JP2509001B2 - Immunological assay for human thrombomodulin, reagent therefor, and kit therefor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION a. Industrial Application The present invention relates to the immunological determination of human thrombomodulin in a sample, in particular a human specimen. More specifically, the present invention relates to a method for immunologically highly sensitively, accurately and easily measuring human thrombomodulin in a trace amount in a sample, a reagent therefor, and a kit therefor.

b. Prior art Thrombomodulin was discovered in 1981 by C. T. Smon et al. as a cofactor protein that markedly promotes the activation of protein C by trobin from rabbit vascular endothelium [CTEsmon, WGOwen: Proc. Nat
l.Acad.Sci.USA, 78 , 2249-2252 (1981)], purified the following year [NLEsmon, WGOwen, CTEsmon: J. Biol. Chem., 25 .
7 , 859-864 (1982)]. Later, human thrombomodulin was also purified from lung and placenta [S. Kurosawa, et al .: Th
romb.Res. 37 , 353-364 (1985)], thrombomodulin from human placenta has a molecular weight of 105,00 in the presence of a reducing agent.
It was reported to be a glycoprotein with 0 and 75,000 in the absence. Thrombomodulin is an artery, vein, capillary,
It is a membrane protein on the cell membrane of endothelial cells of lymphatic vessels. It is known to be present in most organs except the brain, but it is known to be particularly abundant in the lung and placenta. It is also known that its physiological action is a physiologically active substance which converts the blood coagulation promoting action of thrombin into the coagulation inhibiting action on the endothelial cells of blood vessels. That is, thrombomodulin forms a 1: 1 complex with thrombin on the cell membrane, whereby thrombin loses its property as a coagulation factor and expresses a protein C activity-promoting action. Activated factor V and active type in coagulation factors
It is known that by inactivating factor VIII, it exhibits a coagulation-inhibiting effect. It is also known that thrombomodulin binds directly to active factor X and inhibits its activity, thereby suppressing the production of thrombin from prothrombin.

Thrombomodulin, which has the physiological actions described above, has been attracting attention as a new regulator of the blood coagulation / fibrinolysis system. Therefore, the easy and accurate measurement of a very small amount of thrombomodulin in a solution plays a very important role in the research of basic medicine and clinical medicine of the circulatory system.

In particular, highly sensitive measurement of human thrombomodiurin, which is present in a trace amount in a human sample, has the possibility of monitoring the pathological condition of various diseases accompanied by damage to the blood vessel wall.

Recently, an immunological measurement method utilizing an antigen-antibody reaction has been used as a method for measuring a trace component in a sample, particularly in a human sample. When this measurement method is used as a diagnostic agent for the measurement of a large number of samples in a medical field, etc., the following are the main requirements. That is, there is a sensitivity that can measure a trace component in a sample, that there is a property of measuring only a measurement target, that a measurement value can be obtained with good reproducibility, and that measurement operation is easy and simple.

From the above viewpoints, it is the present situation that the conventionally known measuring means for human thrombomodulin is not always satisfactory. In other words, the report of the radioimmunoassay (radiommunoassay, hereinafter abbreviated as RIA) of Etchi Ishii et al.
Ishii et al .: J. Clin. Invest., 76 , 2178 (1985), H. Ishi
i et al., Blood 67 , 362 (1986)), this report describes a competition method using a polyclonal antibody against human thrombomodulin and ¹²⁵ I-thrombomodulin. The sensitivity is about 1 to 2.5 ng / ml, and the measurement time is required to be 19 hours or more.Therefore, this measurement method is an unsatisfactory method for accurately and simply measuring a small amount of thrombomodulin. Atsuta In addition, there is a risk in terms of specificity because only polyclonal antibodies are used as antibodies.

After that, an immunological measurement method by the San-Gerache method was reported. Nishioka et al. Have reported a method using a polyclonal antibody obtained by using thrombomodiurin purified from human lung as an antigen [Junji Nishioka et al., 10th Annual Meeting of the Japanese Society for Thrombosis and Hemostasis, page 51, (1987)]. . Since only a polyclonal antibody is used as an antibody in this method, a problem is expected in terms of the specificity of the polyclonal antibody as in the above method. Further, JP-A-64-45398 and JP-A-64-47391 are disclosed. Here, the Sangerdeut method using only two kinds of monoclonal antibodies is shown, but according to the study by the present inventors, the measurement of human thrombomodulin by the Sangertisch method using only the monoclonal antibody is not practical. It was not completely satisfactory as the measurement method used in the medical field. That is, the sensitivity necessary for measuring a trace amount of human thrombomodulin in the sample could not be obtained in a short time.

c. Problems to be solved and used by the invention Therefore, the present inventors have conducted research to solve the above-mentioned problems of the conventional techniques and provide an immunological measurement method, a reagent and a kit having necessary conditions as a diagnostic agent. I advanced.

A first object of the present invention is to provide an immunoassay method, a reagent and a kit capable of highly sensitively measuring a trace amount of human thrombomodulin in a sample, particularly a human sample.

The second object of the present invention is to provide a simple and short-time immunological assay method and reagent that can be used in actual medical treatment, especially in clinical settings, for human thrombomodulin in a sample. And to provide a kit.

Another object of the present invention is to affect human thrombomodiurin in a sample, because it is affected by the person who measures and the measurement conditions (time, temperature), and is not affected as much as possible by the specificity of the antibody used, It is intended to provide an immunological measuring method, a reagent and a kit capable of stably and highly sensitively measuring.

Still other objects of the present invention will become more apparent from the following description.

d. Means for Solving the Problems Thus, according to the studies by the present inventors, the objects and advantages of the present invention are as follows: immunoassay method, immunoassay reagent and immunoassay for human thrombomodulin. It has been found to be achieved by means of a static measurement kit.

[I] Immunological measurement method for human thrombomodulin: In the method for immunologically measuring human thrombomodulin in a sample by the San-Geerti method, an antibody (first antibody) immobilized on an insoluble carrier and labeled One of the antibodies (second antibody) is a polyclonal antibody that recognizes human thrombomodulin, and the other is a monoclonal antibody that recognizes human thrombomodulin. Measuring method.

[II] Reagent for immunoassay of human thrombomodulin; immunoassay of human thrombomodulin in sample using first antibody immobilized on insoluble carrier and second antibody labeled Immunoassay characterized in that one of the antibodies is a polyclonal antibody that recognizes human thrombomodulin, and the other antibody is a monoclonal antibody that recognizes human thrombomodulin. reagent.

[III] Kit for immunological determination of human thrombomodulin: a kit for immunological determination of human thrombomodulin, comprising: (i) human thrombo immobilized on an insoluble solid carrier. An antibody that recognizes modulin (first antibody), (ii) an antibody that recognizes labeled human thrombomodulin (second antibody), provided that one of the first antibody and the second antibody is human -A polyclonal antibody that recognizes thrombomodulin, and the other is a monoclonal antibody that recognizes human thrombomodulin.

