JPH0954092A - Monomer and polymer of immunologically active material and measuring reagent therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable a measurement with high sensitivity and good reproducibility by polymerizing a monomer obtained by reaction of a modifier represented by a specific formula with an immunologically active material, causing a material for inspection to undergo an antigen-antibody reaction, and measuring the reaction product. SOLUTION: A modifier represented by R<1> -(X)m-R<2> (R<1> is a functional group that can bond to a functional group in the material, R<2> is a polymeric group capable of radical polymerization, X is a divalent organic residual group, (m) is 1 or 2) is reacted with an immunologically active material, i.e., an antibody and/or an antigen, to obtain the following monomer: A-[(Y)k-(X)m-R<2> ]n wherein A is a residual group of the active material, Y is a group which the active material and the functional group represented by R<1> together form, (k) is 0 or 1, X is a divalent organic residual group, (m) is 0 or 1, and (n) > 1. The polymer is obtained by polymerization of the monomer, a measuring reagent containing the polymer is put into contact with the material for inspection and made to undergo an antigen-antibody reaction, after that the reaction product is measured using nephelometry or turbidimetry. The active material can thus be measured with high sensitivity and good reproducibility.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunologically active substance polymer, a monomer for polymerizing the same, an immunologically active substance measuring reagent comprising the polymer and an immunologically active substance using the reagent. The present invention relates to a method for measuring an active substance. The immunologically active substance measuring reagent of the present invention is used for qualitative or quantitative determination of an immunologically active substance (antigen or antibody) in the field of clinical examination.

[0002]

2. Description of the Related Art In recent years, immunologically active substances such as proteins appearing in the body in association with various diseases have been widely used for diagnosis by utilizing the immune reaction (antigen-antibody reaction). It is being appreciated. As the measuring method utilizing such an antigen-antibody reaction, radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), latex nephelometry, immunonephelometry (TIA) Various methods have been developed. In any of these RIA, EIA, and FIA, it is necessary to separate the reaction product generated by the antigen-antibody reaction, and therefore the measurement generally requires time and labor, but it is excellent in quantification. It is widely used for such reasons.

In recent years, the turbidimetry method or the nephelometry method for measuring the optical change caused by the antigen-antibody reaction has been attracting attention. In these methods, when the antibody (or antigen) reacts with the test substance, the turbidity of the reaction solution changes before and after the reaction depending on the amount of the test substance. Law,
It is measured by an appropriate means such as an absorbance method. Such nephelometry or nephelometry is easier to operate than radioimmunoassay, enzyme-immunoassay, fluorescence-immunoassay, etc., but it is difficult to measure with high sensitivity and reproducibility. There is.

In order to solve such a problem, a latex turbidimetric method in which an antibody or an antigen is carried on latex particles as a carrier has been proposed. In this method, the antibody or the antigen is carried on a carrier. Since it is necessary, the operation becomes complicated. In addition, a measurement reagent for a nephelometric method to which an additive such as an agglutination aid is added is also known. For example, Japanese Patent Publication Sho 60
No. -4938 describes an immunoassay reagent containing an immunologically active substance, polyethylene glycol and a nonionic surfactant and having an HLB value of about 0.7 to 1.7. Further, JP-A-59-43362 describes an immunological measuring reagent containing an immunologically active substance and a surfactant having a specific structure.

However, even if an immunologically active substance is measured using a measurement reagent containing the above-mentioned additive, a so-called non-specific reaction that causes turbidity due to other substances that are not the test substance coexisting in the sample is caused. Problems such as occurrence of error, occurrence of so-called prozone phenomenon that measurement result is low concentration despite presence of high concentration test substance, or inaccurate measurement result in low concentration region There is a point. Therefore, it is desired to develop an immunological measuring reagent and a method for measuring an immunologically active substance, which are capable of highly sensitively and reproducibly measuring the immunologically active substance.

By the way, "Organic Synthetic Chemistry" Vol. 42, No. 4
No. 283 to 292 (1984), a method for producing a modified protein having reactivity by selectively modifying the protein with a protein modifier such as N- (m-benzoyloxy) succinimide is described. ing. However, the modified protein obtained by this method is a modified protein that is further labeled with an enzyme by utilizing its reactivity and used in an enzyme immunoassay, and it is not described that the modified proteins are polymerized with each other. .

[0007]

SUMMARY OF THE INVENTION The object of the present invention is to provide a immunologically active substance having a high molecular weight while maintaining the property of causing an antigen-antibody reaction with the corresponding immunologically active substance with high specificity. The purpose of the present invention is to provide a combination and a monomer which is a raw material for producing the combination. Another object of the present invention is to provide an immunologically active substance measuring reagent comprising the above immunologically active substance polymer and capable of measuring a corresponding immunologically active substance with high sensitivity and reproducibility, and this reagent. It is to propose the measurement method used.

