JP2569383B2 - Immunological assay method, reagent and kit - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for immunologically measuring a trace amount of protein contained in a specimen, a reagent and a kit therefor. To explain in more detail, a method for accurately measuring immunologically a trace protein contained in a human sample by suppressing non-specific adsorption reaction, especially a target protein to be measured, and a structure similar to the protein The present invention relates to a method for immunologically measuring a trace amount of a target protein in a human sample in which a relatively large amount of the protein is present, a reagent, a kit therefor, and a series of techniques related thereto.

BACKGROUND ART Immunoassays using antibodies are widely used due to their high specificity and high sensitivity. With the discovery of monoclonal antibodies in 1975, further development is expected.

For the immunoassay to be highly sensitive, it is essential that specific reactions derived only from the antigen-antibody reaction be high and other non-specific reactions be as low as possible.

Especially when the amount of antigen present in the sample is extremely small,
Alternatively, when a substance having a similar structure to the antigen coexists in a relatively large amount, it is necessary to suppress the non-specific reaction as much as possible in order to increase the measurement sensitivity.

Controlling this non-specific reaction is an important technical point for completing a highly sensitive immunoassay system. Therefore, various attempts have conventionally been made. A commonly known means is to add an additive for suppressing a non-specific reaction to the immune reaction system.

 Some of them will be described below.

(1) Japanese Patent Application Publication No. 57-182169: This publication discloses that in an immune reaction, a polyanion soluble in a reaction medium, such as dextran sulfate, heparin, polystyrenesulfonic acid, cellulose phthalate acetate, hyaluronic acid or chondroitin. A method for treating a sample to be measured with sulfuric acid is described.

(2) Japanese Patent Application Publication No. 58-187862: In this publication, a nonionic surfactant, an anionic surfactant or a cationic surfactant is added to an immunoreactive system in the measurement of a physiologically active substance by an immunoassay. A method for improving the measurement sensitivity by adding a synthetic surfactant such as

(3) Japanese Patent Application Publication No. 60-36964: This publication discloses (i) a surfactant containing one aromatic residue and having an HLB number of 16 or more, and (ii) a zwitterionic interface. Treating the surface with at least one of an activator or (iii) a solution containing a salt of a polyvalent anion at a concentration of 100 mM or more, which limits the non-specific binding of a ligand or antiligand to another substance. Biochemical detection methods have been described.

(4) Japanese Patent Application Publication No. Sho 62-71861: This publication states that "a component of an immune reaction in which one of the reaction components is present on a solid phase at a temperature of 15 to 40 ° C. by the immunoassay principle. A method for measuring an immunoreactive component ", which comprises adding a surfactant having an HLB value of more than 20 in the method. The publication discloses that the addition of a surfactant having the above-mentioned HLB value promotes an immune reaction and further enhances the elimination of the condensation reaction components, particularly unfavorable such as nonspecific addition of a conjugate to a solid. It is stated that side reactions are suppressed.

(5) Japanese Patent Publication No. 59-25184 Japanese Patent Publication No. 59-25184 states that "When determining the amount of a trace substance in a living body by an immunoassay, 0.1% or more of a hydrophobic protein and 0.2 M to 1.0 M of a salt coexist. A method for removing non-specific inhibitory action in an immunoassay characterized by suppressing the influence of an interfering substance contained in a biological sample ", wherein the hydrophobic protein is collagen hydrolyzate, gelatin, lipoprotein lipase. And salts, and salt, phosphate and the like.

(6) Japanese Patent Application Publication No. 61-65162 In this publication, in an immunological measurement method using a mouse-derived monoclonal antibody, mouse serum, mouse ascites, rat serum or rat ascites are used as nonspecific reaction absorbents. And a non-specific reaction absorbent containing serum or ascites described above.

In the method, when a monoclonal antibody (IgG or IgM) derived from a mouse is used as a solid phase and / or a labeled antibody, if an anti-mouse immunoglobulin (for example, anti-mouse IgG or IgM) is present in a specimen, Solid phase and / or
Alternatively, it reacts with the monoclonal antibody of the labeled antibody, and even if the antigen-negative sample shows a positive result, the mouse serum, mouse ascites, rat serum Or use rat ascites. Furthermore, the above publication discloses that other additives such as sheep serum, horse serum, bovine serum, pig serum, rabbit serum, dog serum, chicken serum, saline, PBS or gelatin absorb the non-specific reaction described above. It is stated that there was almost no effect.