An immunoassay kit comprising (iii) a substrate and a reaction terminator for measuring enzyme activity when labeled with an enzyme, (iv) a diluent, (v) a detergent, and (vi) a standard substance.

 The present invention will be described in more detail below.

[A-1] Preparation and Purification of Antigen Human thrombomodiurin as an antigen for obtaining the polyclonal antibody and the monoclonal antibody used in the present invention is, in principle, a natural type extracted from a natural material. It may be used, but it may be obtained by protein engineering or genetic engineering techniques as long as it has immunological properties equivalent to those of natural human thrombomodulin. For example, it may be a hapten, ie, a peptide containing the antigenic determinant site of naturally occurring human thrombomodulin. Further, human thrombomodulin may be a fragment obtained by proteolytic enzymes such as elastase and trypsin.

Examples of materials for obtaining natural human thrombomodulin include human lung, human blood vessels, and cultured human
Examples thereof include vascular endothelial cells and culture supernatants thereof, and human placenta is preferable from the viewpoint of easy availability. Separation
The purification can be carried out by a combination of commonly used protein separation techniques such as salting out, extraction, centrifugation, ultrafiltration, various chromatographies, etc., but the method described in the above-mentioned S. Kurosawa et al. , That is one example.

Using the purified human thrombomodulin thus obtained as an antigen, a polyclonal antibody and a monoclonal antibody can be prepared by the method described below.

[A-2] Preparation of polyclonal antibody recognizing human thrombomodulin: The polyclonal antibody recognizing human thrombomodulin used in the present invention is human thrombomodulin or the above-obtained human thrombomodulin. The fragment can be obtained as an antigen according to a method known per se.

For example, "The Biochemical Society of Japan, ed.
1-10, Tokyo Kagaku Dojin, 1986 ”.

The immunized animal is not particularly limited as long as it is a mammal. For example, when human thrombomodulin is used as an antigen, goat, rabbit, guinea pig, rat or mouse is preferably mentioned.

After immunization, blood is collected to obtain antiserum. The obtained antiserum is used in a commonly used method such as salting out, extraction, centrifugation,
A purified polyclonal antibody can be obtained by combining ultrafiltration and various chromatographies.

[A-3] Preparation of monoclonal antibody recognizing human thrombomodulin: the monoclonal antibody recognizing human thrombomodulin used in the present invention uses human thrombomodulin or a fragment thereof as an antigen. , G.Kkler and Milstein, Nature (Londo
n), 256 , 495-497 (1975)), the hybridoma prepared by culturing is secreted and separated from the culture solution. That is, after immunizing a mouse with human thrombomodulin, a lymphocyte of this mouse is fused with a mouse myeloma cell to prepare a hybridoma. The hybridoma thus obtained produces various monoclonal antibodies according to each of the various fused lymphocytes, so that the hybridoma producing the desired monoclonal antibody is cloned by hybridizing as a hybridoma. To isolate. The cloned hybridoma is cultured in vitro to secrete the monoclonal antibody. The anti-human thrombomodulin monoclonal antibody is separated from this culture supernatant.

In the present invention, it is preferable to use, as the monoclonal antibody, an antibody having an inhibitory effect on the complex formation between human thrombomodulin and thrombin and an inhibitory effect on the activation of human protein C. Such monoclonal antibodies are known and are described in, for example, I. Maru.
yama et al .: J. Biol. Chem., 260 , 15432 (1985), monoclonal antibody TM-A73 described in JP-A-64-45398.
There is a monoclonal antibody displayed as.

The reason why it is preferable to use the monoclonal antibody having the above-mentioned inhibitory action is that it does not recognize the complex of human thrombomodulin and thrombin in the sample or the fragment thereof, and does not recognize the free human thrombomodulin or its fragment. This is because it can be directly recognized and measured.

[B] Preparation of first antibody and second antibody: In the present invention, the polyclonal antibody or the monoclonal antibody is used as an antibody (first antibody) immobilized on an insoluble carrier or a labeled antibody (second antibody). , One of them is used as a polyclonal antibody and the other is used as a monoclonal antibody. By this combination, it is possible to simultaneously express the high affinity possessed by the polyclonal antibody and the high specificity possessed by the monoclonal antibody in the immunological assay method for human thrombomodulin. As a result, a simple immunological measurement method having excellent sensitivity, specificity and reproducibility can be obtained.

However, according to the study by the present inventors, the immobilized antibody (first antibody) is a polyclonal antibody that recognizes human thrombomodulin, and the labeled antibody (second antibody) is human thrombomodulin. It has been found that a combination which is a monoclonal antibody that recognizes yurin gives particularly favorable results.

[B-1] Preparation of immobilized antibody The anti-human thrombomodulin antibody (first antibody) immobilized on an insoluble (solid) carrier can be obtained as follows.

Examples of the insoluble solid carrier include polymers obtained from nature and derivatives thereof, and synthetic polymers and derivatives thereof. The former includes polysaccharides and their derivatives such as cellulose, cephadex, sepharose, carboxymethyl cellulose, nitrocellulose, cellulose acetate, dextran, and inorganic polymers such as glass and silica gel. The latter includes vinyl polymers such as polystyrene, polyethylene, polypropylene and AB.
S, polyvinyl fluoride, polyamine-methyl vinyl ether-maleic acid copolymer, ethylene-maleic acid copolymer, etc .; condensation type polymers such as 6-nylon, 6,6-
Examples include polyamides such as nylon, polyesters such as polyethylene terephthalate, and amino acid polymers.
In addition, the shape is a test tube, microtiter plate,
Beads or membranes are not particularly limited. As the antibody immobilized on the insoluble solid carrier which becomes fragile, the antibody molecule of the above-mentioned anti-human thrombomodulin antibody, or a fragment thereof which does not lose the antigen-binding ability, for example, F (ab ′) 2
₂ , Fab ′, Fab, Facb, etc .; or a derivative of an antibody molecule or a fragment thereof that does not lose its antigen-binding ability. A method for immobilizing these antibodies on an insoluble solid carrier is a physical adsorption method, for example, a method in which a polystyrene solid carrier is immersed in a solution of an anti-human thrombomodulin antibody; an ion binding method, for example, an ion exchange resin or an amino group. , A method using a solid carrier having an ionizable functional group such as a carboxylic acid group, a sulfonic acid group and a phosphoric acid group;
Alternatively, a covalent bond method by a chemical reaction, for example, a carboxy chloride method, a carbodiimide method, a maleic anhydride derivative method, an isocyanate derivative method, a cyanogen bromide activated polysaccharide method, a diazo method, an active ester method, a carrier bonding method by a crosslinking reagent (crosslinking As reagents, glutaraldehyde, hexamethylene isocyanate, succinimide, maleimide compounds, etc.); Furthermore, it has no binding ability to human thrombomodulin, but biological reaction to anti-human thrombomodulin antibody. A method of binding via a substance capable of binding by, for example, a method of using a protein A-bound solid support and the like.

