 3 and 14 having 5 sulfate residues with the results for the polypeptides 23 and 24 having 5 phosphate residues, the polypeptide having a sulfate residue is the eluted salt. It can be seen that the concentration becomes high. From these results, by appropriately selecting the type and number of acid residues to be introduced, the type of constituent amino acid residues, the type of column packing, etc., it is possible to select a polypeptide having any concentration of eluted salt. I understand. The type and number of amino acid residues that introduce an acid residue derived from a strong acid, and even if the type and number of the acid residues are the same, due to the presence of other amino acid residues in the polypeptide, an anion exchange column The degree of strength held to varies.
For example, a polypeptide having a sulfated tyrosine residue 12-
From the results of 19, even if the number of sulfated tyrosine residues is the same, β-
The concentration of eluted salt varies depending on the presence or absence of an alanine residue (β
-Introducing an alanine residue lowers the concentration of eluted salt). From this result, it can be seen that a polypeptide having an arbitrary elution salt concentration can be prepared by introducing an appropriate amino acid residue in addition to the amino acid residue into which the acid residue has been introduced. Comparative examples pAsp-1 and pAsp-2 (pAs of different production lots
From the results obtained by p), in the case of a polypeptide having a conventional carboxylic acid residue, the concentration of eluted salt changes depending on the production lot, in other words, the tailing situation of the target peak changes, that is, it is uniform. It turns out that it is difficult to obtain a polypeptide having a molecular weight. In contrast,
Since the polypeptide of the present invention can be easily obtained in a homogeneous form by peptide synthesis, such a problem in pAsp does not occur.

Example 9 Preparation of antibody-sulfated polytyrosine conjugate (1) 4- (p-maleimidophenyl) butyryl Ala- (Tyr (SO ₃
H)) ₈ -βAla Preparation Ala- (Tyr (SO ₃ H)) ₈ -βAla 1 mg prepared in Example 4
Dissolve in 500 μl of 1M phosphate buffer (pH 7.0), add 1.2 mg of sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate (manufactured by Pierce Chemical Co.) to this, and let react at 37 ° C. for 3 hours. It was The reaction solution Superdex peptide column: treated with (16 mm ID × 30 cm manufactured by Pharmacia), except for the excess reagent 4-(p-maleimido-phenyl) butyryl _{Ala- (Tyr (SO 3 H)} ) 8 - An aqueous solution of βAla 0.84 mg was obtained (yield 75%). (2) Preparation of Fab ′ fragment Anti-AFP monoclonal antibody A4-4 (hereinafter referred to as “AFP-
A4-4 ”is abbreviated. Wako Pure Chemical Industries, Ltd.) 10 mg was treated according to a conventional method to give an F (ab ') ₂ fragment (5 m
g. (Yield 80%), and further processed into a Fab '
3.1 mg of the fragment (hereinafter abbreviated as "AFP-A4-4.Fab '") was obtained (yield 62%). (3) Preparation of a conjugate of Ala- (Tyr (SO ₃ H)) ₈ -βAla and AFP-A4-4.Fab '4- (p-maleimidophenyl) butyryl Al obtained in (1) above
3.1 mg of a- (Tyr (SO ₃ H)) ₈ -βAla and AFP-A obtained in (2) above
4-4.Fab '3.1mg and 0.1M phosphate buffer (pH 7.0),
The reaction was carried out at 4 ° C for 16 hours. This reaction solution was applied to a Superdex 200 pg column (26 mm ID x 60 cm: manufactured by Pharmacia) and excess 4- (p-maleimidophenyl) butyryl was added.
Ala- (Tyr (SO ₃ H)) ₈ -βAla was removed and DEAE TOYOPEARL
Ala- (Tyr (SO ₃ H)) ₈ -βAla and AFP-A4-4 ・ F were treated by a column (10 mm ID x 2 cm: manufactured by Tosoh Corp.) to collect the adsorbed fraction.
1 mg of a product bound to ab 'was obtained (15% yield).