[0008]

The present invention provides the following immunologically active substance monomer, an immunologically active substance polymer obtained by polymerizing the same, an immunologically active substance measuring reagent containing the polymer, And a method for measuring an immunologically active substance using this reagent. (1) General formula (1) R ^1- (X) m-R ² (1) (wherein R ¹ is a functional group capable of binding to a functional group in the immunologically active substance) Group, R ² is a polymerizable group capable of radical polymerization, X is 2
A valent organic residue, m is 0 or 1. ) At modifying agent is reacted represented obtained, the general formula (2) A - [(Y ) k- (X) m-R 2] n ... (2) ( In the formula, A immunologically active substance Residue, Y is a group formed from the functional group in the immunologically active substance and the functional group of R ¹ , k is 0 or 1, X is a divalent organic residue, m is 0 or 1, R ² is a radically polymerizable polymerizable group, and n is a number of 1 or more.), Which is an immunologically active substance monomer. (2) An immunologically active substance polymer obtained by polymerizing the immunologically active substance monomer according to the above (1). (3) An immunologically active substance measuring reagent comprising the immunologically active substance polymer described in (2) above. (4) A sample containing an immunologically active substance as a test substance,
After contacting with the immunologically active substance measuring reagent described in (3) above, the test substance in the sample and the immunologically active substance polymer in the immunologically active substance measuring reagent are reacted with each other by antigen-antibody A method for measuring an immunologically active substance, which comprises measuring a reaction product. (5) The measurement method according to (4) above, wherein the reaction product is measured by a turbidimetry method or a nephelometry method.

In the present invention, "immunologically active substance" means "antibody and / or antigen". The "immunologically active substance polymer" means "homopolymer and / or copolymer of antibody and / or antigen". Further, “(meth) acry” means “acry and / or methacrylic acid”.

The functional group represented by R ¹ in the general formula (1) is not particularly limited as long as it is a functional group capable of binding to the functional group in the immunologically active substance.
Hydroxyl group, carboxyl group, aldehyde group, amino group, mercapto group, succinimidyloxycarbonyl group, imide ester group, halogenonitroallyl group, pyridinodisulfide group, maleimide group, phthalimidothio group, halogenomethylcarbonyl group, halogenocarbonyl Group, halogenosulfonyl group, nitroazidophenyl group, diazotrifluoroacetyl group, isocyanate group and the like. Among these, a succinimidyloxycarbonyl group, an isocyanate group, a halogenocarbonyl group, a halogenosulfonyl group and the like, which are easily bonded to an amino group in an immunologically active substance, are preferable.

In the above general formula (1) or (2), R
^{As the} radically polymerizable polymerizable group represented by ² ,
The radical-polymerizable unsaturated bond-containing group is not particularly limited, and examples thereof include an acryloyl group, a methacryloyl group, a maleimide group, a styryl group, and a vinyl group. Among these, acryloyl group, methacryloyl group, maleimide group, styryl group and the like, which have good polymerizability between the compounds represented by the general formula (2) or between the compound represented by the general formula (2) and another monomer, A group containing an ethylenically unsaturated bond is preferred.

In the general formula (1) or (2), X
The divalent organic residue represented by is not particularly limited, but an amide group, an ester group, a thioester group, an ether group, an alkylene group, an oxyalkylene group, a polyoxyalkylene group, an alkylene urethane group, a sulfonyl group, etc. Can be given.

Specific examples of the modifier represented by the general formula (1) include the following. Examples of the modifier having a polymerizable group in which R ² is a (meth) acryloyl group include (meth) acrylic acid, (meth) acrolein, (meth) acrylic acid chloride, (meth) acryloyl isocyanate, and (meth) acryloyloxyethyl. Isocyanate, 2-hydroxyethyl (meth) acrylamide, 2-hydroxyethyl (meth) acrylate, 6-((meth) acryloylamino) caproic acid,
Examples thereof include 3-((meth) acryloylamino) propionic acid. Among these, (meth) acrylic acid chloride, which easily reacts with immunologically active substances,
(Meth) acryloyl isocyanate and the like are preferable.

Examples of the modifier having a polymerizable group in which R ² is a maleimide group include N- (6-maleimidocaproyloxy) succinimide, N- (4-maleimidobutyryloxy) succinimide and N- (8- Maleimidocapryloxy) succinimide, N- (11-maleimidoundecanoyl) succinimide, N- (6-maleimidocaproyloxy) sulfosuccinimide sodium salt, N- (4-maleimidobutyryloxy) sulfosuccinimide sodium salt, N- (8-maleimidocapryloxy) sulfosuccinimide sodium salt, N-
(11-maleimidoundecanoyl) sulfosuccinimide sodium salt, N, N'-oxydimethylenedimaleimide, N, N'-o-phenylenedimaleimide, N,
N'-p-phenylenedimaleimide, succinimidyl
4- (N-maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, m-maleimidobenzoyl Examples thereof include -N-hydroxysulfosuccinimide ester, succinimidyl 4- (p-maleimidophenyl) butyrate, and sulfosuccinimidyl 4- (p-maleimidophenyl) butyrate. Among these,
N, which is water-soluble and easily reacts with immunologically active substances
-(6-maleimidocaproyloxy) sulfosuccinimide sodium salt, N- (4-maleimidobutyryloxy) sulfosuccinimide sodium salt, N- (8-
Maleimidocapryloxy) sulfosuccinimide sodium salt, N- (11-maleimidoundecanoyl) sulfosuccinimide sodium salt and the like are preferable.