Further, according to the study by the present inventors, by using an insoluble solid carrier having a mirror-finished surface, that is, a surface having an extremely smooth surface, non-specific adsorption is reduced, and human thrombomodulin is reduced. It was found that the measurement sensitivity of was improved.

Conventionally, as an insoluble solid carrier for highly sensitive measurement, a carrier whose surface has been roughened by polishing to have a large surface area has been used. Certainly, there is a merit that increasing the surface area increases the amount of immobilized antibody, but there is a demerit that non-specific adsorption also increases. According to the research conducted by the present inventors, in the measurement of human thrombomodulin, the amount of immobilized antibody was not high in the mirror-like solid carrier having a center line average roughness (Ra) of 1.5 μm or less. Although it is almost equivalent to the surface-supported solid support, non-specific adsorption is drastically reduced.
It was found to be free for highly sensitive determination of thrombomodulin. The material shape of the mirror-finished insoluble solid is not particularly limited, and examples thereof include polystyrene beads and glass beads.

The center line average roughness (Ra) is obtained by extracting a portion having a measurement length l from the roughness curve in the direction of the center line, and measuring the center line of the portion as the X axis,
When the direction of the vertical magnification is the Y axis and the roughness curve is represented by y = f (x), it means the value of Ra given by the following expression in units of microns.

Regarding this center line roughness (Ra), JIS B 0601-1982
(Japan) <ANSI B46.1-1979 (USA) and R468-1966 (I
SO).

 In the following examples of the present invention, the insoluble carrier is
SURFCOM, a surface roughness meter manufactured by Mikko Co., Ltd. (Surfcom570A)
Was used to measure the surface roughness.

[B-2] Preparation of labeled antibody; The anti-human thrombomodulin antibody (second antibody) labeled in the present invention can be obtained as follows.

As the antibody to be labeled, not only the whole antibody molecule,
A fragment thereof that does not lose its antigen-binding ability, such as Fa
b ′, F (ab ′) ₂ or Facb fragments can be used.

In the case of F (ab ′) ₂ or Fab ′, such a fragment is obtained by digesting the polyclonal antibody or monoclonal antibody obtained as described above by a known method, for example, digesting the antibody with pepsin to obtain F (ab ′) 2. ₂ fragment or by further reducing the F (ab ′) ₂ fragment.
(II. Nison
off et al., Arch. Biochem. Biophys., 89 , 230 (1960): P.
Parham., J. Immunol., 131 , 2895 (1983)).

A radioisotope is well known as a labeling substance used for labeling, but considering industrial production, it may lead to pollution, has a period of use, and has special training for users. The enzyme which does not have these drawbacks and has a broadening effect is suitable in that

Examples of the enzyme bound to such an anti-human thrombomodulin antibody include lysozyme, malate dehydrogenase, glucose-6-phosphatodehydrogenase, peroxidase, glucose oxidase, alkaline phosphatase, luciferase,
Examples include β-galactosidase, alcohol dehydrogenase, invertase, and the like. The binding method of the binding antibody between these enzymes and the above-mentioned anti-human thrombomodulin antibody can follow the usual methods such as the glutaraldehyde method, the periodoic acid method and the maleimide method.
For example, it can be carried out by reacting the maleimidated antibody or antibody fragment with the SH-enzyme in a solution. Maleimidation of antibodies or fragments of antibodies can be performed, for example, by succinimidyl 4- (N-maleimidomethyl) cyclohexane carbonate (SMCC), sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane carbonate (sulfo SMCC), succinimidyl-metamaleimidobenzoate. (MBS), succinimidyl 6-maleimidohexanoate (EMCS) and the like can be used for maleimidation. The SH group can be introduced into the enzyme by a known method (see, for example, “Enzyme Immunoassay”, edited by Ishikawa, Medical Institute). For example, an enzyme and S-acetylmercaptosuccinic anhydride (AMSA) or N-succinimidyl-3- (2-pyridylthio) propionate (SPD
It can be obtained by reacting with P) or the like.

The binding antibody (second antibody) between the enzyme thus obtained and the anti-human thrombomodulin antibody, that is, the number of enzymes labeled in the enzyme-labeled antibody, is determined by measuring the molecular weight, the absorbance, the enzyme activity, etc. You can For example, when the enzyme is peroxidase, the number of labels can be determined by measuring the absorbance at 280 nm derived from the antibody and peroxidase and the absorbance at 403 nm derived from peroxidase (E. Ishikawa et al., J. Im
munoassay., 4 , 209-327, (1983), p243).

That is, at 403 nm, there is no absorbance of the antibody, and since it is the absorbance derived only from peroxidase, the concentration of peroxidase is calculated from the absorbance. Since the absorbance at 280 nm is derived from both the antibody and peroxidase, the antibody concentration is calculated from the measured absorbance at 280 nm, the concentration of peroxidase calculated by the absorbance at 403 nm, and the molecular extinction coefficient of peroxidase at 280 nm. The value obtained by subtracting the contribution of peroxidase at 280 nm and 280 n
It is calculated from the molecular extinction coefficient of the antibody. The number of peroxidase labeled on the antibody can be determined from the concentration of peroxidase and antibody thus obtained.

Thus, the labeled number of the labeled anti-human thrombomodulin antibody of the present invention was obtained, and the correlation with the measurement sensitivity was investigated. As a result, the whole antibody molecule, Fab ′, F (ab ′) ₂ or Facb fragment was labeled with an enzyme. It has been found that an average of 1.0 or more molecules are bound per molecule, but it is clear that 1.5 or more molecules bound (multi-labeled antibody) produce more preferable results. In order to obtain a sufficient measurement sensitivity for measuring human thrombomodulin in a sample, the multi-labeled antibody is a whole antibody, Fab ', F (ab') ₂ or Facb.
An average of 1.5 or more molecules are preferably bound per molecule of fragment, more preferably 2 or more molecules.

Such a multi-labeled antibody is produced, for example, by reacting the maleimidated antibody or antibody fragment with the SH-enzyme. The reaction conditions include, for example, a molar ratio of the antibody to the enzyme of 1: 1 or more, preferably 1: 3.
Above, reaction temperature 4-40 ℃, preferably 4-30 ℃, reaction pH
Conditions of 5.5 to 7.5 and reaction time of 5 to 48 hours can be followed, but the reaction conditions are not limited as long as the multilabeled antibody of the present invention can be obtained. The multi-labeled antibody thus obtained can be separated and purified from the reaction solution using, for example, gel chromatography.

The thus-obtained antibody labeled with the enzyme of the present invention can be correctly measured with the measurement sensitivity necessary for measuring a trace amount of human thrombomodulin.