Example 10. Preparation of antibody-sulfated polytyrosine conjugate (1) 4- (p-maleimidophenyl) butyryl Ala- (Tyr (SO ₃
Preparation of H)) ₈ Ala- (Tyr (SO ₃ H)) ₈ (Polypeptide 1 prepared in Example 4)
9) 25 mg was dissolved in 3 ml DMF, 10 mg of sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate (manufactured by Pierce Chemical Co.) was added thereto, and the mixture was reacted at room temperature for 1 hour. This reaction mixture was added to an ODS column [column: Wakosil ₅ C
₁₈ (2.0φx25cm) (manufactured by Wako Pure Chemical Industries, Ltd.), elution conditions:
50 mM ammonium acetate pH6, 2 → 60% acetonitrile], and the obtained fraction containing the desired product was concentrated to dryness and 4- (p-maleimidophenyl) butyryl Ala- (Tyr (SO ₃ H)) ₈ 26.5 mg was obtained (yield 95%). Obtained 4- (p-maleimidophenyl)
The NMR data of butyryl Ala- (Tyr (SO ₃ H)) ₈ is shown below. ¹ H-NMR (270 MHz, DMSO-d ₆ ) δppm: 7.16 (s, 2H maleimidproton) In comparison with the results of Example 9 (1), this method introduced a maleimide group at the N-terminal. It is understood that the obtained polypeptide can be obtained in good yield. Furthermore, 4- (p-maleimidophenyl) butyryl Ala- (T
It was also found that yr (SO ₃ H)) ₈ can be stably stored without being decomposed if stored at 15 ° C. or lower. Therefore, yr (SO ₃ H) ₈ obtained by the method of Example 9 (1) (in an aqueous solution state) It decomposes in about a day.) (2) Preparation of Fab ′ fragment Anti-AFP monoclonal antibody A4-4 (hereinafter referred to as “AFP-
A4-4 ”is abbreviated. 30 mg manufactured by Wako Pure Chemical Industries, Ltd. was treated according to a conventional method to give an F (ab ') ₂ fragment (16
mg. (Yield 80%), and further processed into a Fab '
11.1 mg of the fragment (hereinafter abbreviated as "AFP-A4-4.Fab '") was obtained (yield 70%). (3) Preparation of a conjugate of Ala- (Tyr (SO ₃ H)) ₈ and AFP-A4-4 · Fab ′ 4- (p-maleimidophenyl) butyryl Al obtained in (1) above
_{a- (Tyr (SO 3 H)} ) and ₈ 1 mg, the (2) AFP-A4-4 · Fab obtained in '
11.1 mg was reacted with 50 mM phosphate buffer pH 6.5 at 4 ° C. for 16 hours. This reaction solution was added to the POROS DEAE column (6 mmI
D × 1 cm: manufactured by Perceptive) with Ala- (Tyr (SO ₃ H)) ₈
The bound product with AFP-A4-4.Fab 'was fractionated to obtain 6 mg (yield 60
%). From the comparison between this result and the result of Example 9 (3),
It can be seen that a conjugate of the polypeptide of the present invention and Fab 'can be efficiently obtained by using the polypeptide having the maleimide group introduced at the N-terminal obtained by the method of Example (1). Although the reason for this is not clear, in Example 9 (1), the removal of free sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate was carried out using a Superdex peptide column,
In this Example (1), it is considered that this is because the ODS column is used. That is, the peptide of the present invention having a maleimide group introduced therein and free sulfosuccinimidyl
Due to the small difference in the molecular weights of -4- (p-maleimidophenyl) butyrate, it was not possible to separate them sufficiently on the Superdex peptide column, and free sulfosuccinimidyl- 4- (p-maleimidophenyl) butyrate reacted with Fab ', resulting in Ala- (Ty
It is considered that the yield of the bound product of r (SO ₃ H)) ₈ -βAla and AFP-A4-4 · Fab ′ was reduced.

Example 11. Examination of Elution Position of Anion Exchange Chromatography of Conjugate of Anionic Polypeptide and Antibody (Sample) Using the same reagent as in Example 9, the same operation was performed to obtain various anionic polypeptides and AFP-described in Table 1. A4
A -4.Fab'-bound product was prepared, and an aqueous solution containing this at 1 mg / ml was used as a sample. However, since the polypeptide 24 has a maleimide group bonded to the N-terminus, 4- (p-maleimidophenyl) butyrate treatment is not performed, and AFP-A4-4.
Combined with Fab '. As a control, commercially available pAsp (2L
ot) was also subjected to the same operation using the same reagents as in Example 9 to obtain a conjugate with AFP-A4-4.Fab ′, which was 1 mg / ml.
An aqueous solution containing it was prepared and used as a sample. As for the polypeptide 22, the desired bound product with AFP-A4-4 · Fab ′ was not obtained. This is presumably because the presence of an anionic amino acid residue at the N-terminus of the polypeptide reduces the efficiency of binding to the antibody (more precisely, the binding between the polypeptide and the cross-linking agent) due to a charge. (Analysis conditions and measurement operation) The same procedure as in Example 8 was performed. However, the detection was performed at UV280 nm in all cases. (Results) FIG. 2 shows the eluted salt concentrations and peak widths of the bound products of various polypeptides and AFP-A4-4.Fab ′. Incidentally, FIG.
In the figure, ● indicates the salt concentration at the apex of the elution peak, and I indicates the range of the salt concentration at the beginning and the end of elution (that is, the peak width). Further, each symbol on the horizontal axis of the figure indicates the type of polypeptide (polypeptide No. and abbreviation in Table 1). The following can be seen from the results shown in FIG. By binding with an antibody, the elution salt concentration is slightly lower than that of the polypeptide alone, but the relative relationship between various polypeptides and the elution salt concentration of pAsp is shown in Example 8.
It turns out that it is the same as that obtained in. Although not shown in Fig. 2, POROS-DEAE (4.6φx10mm,
When the serum components were analyzed under the analysis conditions of Example 8 using an anion exchange column), the salt concentration at the peak of the elution peak of the coexisting substance in the serum was around 0.3 M. From this, when the conjugate of polypeptide 1 (sulfate residue number: 1) and the antibody is used as the separation-improving substance, the target peak overlaps with the peak of the coexisting substance in the serum and the It was found that the accuracy was reduced. From the results of FIG. 2, although not as high as that of polypeptide 1, polypeptide 2 (sulfate residue number: 3), polypeptide 10 (sulfate residue number: 1)
It is also suggested that the accuracy of measurement (analysis) may be slightly reduced by the effect of coexisting substances in serum even when a conjugate of polypeptide 11 (sulfate residue number: 3) and antibody is used as a separation improving substance. Was done. In addition, POR which is the filler used this time
Since OS-DEAE has a slightly hydrophobic base material, the conjugate between the polypeptide 10 to 20 having a sulfated tyrosine residue and the antibody is more likely to be a polypeptide having a sulfated serine residue. The apparent dissolved salt concentration is higher than that of the bound substance with the antibody. Therefore, the apparent elution salt concentration of the conjugate of the antibody 10 with the polypeptide 10 having only one sulfate residue but having a tyrosine residue is the polypeptide 2 having three sulfated serine residues. And the degree of binding to the antibody. However, when the filler used was changed to one having a hydrophilic base material and the same experiment as above was carried out, a conjugate product of the polypeptide 10 having only one sulfated tyrosine residue and the antibody was formed. The apparent elution salt concentration of was similar to that of the conjugate of Polypeptide 1 having only one sulfated serine residue and the antibody, and the peak of the coexisting substance in serum was measured (analysis). Is expected to decrease in accuracy. From these results, it is also found that a polypeptide having at least 3, preferably 4 or more, more preferably 5 or more acid residues derived from a strong acid is desirable as the separation improving substance.