The above-described methods for suppressing the non-specific adsorption reaction as described above each have disadvantages, and cannot be said to be a satisfactory method particularly when it is necessary to accurately measure a minute amount of a target substance.

That is, the methods described in the above publications (1) to (4) use various surfactants which are non-proteinaceous substances. However, it is not suitable as a method for measuring a trace amount of a target protein, because it also interferes with the specific adsorption reaction intended originally. On the other hand, the method using the hydrophobic protein described in the above-mentioned publication (5) together with salts did not show the effect of suppressing the nonspecific adsorption reaction in the immunoassay system intended by the present inventors. Further, the method using serum or ascites of mouse and rat described in the above-mentioned publication (6) has a problem in reproducibility and homogeneity especially when ascites is used, and the specific adsorption reaction is reduced depending on the source. In some cases, which is contrary to the original purpose of the immunoassay.

In general, animal sera, particularly mammalian sera, are often used for the purpose of suppressing non-specific adsorption reactions in an immunoassay system for human samples.

However, when using mammalian serum for immunoassay of human samples, the prerequisite for using animal serum is that the animal serum does not contain substances having cross-reactivity with the target human protein. Becomes

According to the study of the present inventors, trace proteins in the sample,
For example, in the case of measurement of a coagulated fibrinolytic protein, since a variety of substances that cross-react with the protein are contained in mammalian serum, it is necessary to suppress non-specific adsorption reaction using mammalian serum. A few problems arose. In order to specifically explain these problems, a problem in the case of measuring a complex by taking as an example a thrombin-antithrombin III complex which is a typical and important protein of the coagulation and fibrinolysis system. This will be described in detail below.

Antithrombin III (hereinafter sometimes referred to as “ATIII”) is an important inhibitor of blood coagulation serine proteases. Thrombin and other activated factors XII, XI, X, IX, kallikrein, and plasmin And so on. The reaction between ATIII and the serine protease proceeds in a molar ratio of 1: 1 and the arginine residue of ATIII is ester-bonded to the serine residue, which is the active center of the serine protease, to form a complex. Inhibits the activity of One such complex
One example is a thrombin-antithrombin III complex (hereinafter sometimes referred to as "TAT"). It is thought that the presence and increase of TAT in the blood is an indication that thrombin has been generated by the initiation and activation of the blood coagulation mechanism.

Generally, in the blood of healthy people, prothrombin is about 110,00
0-230,000 ng / ml, ATIII is about 200,000-500,000 ng / ml, whereas TAT is very small, about 1-3 ng / ml. However, the concentration of TAT in the blood of patients with cerebral infarction is about 10 ng / ml, and TA in the blood of DIC patients.
The T concentration is said to be about 30 ng / ml [see, for example, Clinical Pathology 36, No. 6, pp. 623-626 (1988)]. Thus, the TAT concentration in human blood during the formation of a thrombus or in the process thereof is clearly higher than that of a healthy person.

Therefore, it is presumed that by measuring the amount of TAT in blood, it is possible to know a part of the dynamics of the blood coagulation system, thereby elucidating the patient's condition from the blood coagulation surface, for example, thrombus formation or DIC (pan- It is expected that it will be possible to predict the progress of the pathological condition (developed intravascular coagulation) at an early stage and to carry out appropriate treatment.

Conventionally, as an attempt to measure TAT, anti-TAT neoantigen
Measuring methods using antibodies are being studied. That is, He
rbert, K. Lau et al. have attempted to measure TAT by using an anti-TAT neoantigen antibody by an inhibition assay using ¹²⁵ I-labeled TAT (The Journal of Biological Chemistry Vol. 25).
5, 5885 Issue of Jan. 25 (1980)].

Also, Pelzer et al. Are investigating a TAT measurement system using a sandwich system using an anti-thrombin antibody as a solid-phase antibody and an anti-ATIII antibody as an enzyme-labeled antibody (Thrombosis & Hae).
mostsis, July 14 (1985)).

However, these conventional methods have not always met the current medical needs. For example,
Lau et al.'S method is concerned with the selectivity of the anti-TAT neoantigen antibody, which cross-reacts with free thrombin, ATIII, or prothrombin, and the measured values may vary significantly. Was. The reason is that the TAT concentration in a human sample is generally about 1 / as described above in a healthy person compared to the concentration of free ATIII or prothrombin.
For it is only an amount of 10 ⁵ . In addition, the method of Pelzer et al. Is not sensitive enough, so that it is difficult to dilute plasma to a sufficiently low concentration, and it has a drawback that it is susceptible to plasma interference usually observed in a measurement system using a so-called plasma system. .