[C] Immunological measurement method: As a sample in the measurement method of the present invention, particularly a human sample, an ordinary clinical sample such as blood in serum or plasma form, internodal fluid, lymph, thymus fluid, ascites fluid, amniotic fluid, Any liquid containing human thrombomodulin such as cell tissue fluid, bone marrow fluid or urine may be used. Preferred is blood in serum or plasma form.

In the immunological measurement method of the present invention, the first antibody and the second antibody are used in combination, and human thrombomodulin is measured in a sample by the so-called Sangerti method.

The Saint-Germany method can be broadly classified into a one-step and one-step method. The 1 step method is a method in which the sample containing human thrombomodulin, the antibody immobilized on an insoluble carrier (first antibody) and the labeled antibody (second antibody) are used in the same reaction system as the antigen The antibody reaction is performed, and the immobilized antibody / antigen /
In this method, a complex of labeled antibody is formed, and after washing operation, the amount of labeling substance is measured. In this case, the sample (specimen), the immobilized antibody and the labeled antibody may be allowed to react at the same time, or the sample and the immobilized antibody may be reacted first, and then the labeled antibody may be added and reacted. Good. Also,
The immobilized antibody may be added after previously reacting the sample with the labeled antibody. In any case, it is only necessary to form a complex of immobilized antibody / antigen / labeled antibody before the washing operation. On the other hand, in the 2-step method, the sample is first reacted with the immobilized antibody to form a complex of the immobilized antibody / antigen, and then the sample is removed and washed, and then the labeled antibody is added. Form a complex of immobilized antibody / antigen / labeled antibody. Then, after the washing operation, the amount of the labeling substance is measured. According to the present invention using a polyclonal antibody and a monoclonal antibody, a minute amount of human thrombomodulin in a sample can be easily measured with high sensitivity and specificity with good reproducibility by the 1-step or 2-step method. You can do it.

The solvent used in the above-mentioned immune reaction may be any of various ordinary solvents that do not adversely affect the reaction. For example, it is preferable to use a phosphate buffer solution, a Tris / hydrochloric acid buffer solution, an acetate buffer solution or the like having a pH of about 6.0 to 8.0.

There are no particular restrictions on the temperature of the immune reaction during the measurement as long as it does not denature the properties of the protein as a constituent and does not significantly suppress the immune reaction, but it is generally 50 ° C or lower, preferably about 4 ° C to 45 ° C. The reaction may be carried out under the temperature condition of about 5 minutes to 5 hours, preferably about 30 minutes to 3 hours.

[D] Immunological measurement kit: In the present invention, an anti-human thrombomodulin antibody (first antibody) immobilized on an insoluble solid carrier and a labeled anti-human thrombomodulin antibody (second antibody) are used. The kit for measuring the amount of human thrombomodulin can be constructed by combining other substances necessary for immunological measurement.

In the immunological assay kit of the present invention, other reagents combined with (i) the first antibody and (ii) the second antibody include the following (iii) to (vi).

(Iii) In the case of labeling with an enzyme, a substrate and a reaction terminator for measuring the enzyme activity (iv) a diluent (v) a detergent and (vi) a standard substance will be explained below. Any one can be used as long as it is used for immunological measurement.

Substrate and reaction terminator for measuring enzyme activity: In the assay kit and method of the present invention, the substrate and reaction terminator used when an enzyme is used as a labeling substance is the kind of enzyme as a labeling substance. Corresponding to the above, those commonly known in immunological assay can be used. Examples thereof include 2,2'-azino-di- [3ethylbenzthiazoline sulfonic acid] diammonium salt (ABTS), orthophenylenediamine (OPD), 3,3 ', 5,5 as a substrate for peroxidase. There is ′ -tetramethylbenzidine (TMB), etc., and H ₂ S is used as a terminator.
O ₄ , HCl, acetic acid, glycine buffer (pH 10.3), sodium fluoride solution, etc. are available.

Substrates for alkaline phosphatase include 4-nitrophenyl phosphonate, 4-methylumbelliferyl phosphonate, NADP and the like.

Substrates for β-glass tosidase include 2-nitrophenyl-β-D-galactoside and 4-methylumbelliferyl-β-D-galactoside. Examples of the terminator include 0.1M Na ₂ CO ₃ .

Diluent: The diluent used in the assay kit and assay method of the present invention may be any diluent commonly used in immunological assay. Any diluent may be used as long as it does not adversely affect the immune reaction, and for example, a phosphate buffer, a Tris-HCl buffer, an acetate buffer or the like having a pH in the range of 6.0 to 8.0 is mainly used.

Detergent In the present invention, a detergent generally used in immunoreaction measurement can be used as it is. However, according to the present inventors, a substance which does not participate in the immune reaction by combining the first antibody and the second antibody as described above and the specific surfactant does not suppress the immune reaction in the measurement of human thrombomodiurin. It was found that it has the effect of suppressing only nonspecific adsorption of labeled antibody.

In the present invention, the presence of such a surfactant in the immune reaction solution or in the washing solution is advantageous for highly sensitive measurement. When the immune reaction is a two-step reaction, it can be present in any of the reactions, but it is preferable that it is present in the second reaction.

The surfactant used in the present invention is a double-sided surfactant and / or a nonionic surfactant having an HLB value (Hydraphile Lipophile Balance) of 16 or more.

A surfactant having an HLB of less than 16 suppresses nonspecific adsorption and simultaneously suppresses an immune reaction, which is not preferable.

As the nonionic surfactant having an HLB value of 16 or more, for example, polyoxyalkylene alkylaryl ether type, polyoxyalkylene alkyl ether type, polyoxyalkylene polyhydric alcohol fatty acid ester type, polyoxyethylene polyoxypropylene polyol type, etc. HLB value of 16 or more, for example, Triton X-305 (HLB17.3), Triton X-405 (HLB17.9), Emulgen 950 (HLB18.2), Emulgen 985 (HLB18.9),
Tween20 (HLB16.7), Bourronick F68 (HLB29), Tetranik 707 (HLB> 20) and the like.

 Examples of the amphoteric surfactant include carboxylate type
(Betaine type), sulfonate type (sulfobetaine type)
And phosphate type. Sulfobeta
The carcinogenicity of the in type may cause a problem, so its use is
It cannot be said that it is always favorable in the business. Preferably
Tyne-type amphoteric surfactant with the general formula R₁R₂R₃NR_Four[formula
Medium, R₁Is C_Five~ C_{twenty two}Alkyl group, R₂And R₃Is C₁~ C_FiveAl
Kill group, R_FourIs COO C with a substituent₁~ C_FiveAlkyl group
Betaine type. More preferably
Is R₂, R₃Are each a methyl group, and R_FourIs CH₂COO so
is there. Suitable surfactants of this type include R₁Lauri
Kao shares "Amphithor 24B" (trade name)
Sold by the company.

These surfactants can be used alone or in combination of two or more kinds.

The concentration of the surfactant in the cleaning liquid or the like is 1% by weight or less, preferably 0.0025 to 1% by weight.