Example 12 Examination of stability of sulfated serine-containing polypeptide and sulfated tyrosine-containing polypeptide in aqueous solution Ala- (Ser (SO ₃ H)) ₈ -βAla (polypeptide 4) or Ala- (T
yr (SO ₃ H)) ₅ -βAla (polypeptide 14) was stored at 40 ° C in a buffer solution of pH 6 to 10 and stability was examined. (Sample) Polypeptide 4 or 14 was dissolved in each of the following buffers to a final concentration of 1 mg / ml, and used as a sample. Buffer: pH 6.0 2-morpholinoethanesulfonic acid (MES). pH 7.0 3-Morpholinofluorosulfonic acid (MOPS). pH 8.0 N-tris (human oxymethyl) methyl-3-aminopropylsulfonic acid (TAP
S). pH 9.0 TAPS. pH 10.0 N-Cyclohexyl-2-human oxy-3-aminopropylsulfonic acid (CAPS
O). The buffer concentration was 50 mM in all cases. (Storage method) Each sample was stored at 40 ° C for a predetermined number of days. (Results) Polypeptide residual ratio (%) in the sample on the 6th and 19th days of storage was determined by the analysis conditions and the operating method of Example 8, and the peak area of the polypeptide in the sample immediately after preparation And the peak area of the polypeptide in the sample after storage for a predetermined number of days. The measurement results for polypeptide 4 are shown in FIG. 3, and the measurement results for polypeptide 14 are shown in FIG. In each figure, the black bar indicates the result obtained for the sample on the 6th day of storage, and the white bar indicates the result obtained for the sample on the 19th day of storage. From the results of FIG. 3, Polypeptide 4 was the most stable in that its decomposition was promoted as the pH during storage increased.
It can be seen that, even at pH 6, 10% or more is decomposed after storage for 16 days. On the other hand, from the results of FIG.
It can be seen that 14 is stable at all pH. From these results, the sulfate group introduced into the tyrosine residue is stable and less susceptible to pH than the sulfate group introduced into the serine residue, that is, it has more preferable properties when used as a separation improving substance. You know that you have.

Example 13 TSH (Thyroids
Measurement of stimulating hormone (thyroid stimulating hormone) (peroxidase-labeled anti-TSH antibody F)
Preparation of ab 'fragment) An anti-TSH antibody (hereinafter abbreviated as "TSH-1", manufactured by Wako Pure Chemical Industries, Ltd.) is treated by a conventional method to obtain a Fab' fragment, which is then treated with a peroxidase (a conventional method). (Toyobo Co., Ltd.) combined with peroxidase labeled anti-TSH
Antibody Fab 'fragment (hereinafter referred to as "TSH-1.Fa
b'-POD "is abbreviated. ) Was prepared. (Antibody solution 1) 50m containing 5 nM of TSH-1 / Fab'-POD
An M MOPS buffer solution (pH 7.5) was prepared and used as antibody solution 1. (Antibody solution 2) An anti-TSH monoclonal antibody (hereinafter abbreviated as "TSH-2", which has been confirmed to have a recognition site different from that of TSH-1. Wako Pure Chemical Industries, Ltd.) was treated by a conventional method. Fab 'fragment (hereinafter referred to as "TSH-2.Fa"
b '"is abbreviated. ), And this and Ala- (Tyr
A conjugate with (SO ₃ H)) ₅ -βAla (polypeptide 14) or Ala- (Ser (SO ₃ H)) ₅ -βAla (polypeptide 3) was prepared in the same manner as in Example 9 (3). Prepared. 5 containing 50 nM of any of these
Prepare 0 mM MOPS buffer (pH 7.5),
Antibody solution 2 was used. (Sample) Commercially available TSH (manufactured by genzyme) was added with 50 mM MO.
A sample was prepared by adding 70 pM to a PS buffer solution (pH 7.5, containing 0.5% bovine serum albumin). (Use condition of HPLC) Column: 0.46 φ × 1.0 cm. Filler: POROS-DEAE gel (trade name of Perceptive Co.). Eluent A: 50 mM MOPS buffer pH 7.5. Eluent B: 50 mM MOPS buffer pH 7.5,
3M sodium chloride. Substrate solution: 25 mM 4-N-acetylaminophenol (Dojindo Laboratories Co., Ltd.) aqueous solution. Flow rate: eluent A + eluent B; 1.0 ml / min, substrate solution; 0.1 ml / min. Reaction part: 0.025 φ × 1000 cm (60 ° C. heat retention). Detection: Measured at an excitation wavelength of 328 nm and a fluorescence wavelength of 432 nm. Gradient conditions: 0 → 10 min B = 0 → 100% (measurement operation) 100 μl of antibody solution 1, 50 μl of sample and 2 5 of antibody solution
After mixing 0 μl and leaving it at 25 ° C. for 30 minutes, 10 μl of the mixed solution was measured (analyzed) by HPLC under the above conditions. (Results) As a result of HPLC analysis, the concentration of eluted salt (concentration of sodium chloride) of each substance was as follows.・ TSH-1 ・ Fab'-POD and TSH-1 ・ Fa
Complex of b′-POD and TSH: 0-0.1M.・ TSH-1 ・ Fab'-POD, TSH, and TSH
-2 · Fab 'and _{Ala- (Tyr (SO 3 H)} ) 5 -
Complex with βAla (polypeptide 14) conjugate:
0.5-1.2M.・ TSH-1 ・ Fab'-POD, TSH, and TSH
-2 · Fab 'and _{Ala- (Ser (SO 3 H)} ) 5 -
Complex with βAla (polypeptide 3) conjugate: 0.
25-0.45M. From the above results, it becomes possible to more clearly separate the coexisting free POD-labeled antibody and the complex containing the substance to be measured by using the conjugate of the sulfated polypeptide and the antibody. It is also understood that the elution position of the target complex can be freely adjusted by appropriately selecting the type of sulfated polypeptide. It was also found that a good calibration curve for TSH in the sample can be obtained by using the peak of the complex bound with the sulfated polypeptide, in other words, the amount of TSH can be quantified.