Further, examples of the modifier having a polymerizable group in which R ² is a styryl group include carboxystyrene, formylstyrene, hydroxystyrene, aminostyrene, styrenecarboxylic acid chloride, and styrenesulfonic acid chloride. Among these, styrene carboxylic acid chloride, styrene sulfonic acid chloride and the like, which are easy to react with the immunologically active substance, are preferable.

The immunologically active substance (hereinafter sometimes referred to as a substance to be modified) which is reacted with the modifying agent represented by the general formula (1) is obtained by ring-opening a hydroxyl group, a carboxyl group, an amino group or various sugar chains. It has a reactive functional group such as an aldehyde group and can be an antibody or an antigen. Examples of such a substance to be modified include C-reactive protein (CR
P), antibodies against plasma proteins such as rheumatoid factor (RF) and transferrin; thyroid stimulating hormone (TS
H), triiodothyronine (T ₃ ), thyroxine (T ₄ ), thyroxine binding protein (TBG), thyroglobulin, insulin, estriol (E ₃ ), chorionic gonadotropin (HCG), human placental lactogen (HPL). ) And other hormones; carcinoembryonic antigen (CEA), B ₂ -microglobulin, α-fetoprotein (AFP) and other tumor-related substances;
Antibodies or antigens against viral hepatitis antigens or antibodies such as Bs antigen, HBs antibody, HBe antigen, HBe antibody;
Antibodies or antigens against various biological components such as viruses such as mumps, herpes, measles, rubella, cytomegalo and anti-AIDS antibody (HIV); antibodies against various drugs such as phenobarbital, acetaminophenone, salicylic acid and cyclosporine; enzymes, etc. Protein; examples include antibodies against enzymes. The enzyme as the immunologically active substance is
It is used as a protein having the property of immunological activity, not as a protein having the property of biocatalysis. That is, the enzyme as an immunoactive substance is not used to exert the enzyme activity. The antigen or antibody against the above-mentioned antibody or antigen becomes the immunologically active substance (test substance) to be measured in the immunologically active substance measuring reagent or measuring method described later.

By reacting the above-mentioned immunologically active substance (substance to be modified) with the modifying agent represented by the general formula (1), the functional group in the immunologically active substance and the modifying agent in the modifying agent are reacted. When the reactive functional group is bonded to the compound represented by the general formula (2)
An immunologically active substance monomer represented by is obtained.

The group represented by A in the general formula (2) is a residue of the substance to be modified. The group represented by Y in the general formula (2) is a group (bond) formed from the functional group in the substance to be modified and the functional group (R ¹ ) in the modifier, Examples thereof include an amide group, a dicarbamide bond, a urea bond, a urethane bond, a disulfide bond, an imidoamide bond, and a 3-thiosuccinimide group (a bond formed by reacting a maleimide group with a thiol group).

In the general formula (2), n is a number of 1 or more, preferably 1 to 1, depending on the type of immunologically active substance.
90, more preferably 1-10. When n exceeds 90, the immunological activity may decrease depending on the type of immunologically active substance. The number of n in the synthesized immunologically active substance monomer can be determined by quantifying the functional group in the synthesized immunologically active substance monomer. For example, when the functional group is an amino group, the amino group in the monomer before and after the reaction is quantified, the ratio (modification rate) of the amino group modified by the modifying agent is determined, and n is calculated from the modification rate. The number of can be determined.

The reaction between the immunologically active substance and the modifying agent represented by the general formula (1) can be carried out as follows. For example, a solution prepared by dissolving an immunologically active substance in a phosphate buffer solution, a carbonate buffer solution, an acetate buffer solution, a Tris / hydrochloric acid buffer solution, or various physiological saline solutions is mixed with water, methanol, ethanol, propanol, dimethylformamide, tetrahydrofuran. Alternatively, a solution in which a modifier is dissolved is added to these mixed liquids, and the reaction temperature is 0 to 50 ° C., preferably 4 to 2
5 ° C., reaction time 15 minutes to 24 hours, preferably 1 to 1
Incubate for 2 hours. The reaction product can be used as a monomer of an immunologically active substance without purification, and can be purified by a method such as dialysis, salting out and gel filtration, if necessary.