In the present invention, even a surfactant amount of 0.1% by weight or less is excellent in cleaning effect, and further less foaming during cleaning, resulting in severe foaming, and removal of foam becomes complicated,
This is preferable because the problem that the removal becomes insufficient and the cleaning becomes insufficient on the contrary is reduced.

The solvent of these surfactants may be any of those which do not adversely affect the measurement, such as water, physiological saline, and a buffer solution such as a phosphate buffer solution.

Standard substance As the standard substance used in the assay kit and assay method of the present invention, a protein or polypeptide having at least one antigen-determining site recognized by each of the first antibody and the second antibody is used.

One of the typical ones is a natural form of human thrombomodulin or a fragment thereof extracted from a natural material.

Examples of the material for obtaining natural human thrombomodulin include human placenta, human lung, human blood vessels or cultured human / vascular endothelial cells and culture supernatant thereof, but they are relatively easy to obtain. Human placenta is preferred. Separation / purification can be performed by a combination of commonly used protein separation techniques such as salting out, extraction, centrifugation, ultrafiltration, various chromatographies, etc. The method is one example.

In addition, the fragment of human thrombomodulin is
The natural human thrombomodulin obtained as described above can be obtained by a method known per se using a proteolytic enzyme such as esterase or trypsin or a proteolytic reagent such as cyanogen bromide. can be [for example S.Kurosawa et al., J.Biol.Chem., 262 .2206 (1
987)].

Furthermore, not only the above-mentioned natural type, but also a polypeptide having the above-mentioned properties obtained by a protein engineering or genetic engineering technique may be used.

Use of protein According to the study by the present inventors, in the determination of human thrombomodulin, the molecular weight of the immune reaction solution was 16,000 to 5.0.
The presence of a protein or a mixture containing it having an isoelectric point of 1.0 to 5.0 and adjusting the final concentration of these immunoreaction solutions to 0.005 to 0.8% by weight suppresses nonspecific adsorption, and It was found that the back ground was extremely low and high measurement sensitivity was obtained. Such a protein or a mixture containing the protein may be contained in the immunoassay reagent or kit of the present invention in the immunoreaction solution in a predetermined amount. Examples of such proteins include casein, pepsin, ovoglycoprotein, orosomucoid and the like. The results show that the specific adsorption increases when a protein with a molecular weight of 16,000 or less is used, and that with a molecular weight of 50,000 or more, the immune nonspecific reaction is insufficiently reduced and the specific immune response is reduced. The molecular weight was determined to be 16,000 to 50,000. Regarding the isoelectric point, the non-specific adsorption was increased when a protein of 5.0 or more was added, and the specific reaction was suppressed when the isoelectric point was lower than 1.0, so the isoelectric point was set to 1.0 to 5.0. .

In addition, when the immunoassay was carried out using various concentrations of the protein solution such as skim milk, the non-specific reaction was significantly increased even when the amount of the antigen was 0 at less than 0.005% by weight. During this period, a storage test of the protein, such as skim milk solution, was carried out in a refrigerator. As a result, when the content of the protein was more than 0.8% by weight, a precipitate which could not be redissolved was generated in the refrigerator. Considering the above two facts, it is appropriate that the protein concentration is in the range of 0.005 to 0.8% by weight in order to satisfy the stability of the reagent and effectively reduce the nonspecific reaction. Such a mixture includes, for example, 10 to 60% by weight of the protein as a main component, preferably 20 to 50% by weight, sugar (e.g., lactose) 30 to 80% by weight, preferably 40 to 60% by weight, other fats (e.g., 0.5-2% by weight), ash (eg 5-12)
% By weight, water (for example, 2 to 8% by weight), and the like. A typical example of such a mixture is skim milk. Skim milk contains casein as a protein, but skim milk has better dispersibility in the immune reaction solution, has a higher effect of suppressing non-specific reactions, and is stored at a temperature of 4 ° C as compared with the case of using casein alone. It has a good property (precipitation does not easily occur). The skim milk used in the present invention may be milk of any origin as long as it is defatted milk. One of the most typical is the commercially available skim milk from Difco.

In the present invention, the "molecular weight" of a protein means a molecular weight measured by an osmotic pressure method. Specifically, a polymer solution, a pure solvent and a solvent molecule are freely permeable, but an eluting polymer is not permeable. The osmotic pressure difference between both solutions when contacting with a membrane as a boundary is used to measure the molecular weight of a protein by utilizing the fact that it serves as a parameter of the molecular weight of a polymer.
It is a value measured at 4 ° C. using a urea solution. The "isoelectric point" refers to the value measured by the chromatofocusing method that separates proteins according to their isoelectric point. Specifically, a column (0.5 cmφ x 45 cm) packed with PBE94 (manufactured by Pharmacia) is used. Eluent The value measured with 0.025M imidazole hydrochloric acid (pH 7.4).

The protein is used so as to have the above concentration in the immune reaction solution, but in the case of a kit, it is preferable that at least the protein is contained in the standard substance, the second antibody or the diluent. The content should be appropriately determined so as to have the above concentration in the immune reaction solution.

In the measurement of human thrombomodulin in the method of the present invention, the above-mentioned molecular weight of 16,000 to 50,0 in the immune reaction solution
00 and a protein having an isoelectric point of 1.0 to 5.0 or a mixture containing the same and γ-globulin or a γ-globulin derivative at a final concentration of 0.0025 to 0.1% by weight in an immunoreaction solution.
When it is allowed to exist at a concentration of 1, the non-specific adsorption is suppressed, that is, the background is further reduced, and higher measurement sensitivity is obtained as compared with the case where the protein is used alone, which is preferable. Examples of γ-globulin include those separated from the serum of humans or mammals such as cow, rabbit, goat, etc., and human γ-globulin is particularly preferable. Examples of the γ-globulin derivative include chemically modified γ-globulin such as S-alkylated γ-globulin and S-sulfonated γ-globulin. Next, if the addition amount is less than 0.0025% by weight, the decrease in the background due to the addition of γ-globulin or its derivative is small, and a substantially sufficient addition effect cannot be obtained. Further, even if added in excess of 0.1 wt%, the effect above 0.1 wt% is difficult to recognize, and the upper limit of the addition amount is appropriately 0.1 wt% from the viewpoint of manufacturing cost because γ-globulin is relatively expensive. Is. For the above reasons, the addition amount is appropriately within the above range.

e Effects of the Invention Thus, according to the present invention, a very small amount of human thrombomodulin, such as a sample, for example, a clinical sample, can be measured with high sensitivity, accuracy, and simple operation. Moreover, the measurement operation can be performed in a relatively short time.

In particular, according to the present invention, even if the complex of human thrombomodulin and thrombin is present in the sample specimen, the presence of the complex does not affect the accuracy of the measurement of free human thrombomodulin. Absent.