Therefore, a measurement system that can specifically measure TAT with high sensitivity using a small amount of sample has been desired.

Therefore, the present inventors added bovine serum to the known TAT measurement system for the purpose of increasing sensitivity, and the bovine serum showed no sensitivity even when added to the TAT measurement system in human samples. No enhancement effect was observed. This is presumably because bovine serum contains components having extremely high cross-reactivity with human serum components, such as bovine ATIII and bovine TAT at high concentrations.

Means for Solving the Problems Accordingly, the present inventors have found that a small amount of a protein such as TAT in a human sample, in particular, a human sample in which a large amount of the small amount of the protein and other proteins having a structure similar thereto are present in a human sample. In order to measure with high sensitivity, research was advanced on an additive for immunoassay which has no cross-reactivity with human sample components and can effectively remove non-specific adsorption.

As a result, in the immunological measurement method, the serum of the bird that has not been subjected to a specific treatment (for example, treatment with a high-concentration saline solution, a low-pH solution, or a high-pH solution) is present in the reaction solution of the immune reaction. It was found that the target specific adsorption reaction was not inhibited, and the non-specific adsorption reaction was sufficiently suppressed, so that a trace amount of protein in the sample could be measured with high sensitivity.

Thus, according to the present invention, there is provided a method for measuring a trace protein in a sample by an immunoreaction using a solid-phase antibody having an antibody immobilized on an insoluble carrier and a labeled antibody obtained by binding a label to the antibody. An immunoassay method characterized by being carried out in the presence of avian serum is provided, and reagents and kits used for the method are provided.

According to the present invention, it is possible to immunologically measure a target trace protein with high sensitivity from a human specimen in which a trace protein in a sample, particularly a trace protein and another protein having a structure similar to the trace protein are present in a large amount. Can be.

 Hereinafter, the present invention will be described in more detail.

In the immunological assay system of the present invention, the target trace protein as the substance to be measured can be various proteins, but as described above, a large amount of other proteins having a similar structure to the target trace protein coexist. The present invention is particularly suitable for the measurement of a target trace protein in a human sample that is present as a target.

"A protein having a similar structure to the target trace protein" refers to a protein having a region having the same amino sequence as the target trace protein and having a strong affinity for a solid phase antibody or a labeled antibody in an immunoassay system. Means

Such target proteins, for example, alpha ₂ - plasmin inhibitor _{(α 2 PI), α 2} - plasmin inhibitor-plasmin complexes, Protein C, Protein S, tissue plasminogen activator, plasmin activator inhibitor complex, Coagulated fibrinolytic proteins such as thrombin-antithrombin complex (TAT); hormones such as human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH): α-fetoprotein (AFP), cancer Fetal antigen (C
Tumor markers such as EA); pulmonary surfactant apoprotein: chondrocalcin and the like. Among these target trace proteins, the coagulated fibrinolytic protein, particularly the thrombin-antithrombin complex (TAT), can be measured with extremely high sensitivity by the immunological assay method of the present invention. The invention applies advantageously.

Avian serum used in the immunoassay system of the present invention includes:
For example, serum obtained from birds such as chicken, goose, turkey, quail or pigeon, particularly from poultry birds may be used, and among them, chicken, goose or turkey serum is preferable.

In the present invention, the avian serum may be present in an immune reaction system for immunologically measuring a target protein in a human specimen. To explain this point in detail, a typical method of immunological measurement is generally called a sandwich method, and the sandwich method is roughly classified into a one-step method and a two-step method. The present invention relates to a one-step method and a two-step method in this sandwich method.
It applies to any of the column methods. That is, the sandwich method by the one-step method is a method in which a human sample containing a target protein, a solid phase antibody immobilized on an insoluble carrier, and a labeled antibody are immunologically reacted in the same reaction system to form a solid phase antibody-target protein-labeled antibody. This is a method for determining the amount of the target protein by forming a complex and measuring the amount of the labeling substance in the complex. On the other hand, the sandwich method according to the two-stage method involves immobilization on a human specimen containing the target protein and an insoluble carrier. First, a solid phase antibody-target protein complex is formed by reacting immunologically with the immobilized solid phase antibody (primary immune reaction), and then a solid phase antibody-target protein-labeled antibody complex is added by adding a labeled antibody. This is a method in which a body is formed (secondary immune reaction) and the amount of the target protein is determined by measuring the amount of the labeling substance in the complex.