The immunologically active substance monomer thus obtained has a radically polymerizable polymerizable group, and therefore can be easily polymerized. Therefore, it can be used as a monomer for producing a polymer of an immunologically active substance.

The immunologically active substance polymer of the present invention is obtained by radical polymerization of the immunologically active substance monomer represented by the general formula (2). Homopolymers or copolymers of two or more kinds, or 1
It is a copolymer of one or more immunologically active substance monomers and other monomers.

The immunologically active substance polymer of the present invention has a structural unit represented by the following general formula (3).

Embedded image (In the formula, R ³ is a group formed by radical polymerization of R ² of the general formula (2), and represents a residue of R ^2. q
Is a number of 1 or more, r is a number of 0 or more, and q + r = n is satisfied. Here, n is the same as n in the general formula (2). A,
R ² , X, Y, m, and k are the same as those in formula (2). )

The group represented by R ³ in the general formula (3) is formed from R ² by radical polymerization of the immunologically active substance monomer represented by the general formula (2). A group, which is the residue of R ² . That is, R ³ is R ²
Is a group formed as a result of radical polymerization of the polymerizable unsaturated bond present in the. Specific examples of R ³ include the following, but are not limited to these.

[0025]

Embedded image Each of the above groups is a group formed of an acryloyl group, a methacryloyl group, a maleimide group, a styryl group and a vinyl group (these groups are the groups already exemplified as R ² ).

The other monomer copolymerizable with the immunologically active substance monomer is not particularly limited as long as it is a radical copolymerizable monomer, and examples thereof include butyl (meth) acrylate and (meth) acrylic. (Meth) acrylic acid esters such as methyl acidate, ethyl (meth) acrylate, hexyl (meth) acrylate, and 2-hydroxyethyl methacrylate, (meth) acrylic acid amide, N-hydroxy (meth) acrylic acid amide, N, N-dialkyl (meth) acrylic acid amide, N, N-dialkylaminoethyl (meth) acrylic acid amide, (meth) acrylic acid, polyethylene glycol mono (meth) acrylic acid ester, pyrrolidone, styrene, α-methylstyrene, methyl Nuclear substituted styrene, chloro nucleus substituted styrene, halogen nucleus substituted styrene, styrene Examples of vinyl monomers such as sodium sulfonate, vinyl chloride, vinylidene chloride, ethylene, propylene, isobutylene, vinyl acetate, vinyl propionate, ethyl vinyl ether, n-butyl vinyl ether, diethyl itaconate, and di-n-butyl itaconate. To be These may be used alone or in combination of two or more. As the other monomer, a water-soluble vinyl monomer is preferable.

The degree of polymerization of the immunologically active substance polymer of the present invention is usually 2 to 1000, preferably 2 to 100.
The molecular weight of the immunologically active substance polymer cannot be generally defined depending on the kind of the immunologically active substance as a starting material, but for example, in the case of an antibody polymer, the average molecular weight is 300.
It is about 000 to 1,000,000. The proportion of the immunologically active substance monomer in the immunologically active substance polymer is not particularly limited, but is 0.01 to 100 mol%, preferably 0.1 to 90 mol%. is there.

The immunologically active substance polymer of the present invention is obtained by radical polymerization of, for example, an immunologically active substance monomer represented by the general formula (2) and optionally other monomers in the presence of an initiator. It can be manufactured by As the above-mentioned initiator, a usual radical initiator can be used without limitation. Specifically,
2,2'-azobisisobutyronitrile (AIBN),
Benzoyl peroxide, diisopropylperoxydicarbonate, t-butylperoxy-2-ethylhexanoate, t-butylperoxypivalate, t-butylperoxydiisobutyrate, 2,2'-azobis [2-
(5-Methyl-2-imidazolin-2-yl) propane] dihydrochloride, 2,2′-azobis [2- (2
-Imidazolin-2-yl) propane] dihydrochloride, 2,2'-azobis [2- (4,5,6,7-tetrahydro-1H-1,3-diadipin-2-yl) propane] dihydrochloride, 2 , 2'-azobis [2-
(3,4,5,6-Tetrahydropyrimidin-2-yl) propane] dihydrochloride, persulfate and persulfate monohydrogen sulfite. The initiator is used in an amount of 0.01 to 10 parts by weight, preferably 0.1 to 5 parts by weight, based on 100 parts by weight of all the monomers.

In producing the immunologically active substance polymer, a polymerization degree adjusting agent such as alkyl mercaptan or alkyl dithiol may be used. Specific examples include ethyl mercaptan, mercaptoethanol, mercaptoacetic acid, aminoethanethiol and ethanedithiol. The amount of the polymerization degree adjusting agent used is 0.01 to 100 parts by weight, preferably 0.1 to 10 parts by weight, based on 100 parts by weight of all the monomers.