DESCRIPTION OF THE FIGURES FIG. 1 shows a calibration curve in the case of measuring human thrombomodulin in combination with the polyclonal antibody and the monoclonal antibody of the present invention. For reference, an antibody other than the present invention was combined. A calibration curve is also shown.

FIG. 2 shows the relationship between the concentration of added protein and the absorbance in the immunological measurement of human thrombomodulin.

FIG. 3 shows the relationship (calibration curve) between the concentration of human thrombomodulin and the absorbance.

f Example Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 An emulsion of 500 μg of human thrombomodulin purified from human placenta according to the method described in the above-mentioned S. Kurosawa et al. And Freund's complete azimuth band was subcutaneously administered to a rabbit. 14 days later human thrombomodulin
250 μg was subcutaneously administered in the same manner. 2 every 14 days
Subcutaneously administrated in the same manner as above, and blood was collected on the 10th day to make serum
Enzyme-linked immunosorbent assay, i.e., a goat anti-rabbit IgG antibody labeled with human thrombomodiurin as the antigen and horseradish peroxidase-labeled second antibody, and the antibody titer against human thrombomodulin is 3 million times. Recognized. Then, whole blood was collected and used as serum, which was purified with a protein A-Sepharose-4B column to obtain a polyclonal antibody against human thrombomodulin.

Example 2 1. Maleimidation of Anti-Human Thrombomodulin Monoclonal Antibody Monoclonal antibody having inhibitory activity on the complex formation of human thrombomodulin with thrombin and inhibiting protein C activation 13.9 μl of a DMF solution (10 mg / ml) of succinimidyl 4- (N-maleimidomethyl) cyclohexane carbonate (SMCC) was added dropwise at 25 ° C. to 830 μm of a PBS solution of TM-A73) described in JP-A-64-45398. After stirring for 30 minutes, gel filtration was performed on a Sephadex G-25 column (1 cm × 45 cm) using 0.1 M phosphate buffer as an eluent to obtain a maleimidated monoclonal antibody.

2. Introduction of thiol group of peroxidase To 143 mg of 0.1M phosphate buffer (pH6.0) solution of 143 mg of peroxidase (Toyobo), 176.4 μl of DMF solution (60 mg / ml) of S-acetylmercaptosuccinic anhydride was stirred at 25 ° C. The solution was gradually added dropwise to the reaction mixture for 60 minutes. To this solution, 5.72 ml of 0.1 M Tris-HCl buffer (pH 7.0) and 0.1 M EDTA solution (pH 7.
0) 1.14 ml, 1M hydroxylamine solution (pH 7.0) 11.44 m
l was added and stirred at 30 ° C. for 5 minutes. The solution was put into a dialysis tube and dialyzed at 0.1C phosphate buffer (pH 6.0) -5 mM EDTA as an external solution at 4 ° C for two nights to obtain an SH group-introduced peroxidase solution.

3. Binding of maleimidated monoclonal antibody and thiol group-introduced peroxidase 830 µg of maleimidated monoclonal antibody and 1.02 ml of thiol group-introduced peroxidase solution (4.05 mg / ml) were mixed, and the mixture was ultrafiltered to about 600 µl. The mixture was concentrated and reacted at 25 ° C for 24 hours. The reaction solution is analyzed by HPLC (column TS
K Gel3000SW) and eluent PBS (pH 7.2) were used for separation to obtain a peroxidase-conjugated monoclonal antibody.

The molar ratio of the obtained peroxidase-conjugated monoclonal antibody to the peroxidase was 1: 4.6.

Example 3 The anti-human thrombomodiurin polyclonal antibody described in Example 1 was treated with 0.1 M carbonate buffer (pH 9.5) at 5 μg / m 2.
Surface center line average roughness (Ra) measured with mirror beads of polystyrene beads (Immunochemical: Immunobeads, Surfcom570A (manufactured by Tokyo Seimitsu Co., Ltd.): 1.3μ
m) was soaked and left standing at 4 ° C. for 17 hours. Beads 1% BSA-TB
The mixture was allowed to stand at room temperature for 2 hours with S (pH 7.4). The beads were washed again three times with TBS (pH 7.4) to obtain antibody-immobilized beads A. TBS (pH7.4)
Stored at 4 ° C until use.

Example 2 by the same method as the antibody solid beads A
The antibody-immobilized beads B on which the anti-human thrombomodulin monoclonal antibody (TM-A73) described in 1) was immobilized were obtained.

On the other hand, 0.5% BSA-0.05% Tween20-5mMCaCl 2 -TBS (pH7.
0,0.6 of human thrombomodulin with the dilution buffer of 4)
25, 1.25, 2.5, 5.0, 10.0 ng / ml solutions were made. this
0.4 ml of each was put into a polypropylene plastic small test tube to prepare dilution series A and B having a human thrombomodulin concentration of 0 to 10 ng / ml. The antibody-immobilized beads A were added to the test tubes of this dilution series A one by one, and the antibody-immobilized beads B were added one to each dilution series B, and each was subjected to ink incubation at 37 ° C. for 90 minutes.

After washing three times with a solution of Tween 20 dissolved in TBS (pH 7.4) at 0.05% (abbreviated as TBS-T), each test tube in the dilution series A was treated with the peroxidase-labeled anti-human thrombomodi prepared in Example 2. Yurin monoclonal antibody was added at an antibody concentration of 1 μg / ml.
0.4 ml of a solution dissolved in 5% BSA-0.05% Tween 20-5 mM CaCl ₂ -TBS (pH 7.4) was added to each of the test tubes in the dilution column B in the same manner as in Example 2. 1. A peroxidase-labeled anti-human thrombomodulin, prepared from the anti-human thrombomodulin polyclonal antibody described in 1.
0.4 ml of a 4 μm / ml solution of polyclonal antibody prepared in the same manner as described above was added, and each was incubated at 37 ° C. for 30 minutes.

After washing 5 times with TBS-T, 2.5 mM H ₂ O ₂ was added as a color former.
-0.025% Add 0.4 ml of 3,3 ', 5,5' tetramethylbenzidine solution, incubate at 37 ° C for 30 minutes, add 1 ml of 1N sulfuric acid to stop the color reaction, and absorb at 450 nm. It was measured.

 The calibration curve obtained as a result is shown in FIG.

For comparison, a monoclonal antibody having an epitope different from that of the anti-human thrombomodulin monoclonal antibody of Example 2 was immobilized on polystyrene-polished polystyrene beads in the same manner as the antibody-immobilized beads A and B, and antibody-immobilized beads C were immobilized. Got The antibody-immobilized beads A are provided outside the immobilized beads.
A calibration curve for human thrombomodulin was obtained in the same manner as in (Diluent C). The results are shown in FIG. 1 as a comparative example.

As is clear from FIG. 1, the combination of the polyclonal antibody of the present invention and the monoclonal antibody (dilution sequence A) is greater than the combination of two types of monoclonal antibodies (dilution sequence C).
And in B), it is clear that a highly sensitive calibration curve is obtained.