Example 14 Measurement of AFP using a conjugate of Fab 'and anionic polypeptide (preparation of peroxidase-labeled anti-AFP antibody Fab' fragment) Anti-A recognizing an epitope different from AFP-A4-4
FP antibody / WA-1 (hereinafter abbreviated as “AFP-WA-1”; manufactured by Wako Pure Chemical Industries, Ltd.) was treated by a conventional method to obtain a Fab ′ fragment, which was then treated with a peroxidase (TOYOBO) by a conventional method. Peroxidase-labeled anti-AFP antibody Fab ′ fragment (hereinafter referred to as “AF”).
P-WA-1 · Fab′-POD ”is abbreviated. ) Was prepared. (Reagent) 200 nM and AFP-WA-1 * F of the predetermined thing among the binding substances of the various anionic polypeptides shown in Table 1 and AFP-A4-4 * Fab 'prepared in Example 11 were used.
M containing 100 nM of ab'-POD and 0.2 (W / V)% of polyvinyl alcohol (manufactured by Aldrich).
An OPS buffer (pH 7.5) was prepared and used as a reagent. (Sample) Commercially available AFP was used as a 50 mM MOPS buffer (pH 7.
5, 0.2 (W / V)% polyvinyl alcohol) was added and dissolved at 100 ng / ml to give a sample. (HPLC conditions) Column: POROS-DEAE (4.6φ x 10 m
m). Eluent A: 50 mM MOPS buffer (pH 7.5). Eluent B: 50 mM MOPS buffer (3M NaCl
Inclusion. pH 7.5). Substrate solution: 50 mM MOPS buffer (90 mM 4-N
-(4-carbobutyryl) aminophenol and 20 m
Contains MH ₂ O ₂ . pH 7.5). Flow rate: A + B solution 1 ml / min (substrate solution 0.1 m
l / min). Reaction part: 0.025φ × 1000 cm. Temperature: 60 ° C. Detection: excitation wavelength 328 nm / fluorescence wavelength 432 nm. Gradient conditions: the same as in Example 8. (Measurement method) 100 μl of the reagent and 10 μl of the sample were mixed, and the mixture was mixed at 8 ° C. for 10
After reacting for 20 minutes, add 20 μl of the mixed solution to HP under the above conditions.
Analyzed by LC. (Results) Conjugates of various polypeptides with AFP-A4-4.Fab ', AFP-WA-1.Fab'-POD and AFP
FIG. 5 shows the eluted salt concentration and peak width of the complex with (antigen-antibody complex). In FIG. 5, ● indicates the salt concentration at the peak of the elution peak, and I indicates the range of the salt concentration at the beginning and the end of the elution (that is, the peak width). Further, each symbol on the horizontal axis of the figure indicates the type of polypeptide (polypeptide No. and abbreviation in Table 1). The following can be seen from the results shown in FIG.
Elution salt concentration of the antigen-antibody complex (elution position) depending on the type of polypeptide bound to AFP-A4-4.Fab '
Changes. The order of elution of each antigen-antibody complex was the same as the order of elution of the bound product of AFP-A4-4.Fab ′ and the polypeptide (see Example 11 and FIG. 2). From this result, it is also understood that the concentration of the eluted salt of the target antigen-antibody complex can be appropriately set by appropriately selecting the type of polypeptide. Free AF
The elution salt concentration of P-WA-1 · Fab′-POD is 0 to
Since it was 0.1 M, it was possible to completely separate free AFP-WA-1 · Fab′-POD and the antigen-antibody complex regardless of which polypeptide was used. In particular, when the polypeptides 4 to 9 and 11 to 21 are used, the elution salt concentration of the antigen-antibody complex can be 0.3 M or more. Therefore, by using these polypeptides as a separation improving substance, The influence of coexisting substances in serum on the measurement can be more reliably avoided. As already mentioned, the filler used here, POROS-
Since DEAE has a slightly hydrophobic base material, polypeptides 10 to 2 having a sulfated tyrosine residue are used.
The antigen-antibody complex formed from the conjugate of 0 and the antibody has a higher apparent elution salt concentration than those formed from the conjugate of the polypeptide having a sulfated serine residue and the antibody. Therefore, the apparent elution salt concentration of the antigen-antibody complex formed from the conjugate of polypeptide 10 having only one sulfate residue but having a tyrosine residue and the antibody is It is almost the same as that of the antigen-antibody complex formed from the conjugate of the polypeptide 2 having three activated serine residues and the antibody. However, when the filler used was changed to one whose base material is hydrophilic and the same experiment as described above was performed, a conjugate of the polypeptide 10 having only one sulfated tyrosine residue and the antibody was obtained. The apparent eluting salt concentration of the antigen-antibody complex formed from the same as that of the antigen-antibody complex formed from the conjugate of the polypeptide 1 having only one sulfated serine residue and the antibody is the same. Therefore, it is expected that the accuracy of measurement (analysis) overlaps with the peak of the coexisting substance in the serum. From the above results, in order to use the polypeptide as a separation-improving substance, the number of acid residues derived from the strong acid in the polypeptide is considered to be 3 to 20, preferably 4 to 20 , and more preferably 5 to 15. (If the number of acid residues is increased, the concentration of the salt eluted from the column becomes too high, which is not preferable in the measurement operation.). Two or more kinds of polypeptides having different elution salt concentrations are combined (for example, any one of polypeptides 4 to 7, 8, 9, 11 to 14 and any combination of polypeptides 7 and 16 to 21), and each is suitable. When used in combination with a binding substance, two or more substances having a similar structure (for example, two or more substances that differ only in a part of the structure such as sugar chain structure, isozyme, recognize different epitopes for one antigen) It is possible to carry out a differential measurement of two or more antibodies). PAsp-1 and pAsp as polypeptides
-2 (pAsp from different production lots) shows that, in the case of a polypeptide having a conventional carboxylic acid residue, the concentration of eluted salt changes depending on the production lot, and the tailing situation of the target peak changes. ,In other words,
It was suggested that there is a problem that it is difficult to obtain a polypeptide having a uniform molecular weight. On the other hand, since the polypeptide of the present invention can be easily obtained as a homogeneous one by peptide synthesis, such a problem in pAsp does not occur. In addition, 100 μ of the antibody solution 1 prepared in Example 11 was used.
1, sample 50 μl and antibody solution 2 [TSH-2 · Fab ′
And Ala- (Ser (SO ₃ H)) ₅ -βAla (polypeptide 3) bound compound] 50 μl were mixed, and 25
20 μl of the mixed solution obtained by leaving it at 30 ° C. for 30 minutes was measured (analyzed) by HPLC under the above conditions, and the elution salt of the antigen-antibody complex when AFP was measured (analyzed) using polypeptide 3 The antigen-antibody complex of interest was eluted at a concentration (see “Polypeptide abbreviation TSH” in FIG.
The data of the dissolved salt concentration are also shown. ). from this result,
It can be seen that by using the polypeptide of the present invention as a separation-improving substance, the target measurement (analysis) can be carried out using HPLC under constant analysis conditions even when the substances to be measured are different.