According to the present invention, in the above sandwich method, the serum of a bird is allowed to be present in the immune reaction in the one-step method and the primary and / or secondary immune reaction in the two-step method. Specifically, bird serum may be added to the reaction system as it is or in an aqueous solution or buffer, or may be added in advance to a human specimen or a labeled antibody.

In the present invention, the presence of bird serum in the immune reaction system as described above effectively suppresses non-specific adsorption reaction, and the intended specific adsorption reaction is performed without cross-reaction. It becomes possible to measure a very small amount of target protein with high sensitivity.

On the other hand, according to the present inventors, bovine serum, porcine serum, goat serum, rat serum, horse serum, sheep serum, and rabbit serum, which are often used as animal sera for immunological measurement, are trace amounts in human samples. When used for the measurement of proteins, it was revealed that even if the non-specific adsorption was reduced, a cross-reaction was liable to occur and lacked the suitability as an additive for high-sensitivity immunoassay. For example, it has been found that when the measurement target is a protein of the coagulation / fibrinolysis system, a cross-reaction occurs particularly when these animal sera are used in the measurement system of the thrombin / antithrombin III complex (TAT).

Therefore, in the present invention, the trace protein to be measured is not particularly limited, but includes those which easily cause cross-reactivity with antiserum and the like, for example, the coagulation-fibrinolytic protein, and especially the above-mentioned TAT. Next, the present invention, the TAT measurement method as an example, the immunological measurement method, immunological measurement reagents and kits will be specifically described, but this is merely for explanation, the present invention, It is not limited to measuring TAT.

(A) Immunological measurement method of human thrombin / ATIII complex (TAT); an antibody to human thrombin or a fragment equivalent thereto (first antibody) is immobilized on a suitable insoluble carrier (for example, a plastic container) ( This is hereinafter referred to as “solid phase antibody”). Next, the surface of the insoluble carrier is coated with a suitable substance (for example, bovine serum albumin) in order to avoid non-specific binding between the insoluble carrier and the reagent or sample used for measurement.

The thus obtained solid phase antibody is contacted with a human specimen sample for a certain period of time and at a temperature for reaction.

At the time of this immune reaction (primary immune reaction), by adding avian serum to the reaction solution, only non-specific can be reduced without inhibiting the specific reaction. The actual use concentration of bird serum is 1 to 100 v / v%, preferably 5 to 50 v / v%. During this time, the solid-phase antibody and the human / thrombin / ATIII complex (TAT) in the human specimen sample bind.

Then, after washing with an appropriate washing solution, an antibody to human ATIII labeled with an appropriate labeling substance (eg, an enzyme) or a fragment equivalent thereto (second antibody), that is, a solution containing the labeled antibody (eg, an aqueous solution) is solidified. Contact with human-thrombin-ATIII complex (TAT) bound to the phase antibody for a certain period of time and at a temperature for reaction.

By performing this immune reaction (secondary immune reaction) in the presence of avian serum, nonspecific adsorption of the labeled antibody can be effectively suppressed and the specific reaction can be maintained as it is. At this time, the final concentration of the avian serum used in the immunoreaction solution is 1 to 100 v / v%, preferably 3 to 30 v / v%. After the labeled antibody has reacted with the TAT immobilized by the solid phase antibody, the TAT is washed with an appropriate washing solution, and then the amount of the labeling substance labeled on the second antibody present on the insoluble carrier is measured.

Thus, the amount of the human / thrombin / ATIII complex (TAT) in the human sample can be calculated from the value.

As described above, the sandwich method according to the so-called two-step method in which the immune reaction is performed by the two-step reaction of the primary immune reaction and the secondary immune reaction has been described. However, the measuring method of the present invention is not limited to the two-step method, but is described above. The same can be applied to the one-stage method and the like.

(B) Configuration of Measurement Reagent and Kit As measurement reagents for immunological measurement of human / thrombin / ATIII complex (TAT), (a-1) the solid-phase antibody, and (a-2) the labeled antibody Consists of The above (a-
Avian serum or a liquid containing it can be combined with any one or both of 1) and (a-2).

Further, kits for immunological measurement of human / thrombin / ATIII complex (TAT) are described in the above (a-1) and (a-2).
Measuring reagents, and auxiliary agents for efficiently and conveniently using these measuring reagents, for example, (b) a dissolving agent for dissolving a solid reagent or a liquid sample,
(C) an antigen nonspecifically bound to an insoluble carrier, a detergent used for washing the antibody, and (d) a substrate for measuring enzyme activity when using an enzyme-labeled antibody. And its reaction terminator, and (e) other kits commonly used as immunoassay kits.