Example 4 The anti-human thrombomodiurin polyclonal antibody described in Example 1 was treated with 0.1 M carbonate buffer (pH 9.5) at 20 μg / m 2.
The concentration was adjusted to l and polystyrene beads for EIA with various center line average roughness shown in Table 1 below were dipped and allowed to stand at 4 ° C. for 17 hours. Beads with 2% BSA-TBS (pH7.4) at room temperature for 2
Let stand for hours. The beads were washed three times with TBS to obtain various antibody-immobilized beads.

On the other hand, 0.5% BSA-0.2% skim milk-0.015% human-γ-globulin-15 mM CaCl ₂ -TBS (pH 7.4) dilution buffer solution of human thrombomodiurin 0,17ng / ml was prepared, Each 0.2 ml was placed in a small polypropylene plastic test tube. Furthermore, 0.5% BSA-5 having an antibody concentration of 3 μg / ml of the peroxidase-labeled anti-human thrombomodulin monoclonal antibody prepared in Example 2 was added to each test tube.
A mMCaCl ₂ -TBS (pH7.4) solution was added, the above-mentioned antibody-immobilized beads were placed one by one, and incubated at 37 ° C. for 90 minutes. After washing 3 times with TBS-T, 2.5mM as a coloring agent
Add 0.4 ml of H ₂ O ₂ -0.025% 3,3 ', 3,5'-tetramethylbenzidine solution, incubate for 30 minutes at 37 ℃, and add 1 ml of 1N sulfuric acid to stop the color development reaction. .

The absorbance at 450 nm of this solution was measured, and the S / N ratio of various antibody-immobilized beads (where N is human thrombomodiulin 0 ng)
is the absorbance at / ml, S is the absorbance at 17 ng / ml of human thrombomodulin, and the measurement sensitivity (the variation coefficient of 0 ng / ml of human thrombomodulin is 10%,
[Absorbance + 3 x standard deviation] at 0 point obtained from this
Human thrombomodulin concentration is 0 ng / ml and 17 ng / ml
The concentration of human thrombomodulin at the point of intersection with the straight line connecting and is defined as the measurement sensitivity). The results are shown in Table 1.

As is clear from Table 1, the use of the mirror-finished insoluble carrier having Ra of 1.5 μm or less according to the present invention enables the measurement of human thrombomodulin with excellent sensitivity.

Example 5 The anti-human thrombomodiurin polyclonal antibody described in Example 1 was added to 0.1 M carbonate buffer (pH 9.5) at 5 μg / m 2.
Surface centerline average roughness (Ra) measured with polystyrene beads (immunochemicals: immunobeads, Surfcom 570A (manufactured by Tokyo Seimitsu Co., Ltd.), which was adjusted to a concentration of 1 and mirror-finished: 1.3.
μm) was soaked and left at 4 ° C. for 17 hours. Beads in TBS (pH
After washing 3 times with 7.4), the plate was allowed to stand at room temperature for 2 hours with 1% BSA-TBS (pH 7.4). It was washed 3 times again with TBS and stored in TBS at 4 ° C until used.

A solution of human thrombomodiurin at 0, 10 ng / ml was added to 0.5
% BSA-5 mM CaCl ₂ -TBS (pH7.4) containing skim milk at final concentrations of 0, 0.005, 0.012, 0.025, 0.05, 0.1, 0.2%, 0.4 ml each of polypropylene plastic It was put in a small test tube. The above anti-human thrombomodulin immobilization beads were added to each tube one by one, and incubated at 37 ° C. for 90 minutes.

2 with Tween 20 0.05% dissolved in TBS (abbreviated as TBS-T)
After washing twice, 0.5% BSA-5 mM CaCl ₂ -TBS (pH 7. was added to the peroxidase-labeled anti-human thrombomodulin monoclonal antibody prepared in Example 2 at an antibody concentration of 1 μg / ml.
4) skimmed milk to a final concentration of 0, 0.005, 0.012, 0.02
It was prepared with a solution containing 5, 0.05, 0.1, and 0.2%, and 0.4 ml each was added to the same tube as the skim milk concentration, and the mixture was incubated at 37 ° C. for 30 minutes.

After washing 3 times with TBS-T, 2.5 mM H ₂ O ₂ was added as a color former.
-0.025% Add 0.4 ml of 3,3 ', 5,5' tetramethylbenzidine solution, incubate at 37 ° C for 30 minutes, add 1 ml of 1N sulfuric acid to stop the color reaction, and absorb at 450 nm. It was measured.

The relationship between the skim milk concentration in the immunoreaction solution obtained as a result and the absorbance at 0 and 10 ng / ml of human thrombomodiurin is shown in FIG. As is clear from the figure, it is clear that the addition of skim milk suppresses non-specific adsorption without affecting the specific reaction.

Example 6 Anti-human thrombomodulin polyclonal antibody-immobilized beads were prepared in the same manner as in Example 5.

On the other hand, 0.5% BSA-0.015% skim milk-5 mM CaCl-
A solution of human γ-globulin having a concentration shown in Table 2 below was prepared with TBS (pH 7.4), and 0 ng / ml and 14 ng / ml solutions of human thrombomodulin were prepared using these as dilution buffers. 0.2 ml each was added to small polypropylene test tubes. A solution of the peroxidase-labeled anti-human thrombomodiurin monoclonal antibody prepared in Example 2 at an antibody concentration of 3 μg / ml was prepared in the dilution buffer described above, and 0.2 ml was added to the small test tube in correspondence with the human γ globulin concentration. I added them one by one.

Then, add 1 of the above antibody solid beads to each test tube.
The pieces were put in each and incubated at 37 ° C for 90 minutes.

After washing 3 times with TBS-T, 2.5 mM H ₂ O ₂
Add 0.025% 3,3 ', 5,5'-tetramethylbenzidine solution
0.4 ml each was added, and the mixture was subjected to ink incubation at 37 ° C. for 30 minutes, 1 ml of 1N sulfuric acid was added to stop the color reaction, and the absorbance at 450 nm was measured.

From the measured absorbance,
The S / N ratio and measurement sensitivity were calculated in the same manner as in Example 4.
Table 2 shows the results.

From the results in Table 2, it can be seen that the sensitivity of human thrombomodulin measurement can be increased by adding 0.0025% or more of γ-globulin to the immune reaction solution containing skim milk.

Example 7 0.5% BSA-0.2% skim milk-0.015% human γ at 0 ng / ml and 17 ng / ml concentrations of human thrombomodulin.
Globulin _{-5mM CaCl 2 -50mM Tris / HCl-} 0.1M NaCl
(PH7.4) solution (0.2 ml) and 0.5% BSA-5 mM CaCl 2 having the antibody concentration of the peroxidase-labeled anti-human thrombomodulin monoclonal antibody obtained in Example 2 of 3 μg / ml.
₂ 0.2 ml of 50 mM Tris / HCl-0.1 M NaCl (pH 7.4) solution was placed in a small polypropylene test tube, and then anti-human thrombomodulin purine-antibody-immobilized beads prepared in the same manner as in Example 5 were used. Each of them was placed in each of the above small test tubes, and subjected to ink incubation at 37 ° C for 90 minutes.