Example 15. Anti-AFP Monoclonal Antibody / W as an Antibody Using the Polypeptide of the Present Invention
A-2 (hereinafter abbreviated as "AFP-WA-2". Wako Pure Chemical Industries
Made by Co., Ltd. The antigen recognition site of this antibody is AFP-WA-1 and
It is different from that of AFP-A4-4. ) Was also prepared using the same reagents as in Example 9 except that Ala- (Tyr (SO ₃ H)) ₅ -βAla was used as the polypeptide, and the same operation was performed.
AFP-WA-2 ・ Fab 'and Ala- (Tyr (SO ₃ H)) ₅ -βAla conjugate 13
A 50 mM MES buffer (pH 6.5) containing 9 nM, lentil lectin (hereinafter abbreviated as “LCA”, manufactured by Wako Pure Chemical Industries, Ltd.) 1 mg / ml, 1 mM magnesium chloride and 1 mM calcium chloride. It was prepared and used as a test solution 1. (Test solution 2) AFP-WA-1.Fab'-POD prepared in Example 12
7 nM, Ala- (Tyr (SO ₃ H)) ₈ -βAla and AFP prepared in Example 9
-50nM MOPS buffer (pH 7.5) containing 156nM of the conjugate with A4-4 ・ Fab 'and 0.2 (W / V)% of polyvinyl alcohol
Was used as Test Solution 2. (Sample) Human hepatocellular carcinoma-derived AFP was fractionated into LCA non-binding AFP and LCA binding AFP using an LCA-immobilized column. Either of these was added to human serum not containing AFP at 100 ng / ml to prepare a sample 1 (LCA
Non-binding AFP included and Sample 2 (LCA binding AFP)
Content). (HPLC conditions) -Column: POROS-DEAE (4.6 mm ID x 10 mm). Buffer A: 50 mM TAPS buffer (containing pH 8.5 and 0.25 M NaCl). Buffer B: 50 mM TAPS buffer (pH 8.5, containing 3M NaCl).・ Substrate solution: 50 mM MOPS buffer (pH 7.5, 90 mM 4-N- (4-
Carbobutyryl) aminophenol and 20 mM H ₂ O ₂ ). Gradient conditions: buffer A + B, flow rate 2 ml / min. 0 → 2 minutes B = 0% 2 → 4.5 minutes B = 13% 4.5 → 8 minutes B = 100% 8 → 8.5 minutes B = 0% ・ Post column: POD substrate addition (substrate solution, 0.1 ml / mi)
n). Reaction / detection at 60 ° C for 30 seconds: excitation wavelength 328 nm / fluorescence wavelength 432 nm. (Measurement operation) Reagent 1 100 μl and Sample 1 or Sample 2 10 μl
was mixed with 1 and reacted at 8 ° C. for 10 minutes. Then, 10 μl of the test solution 2 was added thereto, and the reaction was continued for 20 minutes. 80 μl of this reaction mixture was measured (analyzed) by HPLC under the above conditions. (Results) The measurement results are shown in FIG. In the figure, the solid line (-)
Shows the data obtained for sample 1, and also the dotted line (---
-) Indicates the data obtained for sample 2, respectively. Figure 6
From the results of AFP, AFP-WA-2 ・ Fab 'and Ala- (Tyr (SO ₃ H))
The conjugate of _5- βAla and the antigen-antibody complex with AFP-WA-1 · Fab'-POD (complex 1) were at the position of 2.9 minutes, and the complex 1 further contained Ala- (Tyr (SO ₃ H )) The antigen-antibody complex (complex 2) to which the conjugate of _8- βAla and AFP-A4-4 · Fab ′ is bound is eluted at the position of 5.8 minutes, and it is clear that each is clearly separated. Further, from the results of FIG. 6, in Sample 1 containing non-LCA-binding AFP, the produced antigen-antibody complex was Ala- (Tyr (S
O ₃ H)) ₈ -βAla and AFP-A4-4.Fab 'are mainly bound to the complex 2, and in the sample 2 containing LCA-binding AFP, the antigen-antibody complex formed It can also be seen that the body is mainly complex 1. This result shows that Ala- (Tyr (SO ₃ H)) ₈ -βAla
The binding product of AFP-A4-4 · Fab ′ with AFP-A4-4 · Fab ′ shows that the reaction with AFP is inhibited by the competitive reaction with LCA. Further, when the ratio of the amount of Complex 1 (the ratio of Complex 1) to the amount of both complexes produced was determined (see Table 2), this value reflects the ratio of LCA-binding AFP in the sample, in other words, this value. It was also found that the value can be used to determine the LCA-bound AFP ratio in the sample.