The polymerization reaction is preferably carried out in a reaction medium that does not reduce the activity of the immunologically active substance monomer. Examples of the reaction medium include buffer solutions such as phosphate buffer solution, carbonate buffer solution, acetate buffer solution, Tris / hydrochloric acid buffer solution; water; methanol, ethanol, propanol, t-butanol, benzene, toluene, dimethylformamide, tetrahydrofuran, and the like. Examples include organic solvents such as chloroform.
The organic solvent is preferably used by mixing with the buffer solution so that the concentration does not deactivate the immunological activity, for example, the concentration of 0.1 to 40% by volume.

The polymerization reaction is preferably carried out by substituting the polymerization system with an inert gas such as nitrogen, argon or helium. The reaction conditions are such that the polymerization temperature is 15 to 70 ° C., preferably 20 to 60 ° C., and the polymerization time is about 2 to 96 hours, preferably 5 to 72 hours. Polymerization degree (molecular weight) of the immunologically active substance polymer thus obtained
Is the polymerization temperature, the polymerization time, the type or amount of the initiator used,
It can be adjusted by selecting the type or amount of the polymerization degree adjusting agent. After the completion of the reaction, it can be purified by a method such as dialysis, salting out, gel filtration, if necessary.

The reagent for measuring an immunologically active substance of the present invention contains the immunologically active substance polymer obtained as described above, and may be composed of only the immunologically active substance polymer. The additive may be blended. Further, the measuring reagent of the present invention can be stored in a dry state such as a solid or powder, and can be dissolved in an appropriate medium before use, or can be used as a reagent in the form of a solution from the beginning.

As the above-mentioned additive, the additive conventionally used in the measuring reagent utilizing the antigen-antibody reaction can be used without limitation. Specific examples thereof include surfactants such as sodium dodecyl sulfate, Tween 20 (trademark of ICI, trademark), and polyethylene glycol; methanol, ethanol, acetone, N, N'-dimethylformamide,
An organic solvent such as tetrahydrofuran can be used.

As a medium for dissolving the immunologically active substance polymer, any liquid can be used without limitation as long as it can be dissolved without lowering the activity, and specifically, a phosphate buffer, carbonic acid or the like. Buffers, acetate buffers, buffers such as Tris / HCl buffer; water; these buffers or water and organics such as methanol, ethanol, propanol, t-butanol, benzene, toluene, dimethylformamide, tetrahydrofuran, chloroform, etc. Examples thereof include a mixed solution with a solvent, such as a mixed solution having an organic solvent concentration of 0.1 to 40% by volume.

When the measuring reagent of the present invention is in a solution state,
The concentration of the immunologically active substance polymer is 0.001 to 100 m
It is desirable that the amount is g / ml, preferably 0.01 to 10 mg / ml.

The assay system for assaying a test substance using the assay reagent of the present invention is not particularly limited, and known radioimmunoassays, enzyme immunoassays, fluorescence immunoassays, nephelometry, or nephelometry are used. The law etc. can be adopted. Among these, the turbidimetric method for measuring the transmitted light of the reaction solution containing the antigen-antibody reaction product or the nephelometric method for measuring the intensity of scattered light is preferable. Whichever measurement system is adopted, a sample containing an immunologically active substance as a test substance is brought into contact with the measurement reagent of the present invention,
After the test substance in the sample and the immunologically active substance polymer in the measuring reagent of the present invention are reacted with each other by an antigen-antibody reaction, the reaction product is measured according to the measuring system adopted. This makes it possible to quantify or qualify the test substance.

The concentration of the immunologically active substance polymer in the reaction system when carrying out the antigen-antibody reaction is 0.001 to 100 mg / m 2.
1, preferably 0.01 to 10 mg / ml, the reaction temperature is 0 to 70 ° C., preferably 4 to 40 ° C. at which the immunologically active substance polymer or the test substance is not inactivated, and the pH of the reaction system is 2 to
It is desirable to set it to 13, preferably 4 to 11. The reaction time is 0.1 minute to 1 hour, preferably 1 minute to 20 minutes.

As the reaction medium, the same buffer solution, water or a mixed solution of these and an organic solvent as exemplified as the medium used when the measurement reagent of the present invention is used as a solution-state reagent can be used. Further, the same additives as those that can be added to the measuring reagent of the present invention can be added to the reaction system.

When the nephelometric method or the nephelometric method is adopted as the measuring system, the turbidity or scattered light of the reaction solution after completion of the reaction is measured by a known means such as a turbidimeter or an absorptiometer to obtain the antigen. The antibody reaction product can be measured. In this case, since the immunoreactive substance is polymerized and polymerized in the measuring reagent of the present invention, turbidity or scattered light can be measured with high sensitivity and reproducibility. Therefore, it is possible to omit the conventional operation of loading the immunologically active substance on the carrier such as latex.