After removing the solution in each small test tube by suction, 3 ml of physiological saline containing 0.05% of the surfactant in the documents shown in Table 3
And washed three times. After washing, 2.5mM H ₂ O ₂ -0.025% 3,3 ', 5,5'-tetramethylbenzidine solution as a color-developing agent was added in 0.4ml aliquots, and the mixture was subjected to ink incubation at 37 ° C for 30 minutes and 1N sulfuric acid 1ml. To stop the color reaction and add 450 nm
The absorbance of was measured. The S / N ratio was calculated from this absorbance in the same manner as in Example 4. The results are shown in Table 3.

As is clear from Table 3, when using a detergent containing an amphoteric surfactant and a surfactant having an HLB value of 16 or more, human thrombomodulin can be measured with high sensitivity.

Example 8 Tetronic 70 of each concentration shown in Table 4 as a cleaning agent
The physiological saline solution of 2 was used and the same operation as in Example 7 was carried out to examine the effect of the concentration used. Table 4 shows the results.

As is clear from the above results, S in the range of 0.0025 to 1.0%
The / N ratio is large, that is, human thrombomodulin can be measured with high sensitivity. At 2%, foaming was severe and the absorbance at a human thrombomodulin concentration of 17 ng / ml was markedly reduced.

Example 9 Solid beads of anti-human thrombomodulin polyclonal antibody were prepared in the same manner as in Example 5.

On the other hand, 0.5% BSA-0.2% skim milk 0.015% human
γ-globulin-5mM CaCl-50mM Tris / HCl-0.1M Cl
A human thrombomodulin solution (0-33.3 ng / ml) serially diluted with NaCl (pH 7.4) was prepared, and 0.2 ml of each solution was added to a polypropylene test tube. Next, the peroxidase-labeled anti-human thrombomodulin prepared in Example 2
A solution of monoclonal antibody with an antibody concentration of 3 μg / ml was added to 0.5% BSA.
−5 mM CaCl ₂ −50 mM Tris / HCl−0.1M NaCl (pH7.4)
And added 0.2 ml to each of the above small test tubes. Further, the above-mentioned antibody solid beads were put into these small test tubes one by one, and incubated at 37 ° C. for 90 minutes.

After washing three times with 0.005% Tetronic 702-5 mM CaCl-physiological saline, 2.5 mM H ₂ O ₂ -0.025% 3, as a coloring agent,
0.4 ml each of 3 ', 5,5'-tetramethylbenzidine solution was added and incubated at 37 ° C. for 30 minutes, 1 ml of 1N sulfuric acid was added to stop the color reaction, and the absorbance at 450 nm was measured. The calibration curve obtained as a result is shown in FIG.

As is clear from FIG. 3, the background is low and the sensitivity is high (measurement sensitivity calculated by the method of Example 4: 63 pg / ml).
It is clear that the calibration curve of

Continuation of front page (56) Reference JP-A 64-47391 (JP, A) JP-A 50-89525 (JP, A) JP-A 62-272156 (JP, A) JP-A 63-12960 (JP , A) JP 60-36964 (JP, A) JP 57-500845 (JP, A) WO 89/9402 (WO, A)

Claims (8)

(57) [Claims] 1. An antibody immobilized on an insoluble carrier as a first antibody and a labeled antibody as a second antibody in an immunoreaction solution containing skim milk and γ-globulin or a γ-globulin derivative. A method for immunologically measuring human thrombomodulin in a sample according to the method of reacting with human thrombomodulin in the sample, comprising: A. the skim milk in the immunoreaction solution. B. The γ-globulin or γ-globulin derivative is present in the immune reaction solution at a concentration of 0.0025-0.1% by weight so that the final concentration is 0.015 to 0.8% by weight. And C. the first antibody is a polyclonal antibody that recognizes human thrombomodulin and the second antibody is a monoclonal antibody that recognizes human thrombomodulin. An immunological measuring method, which is an antibody and wherein the insoluble carrier has a mirror surface with a center line average roughness of 1.5 μm or less. 2. The monoclonal antibody has an inhibitory effect on the formation of a complex between human thrombomodulin and human thrombin, and an inhibitory effect on the activation of human protein C. The immunological measurement method described. 3. A detergent as an immunological assay method,
The immunological assay method according to claim 1 or 2, wherein a detergent containing an amphoteric surfactant and / or a nonionic surfactant having an HLB value of 16 or more at a concentration of 0.0025 to 1% by weight is used. 4. An antibody immobilized on an insoluble carrier as a first antibody and a labeled antibody as a second antibody in an immunoreaction solution containing skim milk and γ-globulin or a γ-globulin derivative. An immunological assay reagent for human thrombomodulin in a sample to be used, comprising: A. adding the skim milk so that the final concentration of the skim milk in the immunoreaction solution is 0.015 to 0.8% by weight. B. the γ-globulin or γ-globulin derivative is present in the immune reaction solution at a concentration of 0.0025-0.1% by weight, and C. the first antibody is human thrombotic. It is a polyclonal antibody that recognizes modulin, the second antibody is a monoclonal antibody that recognizes human thrombomodulin, and the insoluble carrier has a center line average roughness of 1.5 μm or less. Immunoassay reagent of having a mirror, characterized in that. 5. The monoclonal antibody has an inhibitory action on the formation of a complex between human thrombomodulin and human thrombin and an inhibitory action on the activation of human protein C. The immunological measurement reagent described. 6. A kit for immunologically measuring human thrombomodulin, which is a polyclonal antibody which recognizes human thrombomodulin immobilized on an insoluble carrier and is a polyclonal antibody. A first antibody, (ii) a labeled second antibody that recognizes human thrombomodulin and is a monoclonal antibody, (iii) when the antibody is labeled with an enzyme, to measure the enzyme activity Substrate and reaction terminator, (iv) skim milk, (v) γ-globulin or γ-globulin derivative, (vi) diluent, (vii) detergent, and (viii) standard substance, The skim milk and γ-globulin or γ-globulin derivative are contained in the second antibody, the diluent or the standard substance, and the insoluble carrier has a center line average roughness of 1.5 μm or less. A kit characterized by having a face. 7. The monoclonal antibody has an inhibitory effect on the formation of a complex between human thrombomodulin and human thrombin, and an inhibitory effect on the activation of human protein C. 6. Kit described. 8. The cleaning agent is a double-sided surfactant and / or HL.
0.0025 to 1% by weight of nonionic surfactant with B value of 16 or more
The kit according to claim 6 or 7, wherein the kit is contained at a concentration of.