Comparative Example AFP-WA-2 · Fab ′ was replaced with AFP-WA-2 · Fab ′ and an aspartic acid polymer having an average molecular weight of 6,000 instead of the conjugate of Ala- (Tyr (SO ₃ H)) ₅ -βAla. The bound product of Ala- (Tyr (SO ₃ H)) ₈ -βAla
The same experiment as in Example 15 was carried out except that a binder of AFP-A4-4.Fab 'and an aspartic acid polymer having an average molecular weight of 28,800 was used instead of the binder of AFP-A4-4.Fab'. The composite 1 ratio was determined. It should be noted that the same experiment as above was carried out using the reagent solution 1 used here to which γ-polyglutamic acid having an average molecular weight of 550,000 was added so as to be 100 μg / ml, and the ratio of complex 1 was determined. The results are also shown in Table 2.

[0086]

[Table 2] * ΓPGA: γ-polyglutamic acid with an average molecular weight of 550,000.

From the results in Table 2, it can be seen that in the method of Comparative Example (method using polyaspartic acid), the ratio of Complex 1 tends to be higher in Sample 1, that is, nonspecific reaction tends to occur. , ΓPGA to suppress this phenomenon
It is understood that it is necessary to add an anionic additive such as. On the other hand, when the polypeptide of the present invention is used, such an additive is unnecessary. Therefore, the measuring method using the polypeptide of the present invention as a separation improving substance is superior in this respect also to the measuring method using a polymer having a carboxyl group such as polyaspartic acid which has been conventionally used as a separation improving substance. It turns out that this is a different measurement method. The reason why such a phenomenon occurs is not clear, but is presumed as follows, for example. That is, in an anionic polymer such as polyaspartic acid, since the molecule is relatively large, inhibition of the antigen-antibody reaction due to electric charge, steric hindrance, etc. occurs and the above phenomenon occurs, while the polypeptide of the present invention is Since the molecule is small, it is presumed that there is almost no effect on the antigen-antibody reaction.

Example 16. Study on effect of addition of surfactant to eluent (reagent for measurement) Polypeptide 18 and AF prepared in Example 9
A conjugate with P-A4-4 Fab 'was prepared at 200 nM and Example 14.
A 50 mM MOPS buffer (pH 7.5) containing 100 nM of AFP-WA-1 · Fab′-POD was used as a measuring reagent. (Sample) Same as in Example 14. (HPLC conditions) -Column: POROS-DEAE (4.6 mm ID x 10 mm). Buffer A: 50 mM MOPS buffer (pH 7.5, containing 0.1 (W / V)% of a predetermined surfactant). Buffer B: 50 mM MOPS buffer (containing pH 7.5, 3 M NaCl and a predetermined surfactant of 0.1 (W / V)%). Substrate solution: 50 mM MOPS buffer (pH 7.5, containing 90 mM 4-N- (4-carbobutyryl) aminophenol and 20 mM H ₂ O ₂ ). Gradient conditions: buffer A + B, flow rate 1 ml / min. 0 → 5min A = 100% 5 → 30min B = 0 → 100% 30 → 35min B = 100% ・ Post column: POD substrate addition (substrate solution, 0.1ml / mi)
n). Reaction / detection at 60 ° C for 30 seconds: excitation wavelength 328 nm / fluorescence wavelength 432 nm. Table 3 shows the types of surfactants added to the eluent.