[0040]

EFFECT OF THE INVENTION Since the immunologically active substance monomer of the present invention has a polymerizable group capable of radical polymerization, it can be easily polymerized while maintaining its immunological activity. It can be used as a raw material for producing a biologically active substance polymer.

The immunologically active substance polymer of the present invention is a polymer obtained by radical polymerization of the above immunologically active substance monomer, and has a high specificity with an antigen-antibody reaction with the corresponding immunologically active substance. Since it has a high molecular weight while maintaining the property of causing the above, it can be used as a highly sensitive and reproducible reagent for measuring an immunologically active substance.

Since the immunologically active substance measuring reagent of the present invention comprises the above-mentioned immunologically active substance polymer, the corresponding immunologically active substance can be reproducible with high sensitivity even if it is not carried on a carrier or the like. It can be measured well.

Since the measuring method of the immunologically active substance of the present invention uses the above-mentioned measuring reagent, the corresponding immunologically active substance can be measured with high sensitivity and reproducibility by a simple operation. Such an effect is most exerted when the turbidimetric method or the nephelometric method is adopted as the measuring system.

[0044]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto. Example 1-1 (Preparation of Maleimide Group-Modified Antibody) 50 mg / ml concentration of goat anti-C-reactive protein antibody (Goat Anti CRP IgG, molecular weight approx.) Dissolved in 100 mM sodium carbonate / sodium bicarbonate buffer (pH 9.0). 150,000, hereinafter C-reactive protein may be referred to as CRP) 1200 μl of N- (6-maleimidocaproyloxy) succinimide (EMCS, trademark of Dojindo Co., Ltd.) at a concentration of 50 mg / ml dissolved in dimethylformamide 23 Add 1 μl as modifier and add 1 at 4 ° C
After incubating for 2 hours in the dark, the free amino group in the antibody was reacted with the succinimidyloxycarbonyl group in the modifier, and the modifier was introduced into the antibody by an amide bond.

After completion of the reaction, the reaction solution was treated with 100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (p
H6.0) equilibrated with Sephadex-G25 (Pharmacia
LKB Biotechnology, trademark) packed column (column size: inner diameter 13 mm x 390 mm), flow rate 0.5 ml /
The solution was passed through at min for fractionation (fraction volume = 1.0 ml / each fraction). The peaks of the maleimide group-modified goat anti-C-reactive protein antibody and the unreacted modifier were confirmed by measuring the absorbance of each fraction at 280 nm.
33 was collected to obtain a maleimide group-modified goat anti-C-reactive protein antibody represented by the following formula (4).

Embedded image (A represents the residue of a goat anti-C-reactive protein antibody. N is 7.3.)

The ratio (modification rate (%)) of the free amino group in the antibody forming an amide bond with the succinimidyloxycarbonyl group in the modifier was calculated by using the glycine solution as the standard solution and the modification rate of the unmodified antibody. Was determined to be 0% and determined by a quantitative method for free amino groups (Analytical Biochemistry 14,328-336 (1966)). As a result, the modification rate was 8.1% and n was 7.
It became 3. When the maleimide group-modified goat anti-C-reactive protein antibody was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the maleimide group-modified goat anti-C-reactive protein antibody was found to be unmodified. By comparing with the molecular weight of the C-reactive protein antibody, it was confirmed that no polymerizing such as association or polymerization occurred.

Example 1-2 (Preparation of methacryloyl group-modified antibody) A solution of methacryloyloxyethyl isocyanate having a concentration of 25 mg / ml dissolved in dehydrated 1,4-dioxane was used instead of the modifier solution of Example 1-1. A methacryloyl group-modified goat anti-C-reactive protein antibody represented by the following formula (5) was obtained in the same manner as in Example 1-1 except that 7 μl was used. The modification rate of this methacryloyl group-modified antibody was determined in the same manner as in Example 1-1, and it was 7.8%.
Was 7.0. The methacryloyl group-modified antibody is
By comparing with the molecular weight of the unmodified antibody, it was confirmed that macromoleculeization such as association or polymerization did not occur.

Embedded image (A indicates the residue of goat anti-C-reactive protein antibody. N is 7.0.)

Example 1-3 (Preparation of acryloyl group-modified antibody) Instead of the modifier solution of Example 1-1, 13.6 μl of a 25 mg / ml concentration acrylic acid chloride solution dissolved in dehydrated 1,4-dioxane. An acryloyl group-modified goat anti-C-reactive protein antibody represented by the following formula (6) was obtained in the same manner as in Example 1-1 except that was used. When the modification rate of this acryloyl group-modified antibody was determined in the same manner as in Example 1-1, it was 8.0%, and n was 7.2. In addition, it was confirmed by comparison with the molecular weight of the unmodified antibody that the acryloyl group-modified antibody did not undergo polymerization such as association or polymerization.