[0089]

[Table 3]

(Measurement operation) 100 μl of measurement reagent and sample
10 μl was mixed and reacted at 8 ° C. for 10 minutes. Then
20 μl of this reaction solution was measured by HPLC under the above conditions (analysis)
did. (Results) FIG. 7 shows the eluted salt concentration and peak width of the AFP antigen-antibody complex obtained for the eluates containing various surfactants. In FIG. 7, ● indicates the salt concentration at the peak of the elution peak, and I indicates the range of salt concentration at the beginning and the end of elution (that is, the peak width). Further, each symbol on the horizontal axis of the figure indicates the type of surfactant (surfactant No. in Table 3). From the result of FIG. 7, the following can be understood. It is understood that the peak width can be narrowed by adding the surfactant, in other words, the tailing of the peak at the time of elution can be prevented, and in other words, the measurement accuracy can be improved. It is also found that this effect is prominently exhibited by the cationic surfactant and the amphoteric surfactant. The elution salt concentration (elution position) varies with the addition of a surfactant, in other words, the elution salt concentration of the target antigen-antibody complex can be appropriately set by appropriately selecting the type of the surfactant. I understand.

Example 17. Separation (Sample) by Electrophoresis AFP-A4 ・ Fab ', Ala- (Tyr (SO
₃ H)) ₈ and the combined product of AFP-A4-4 and Fab ', the AF prepared in Example 15
50 mM MOPS buffer solution (pH) containing P-WA2 ・ Fab ', Ala- (Tyr (SO ₃ H)) ₅ -βAla and AFP-WA2 ・ Fab' at 1 mg / ml each
The sample diluted with 7.5) was used. (Measurement procedure) Apply 4 μl of each sample to the sample groove of 1% agarose gel, and use the sample groove side as a cathode for 3 at 200V.
After performing 0 minute electrophoresis, Quick-CBB (Wako Pure Chemical Industries, Ltd.)
The protein was stained using (trade name), and the Rf value of each sample was measured. (Results) The Rf values of AFP-A4 ・ Fab '0.38 and Ala- (Tyr (S
O ₃ H)) ₈ and AFP-A4-4 ・ Fab '0.66, AFP-WA2 ・ Fab'
0.06, Ala- (Tyr (SO ₃ H)) ₅ -βAla and AFP-WA2 ・ Fab '
It was 0.22. From this result, it is found that the binding of the sulfated peptide increases the negative charge and increases the mobility. It is also found that the greater the number of sulfate residues in the sulfated peptide, the greater the mobility.

Example 18. Separation of Sulfated Peptide-Binding Antibody and Antigen Reactant (Sample) Example 0.5 mg / m in 50 mM MOPS buffer (pH 7.5)
50 μl of AFP prepared to have a concentration of 1 was added to a conjugate of Ala- (Tyr (SO ₃ H)) ₈ and AFP-A4-4.Fab ′ prepared in Example 17 (1 mg / m 2
l) 50 μl, or Ala- (Tyr (SO ₃ H)) ₅ -βAla and AFP-WA2 ・ Fab '
(1 mg / ml) or MOPS buffer (pH 7.
5) 50 μl was added and reacted at 37 ° C for 30 minutes, which was used as a sample. (Measurement procedure) Apply 4 μl of each sample to the sample groove of 1% agarose gel, and use the sample groove side as a cathode for 3 at 200V.
After electrophoresis for 0 minutes, antibody affinity transfer (blotting) was performed for 30 minutes at room temperature using a nitrocellulose membrane bound with an anti-AFP antibody. The membrane was washed twice with a washing solution (0.9% NaCl). This membrane was immersed in an anti-AFP antibody solution at 37 ° C. for 30 minutes and then washed twice with a washing solution. Next, this membrane is treated with POD-labeled anti-IgG.
After soaking in the antibody solution at 37 ° C for 30 minutes, the plate was washed twice with a washing solution. Further, this membrane was developed in a coloring solution (0.37 mM nitrotetrazolium blue, 2.6 mM β-nicotinamide adenine dinucleotide reduced form, 0.01% hydrogen peroxide), and the Rf value of the antigen-antibody reaction product was measured. (Results) Ala- (yr (SO ₃ H)) ₈ and AFP-A4 ・ Fab 'conjugate and AFP
Rf value of the antigen-antibody reaction product of 0.63, Ala- (Tyr (SO ₃ H)) ₅ -βA
Rf of AFP-WA2 ・ Fab 'with la and AFP antigen-antibody reaction product
The value was 0.20, and the Rf value of AFP alone was 0.85. As is clear from the above results and the results of Example 17, Ala- (Tyr (SO ₃
H)) ₈ and AFP-A4-4 / Fab 'conjugate and AFP antigen-antibody reaction product
The Rf value and the Rf value of the binding product of Ala- (Tyr (SO ₃ H)) ₈ -βAla and AFP-WA2 ・ Fab 'and the antigen-antibody reaction of AFP are Ala- (Tyr (Syr (Syr (Syr
O ₃ )) ₈ and AFP-A4-4 ・ Fab 'Rf value and Ala- (Tyr (SO ₃
H)) It was almost the same as the Rf value of the conjugate of _5- βAla and AFP-WA2 · Fab '. Thus, the negative charge of AFP is Ala- (Tyr (SO
₃ H)) ₈ and AFP-A4-4 Fab 'conjugate and AFP antigen-antibody reaction product
It is found that there is almost no effect on the Rf value or the Rf value of the combined product of Ala- (Tyr (SO ₃ H)) ₅ -βAla and AFP-WA2 · Fab ′ and the antigen-antibody reaction product of AFP.

[0093]

INDUSTRIAL APPLICABILITY As described above, the present invention is useful for detecting a complex resulting from the interaction between a substance to be measured in a biological sample and a binding substance, a free binding substance, and the complex. A novel novel compound that can be used to more effectively separate the complex and the free binding substance when separating a coexisting substance that may have an influence using a method utilizing a negative charge The present invention provides a method for measuring a substance to be measured in a biological sample using the polypeptide and this polypeptide, and in the case of measuring a trace component in a biological sample such as serum using the measuring method of the present invention. In comparison with conventional measurement methods such as EIA and RIA, or methods using a polymer having a carboxyl group as a separation improving substance, it is possible to easily and highly accurately measure in a very short time. Has a remarkable effect Are those, is a large-made invention a place to contribute to the Shigyo.

[0094]

[Brief description of drawings]

1 shows the elution position of an anionic polypeptide obtained in Example 8 by high performance liquid chromatography (HPLC) using an anion exchange column.

FIG. 2 shows the elution position of the bound product of the Fab ′ fragment of the antibody and the anionic polypeptide obtained by HPLC using an anion exchange column obtained in Example 11.

FIG. 3 shows data on the storage stability of the polypeptide having a sulfated serine residue obtained in Example 12 in various pH solutions.

FIG. 4 shows data on the storage stability of the polypeptide having a sulfated tyrosine residue obtained in Example 12 in various pH solutions.

FIG. 5 shows elution positions of various antigen-antibody complexes by HPLC using an anion exchange column obtained in Example 14.

FIG. 6 shows a measurement (analysis) elution pattern of a sample by HPLC obtained in Example 15.

FIG. 7 shows elution positions of various antigen-antibody complexes, obtained by HPLC using an anion exchange column, obtained in Example 16.

[Explanation of symbols]

1, FIG. 2 and FIG. 5, ● indicates the salt concentration at the peak of the elution peak, and I indicates the range of salt concentration at the beginning and the end of elution (that is, the peak width). Further, each symbol on the horizontal axis of the figure indicates the type of polypeptide (polypeptide No. and abbreviation in Table 1). In FIGS. 3 and 4, the black bar indicates the result obtained for the sample on the 6th day of storage, and the white bar indicates the result obtained for the sample on the 19th day of storage. In FIG. 6, the solid line (−) shows the data obtained for sample 1, and the dotted line (----) shows the data obtained for sample 2. In Figure 7, ●
Indicates the salt concentration at the peak of the elution peak, and I indicates the range of the salt concentration at the beginning and the end of the elution (that is, the peak width). Further, each symbol on the horizontal axis of the figure indicates the type of surfactant (surfactant No. in Table 3).

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shinji Satomura 6-1, Takada-cho, Amagasaki City, Hyogo Prefecture Wako Pure Chemical Industries, Ltd. Osaka Research Institute (72) Inventor Kenji Nakamura Takada-cho, Amagasaki City, Hyogo Prefecture No. 6 No. 1 Wako Pure Chemical Industries, Ltd., Osaka Laboratory (56) Reference International Publication 92/017194 (WO, A1) Chemical Abstracts, Vol. 121, no. 11 (1994), abstract no. 134778 Chemical Abstracts, Vol. 119, no. 1 (1993), abstract no. 3449 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C07K 1/00-1/12 C07K 5/00-7/06 A61K 38/00-38/10

Claims (5)

(57) [Claims] 1. Having 4 to 20 sulfated tyrosine residues,
A polypeptide having a total of 4 to 30 amino acid residues. 2. The polypeptide according to claim 1, wherein the sulfated tyrosine residue has a sulfate residue introduced into a reaction active group of the tyrosine residue. 3. A following general formula ^{[1] A- (R 4)} j-B [1] ( wherein, j is 4 to the inner of .j number of R ⁴ represents an integer of 4-20
20 are sulfated tyrosine residues, the remaining R ⁴ may be the same or different and represent an amino acid residue into which a strong acid-derived acid residue has not been introduced. A protective group may be introduced into the reactive group. A represents a protecting group for the N-terminal amino group of an amino acid into which no hydrogen atom, a sulfated tyrosine residue or an acid residue derived from a strong acid has been introduced, and B represents a hydroxyl group or a protecting group for the C-terminal carboxyl group of an amino acid. ) The polypeptide represented by. 4. The following general formula [2] A- (R ⁵ ) k-B [2] (In the formula, R ⁵ represents an amino acid residue into which an acid residue derived from a strong acid is introduced, and the same or different. May be k is 4 to 2
A represents an integer of 0, A represents a hydrogen atom, an acid residue derived from a strong acid or a protecting group of an N-terminal amino group of an amino acid, and B represents a hydroxyl group or a protecting group of a C-terminal carboxyl group of an amino acid.
However, 4 to 20 of k R ⁵ are sulfated tyrosine residues. ) The polypeptide represented by. 5. The following general formula [3] A- (R ⁵ ) j- (R ⁶ ) IB [3] (In the formula, R ⁵ represents a sulfated tyrosine residue, and j is 4 to 20.
Represents the integer. R ⁶ represents an amino acid residue into which an acid residue derived from a strong acid has not been introduced. Further, (j + 1) is 5 to 21. A represents a protecting group for the N-terminal amino group of an amino acid into which no hydrogen atom, a sulfated tyrosine residue or an acid residue derived from a strong acid has been introduced, and B represents a hydroxyl group or a protecting group for the C-terminal carboxyl group of an amino acid. 4. The polypeptide according to claim 3, which is represented by