Embedded image (A indicates the residue of goat anti-C-reactive protein antibody. N is 7.2.)

Example 1-4 (Preparation of styryl group-modified antibody) Instead of the modifier solution of Example 1-1, a 50 mg / ml solution of p-styrenesulfonic acid chloride dissolved in dehydrated 1,4-dioxane was used. A styryl group-modified goat anti-C-reactive protein antibody represented by the following formula (7) was obtained in the same manner as in Example 1-1 except that 15.2 μl was used. The modification ratio of this styryl group-modified antibody was determined in the same manner as in Example 1-1, and it was 7.7%, and n was 6.9. In addition, it was confirmed that the styryl group-modified antibody did not undergo polymerization such as association or polymerization by comparing with the molecular weight of the unmodified antibody.

[Chemical 6] (A represents the residue of a goat anti-C-reactive protein antibody. N is 6.9.)

Example 2-1 The maleimide group-modified goat anti-C-reactive protein antibody prepared in Example 1-1 was dissolved in 100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 6.0),
An antibody solution for polymerization having a concentration of 25 mg / ml was obtained. Separately, methacrylamide as another copolymerization monomer was dissolved in the above phosphate buffer solution to obtain 160.0 mg / ml.
A methacrylamide solution of Further, 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride as a radical polymerization initiator was dissolved in the above phosphate buffer solution to prepare an initiator solution having a concentration of 7.2 mg / ml. Got

To 800 μl of the above antibody solution for polymerization, 100 μl of methacrylamide solution and 100 μl of initiator solution
Was added, and the mixture was incubated in a nitrogen atmosphere at 35 ° C. for 3 days with shaking in the dark to copolymerize the antibody for polymerization and methacrylamide. After the reaction is completed, the reaction liquid is 30 m
The initiator was removed by dialysis against 100 ml of M disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.5) at 4 ° C. in the dark.

After dialysis, TSK-GEL G4000
A packed column of SWXL (manufactured by TOSOH, trade name) and a packed column of TSK-GEL G3000SWXL (manufactured by TOSOH, trade name) were connected in series in this order and analyzed. That is, the dialyzed reaction solution was treated with 0.
In the above column equilibrated with 3 M NaCl, 50 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 7.0), flow rate = 1.0 ml / min, column temperature = 40 ° C, sample volume = 50 μl, sample Concentration =
The solution was passed under the condition of 1.0 mg / ml. As a result, a peak was confirmed above thioglobulin (molecular weight = 669,000), confirming that the antibody copolymer was prepared.

Example 2-2 In Example 2-1, Example 1-2 was used as the antibody for polymerization.
It carried out like Example 2-1 except having used the methacryloyl group modification antibody obtained by. As a result, a peak was confirmed above thioglobulin (molecular weight = 669,000),
It was confirmed that the antibody copolymer was prepared.

Example 2-3 In Example 2-1, Example 1-3 was used as the antibody for polymerization.
The procedure was carried out in the same manner as in Example 2-1, except that the acryloyl group-modified antibody obtained in 1. was used and 100 μl of an acrylamide solution having a concentration of 133.6 mg / ml was used instead of the methacrylamide solution. As a result, a peak was confirmed above thioglobulin (molecular weight = 669,000), confirming that the antibody copolymer was prepared.

Example 2-4 In Example 2-1, as an antibody for polymerization, Example 1-4 was used.
Example 2-1 except that the styryl group-modified antibody obtained in 1. was used.
I went in the same way. As a result, a peak was confirmed above thioglobulin (molecular weight = 669,000), confirming that the antibody copolymer was prepared.

Example 3-1 The maleimide group-modified goat anti-C-reactive protein antibody prepared in Example 1-1 was dissolved in 100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (pH 6.0),
An antibody solution for polymerization having a concentration of 25 mg / ml was obtained. Separately from this, 2,2′-azobis [2- (2-imidazolin-2-yl) propane] as a radical polymerization initiator
Dissolve dihydrochloride in the above phosphate buffer and add 2.0
An initiator solution with a concentration of mg / ml was obtained.

200 μl of the initiator solution was added to 800 μl of the above antibody solution for polymerization, and the mixture was incubated in a nitrogen atmosphere at 35 ° C. for 3 days with shaking in the dark to polymerize the antibody for polymerization. After completion of the reaction, the reaction solution was added with 30 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer (p
The initiator was removed by dialysis against 100 ml of H7.5) at 4 ° C. in the dark. After the completion of dialysis, analysis was performed in the same manner as in Example 2-1. As a result, ferritin (molecular weight = 440,000) or more, thioglobulin (molecular weight 66
A peak was confirmed at 9000) or less, and it was confirmed that the antibody polymer was prepared.

Example 3-2 In Example 3-1, Example 1-2 was used as the antibody for polymerization.
It carried out like Example 2-1 except having used the methacryloyl group modification antibody obtained by. As a result, peaks were confirmed above ferritin (molecular weight = 440,000) and below thioglobulin (molecular weight 669,000), confirming that the antibody polymer was prepared.

Example 3-3 In Example 3-1, Examples 1-3 were used as the antibody for polymerization.
Example 3 except that the acryloyl group-modified antibody obtained in 1. was used.
It carried out similarly to -1. As a result, ferritin (molecular weight = 440,000) or more, thioglobulin (molecular weight 6
The peak was confirmed below 69000), and it was confirmed that the antibody polymer was prepared.

Example 3-4 In Example 3-1, Examples 1-4 were used as the antibody for polymerization.
Example 3-1 except that the styryl group-modified antibody obtained in 1. was used.
I went in the same way. As a result, ferritin (molecular weight =
440,000 or more, thioglobulin (molecular weight 66
A peak was confirmed at 9000) or less, and it was confirmed that the antibody polymer was prepared.

Example 4-1 Using a single multi-type automatic analyzer (manufactured by Hitachi, Ltd., trade name "Hitachi 7070 automatic analyzer") having a multi-item selection function, C-reactive protein ( CR
P) was measured. First, the standard serum solution (0 mg / d
1, 0.05 mg / dl, 0.2 mg / dl, 0.8 m
g / dl; 20 μl prepared by adding a predetermined amount of CRP to a solution excluding CRP in advance) and 300 μl of the reagent 1 shown in Table 1 and incubated at 37 ° C. for 5 minutes. Then, 50 μl of the reagent 2 shown in Table 1 containing the goat anti-CRP antibody copolymer prepared in Example 2-1 was added,
Incubated at 37 ° C for 5 minutes. After that, the main wavelength 3
The difference between 40 nm and the sub-wavelength 700 nm was used as the measurement count (340 nm-700 nm). The measurement counts for each concentration are shown in Table 2.

Examples 4-2 to 4-4 In Example 4-1, instead of the goat anti-CRP antibody polymer prepared in Example 2-1, Examples 2-2 and 2 were used.
-3 or the goat anti-CRP antibody copolymer prepared in Example 2-4 was used, and the measurement was performed in the same manner as in Example 4-1. The results are shown in Table 2.

Examples 5-1 to 5-4 In Example 4-1, instead of the goat anti-CRP antibody polymer prepared in Example 2-1, Examples 3-1 and 3 were used.
-2, the measurement was performed in the same manner as in Example 4-1, except that the goat anti-CRP antibody polymer prepared in Example 3-3 or Example 3-4 was used. The results are shown in Table 2.

Comparative Example 1 Measurement was carried out in the same manner as in Example 4-1, except that the unpolymerized anti-CRP antibody was used in place of the goat anti-CRP antibody copolymer in Example 4-1. The results are shown in Table 2.

[0065]

[Table 1] * 1 Macrogol 6000 (polyethylene glycol Mw8800) manufactured by Nippon Oil & Fats Co., Ltd., trademark * 2 Anti-CRP antibody (co) prepared in Examples 2-1 to 2-4 and Examples 3-1 to 3-4. Polymer or anti-CRP antibody

[0066]

[Table 2]

From the results in Table 2, goat anti-CRP antibody (co)
Each of the examples using the measurement reagent containing the polymer clearly has a higher measurement count as compared with the comparative example using the measurement reagent containing the non-polymerized anti-CRP antibody, and high-sensitivity measurement is possible. I understand.

Claims (5)

[Claims] 1. An immunologically active substance having the general formula (1) R ^1- (X) m—R ² (1) (wherein R ¹ can bind to a functional group in the immunologically active substance). Functional group, R ² is a radically polymerizable group, and X is 2
A valent organic residue, m is 0 or 1. ) At modifying agent is reacted represented obtained, the general formula (2) A - [(Y ) k- (X) m-R 2] n ... (2) ( In the formula, A immunologically active substance Residue, Y is a group formed from the functional group in the immunologically active substance and the functional group of R ¹ , k is 0 or 1, X is a divalent organic residue, m is 0 or 1, R ² is a radically polymerizable polymerizable group, and n is a number of 1 or more.), Which is an immunologically active substance monomer. 2. An immunologically active substance polymer obtained by polymerizing the immunologically active substance monomer according to claim 1. 3. An immunologically active substance measuring reagent comprising the immunologically active substance polymer according to claim 2. 4. A test substance containing an immunologically active substance as a test substance is brought into contact with the immunologically active substance measuring reagent according to claim 3 to measure the test substance and the immunologically active substance in the sample. A method for measuring an immunologically active substance, which comprises reacting an immunologically active substance polymer in a reagent with an antigen-antibody reaction and then measuring a reaction product. 5. The measuring method according to claim 4, wherein the reaction product is measured by a turbidimetry method or a nephelometry method.