The human thrombin shown as an example of the present invention described above.
Insoluble carriers used in reagents and kits for immunoassay for ATIII complex (TAT) include, for example, polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, and polysaccharide. , Other paper, glass, metal, agarose, and combinations thereof.

The shape of the insoluble carrier may be various shapes such as a tray, a sphere, a fiber, a bar, a disk, a container, a cell, and a test tube.

Further, it is advantageous to use an enzyme, a fluorescent substance, a fluorescent substance, a radioactive substance and the like as a labeling substance of the labeled antibody. Enzymes include peroxidase, alkaline phosphatase, β-D-galactosidase, fluorescent substances such as fluorescein isothiocyanate and phycobiliprotein, luminescent substances such as isorcinol and lucigenin, and radioactive substances such as ¹²⁵ I and ¹³¹ I ,
^{Although 14} C, ³ H and the like can be used, these are not limited to those exemplified, and any other substances can be used as long as they can be used for immunoassay.

When the labeling substance is an enzyme, a substrate and, if necessary, a color former are used to measure its activity.

When peroxidase is used as enzyme, using H ₂ O ₂ as substrate, as a color-developing agent 2,2' Ajinoji -
[3-ethylbenzthiazoline sulfonic acid] ammonium phosphatase as an enzyme such as ammonium salt (ABTS), 5-aminosalicylic acid, O-phenylenediamine, 4-aminoantipyrine, 3,3 ', 5,5'-tetramethylbenzidine, etc. When used, O-nitrophenyl phosphate or the like is used as a substrate. When β-D-galactosidase is used as an enzyme, fluorescein-di- (β-D-galactopyranoside) or 4-methylumbelliferyl-β is used as a base chamber.
-D-galactopyranoside and the like can be used.

In the kit for immunological measurement, the lysing agent (b) may be any of those normally used for immunological measurement, and includes, for example, a phosphate buffer, a Tris-HCl buffer, an acetate buffer and the like. Those having a pH in the range of 6.0 to 8.0 are shown as preferable examples. Further, as the detergent (c), those generally used for immunological measurement are also used as they are. Examples include physiological saline, phosphate buffer, Tris-HCl buffer, and mixtures thereof. These detergents may further contain a nonionic surfactant such as Triton X100, Tween20 or Brig35, and an ionic surfactant such as sodium dodecyl sulfate.

The above-described immunological assay method, assay reagent and kit,
Although the case where the use of avian serum in the present invention is applied to the measurement of thrombin / antithrombin III complex (TAT) in a human sample has been described, the use of avian serum in the present invention is not limited to the measurement of TAT. The same objects and effects are achieved without being limited to the measurement of other coagulated fibrinolytic proteins and to the measurement of other trace proteins.

Thus, according to the present invention, the following immunological assay method, reagent and kit are provided.

Immunological measurement methodA method for measuring a trace amount of protein in a sample by an immunoreaction using a solid-phase antibody having an antibody immobilized on an insoluble carrier and a labeled antibody obtained by binding a label to the antibody. An immunological assay method performed in the presence of serum.

Immunological measurement reagent (a-1) Immunological measurement of a small amount of protein in a sample consisting of a solid phase antibody having an antibody immobilized on an insoluble carrier and (a-2) a labeled antibody having a labeled substance bound to an antibody. The solid phase antibody of (a-1) or (a-
An immunoassay reagent characterized by combining bird serum or a liquid containing the same with the labeled antibody or both of the above 2).

Immunological assay kit (a-1) a solid-phase antibody in which an antibody is immobilized on an insoluble carrier, (a-2) a labeled antibody in which a label is bound to an antibody, (b) a solubilizer, (c) a detergent, and (d) A) when the labeling substance is an enzyme, a kit for immunologically measuring a trace amount of protein in a specimen, which comprises a substrate for measuring enzyme activity and a reaction terminator, wherein A) solid phase antibody,
-2) an immunological assay kit, wherein at least one of the labeled antibody and (b) the lysing agent is further combined with bird serum or a solution containing the same.

In the above-described measurement system including the immunological measurement method, reagent and kit according to the present invention, the antibodies in the solid phase antibody and the labeled antibody may be either polyclonal antibodies or monoclonal antibodies. Furthermore, these antibodies can be not only whole antibodies, but also their fragments, ie, fragments thereof having the same recognition site. It is preferable to use a Fab ′ fragment or an F (ab ′) ₂ fragment as the fragment.

In the immunoassay system, the insoluble carrier, the labeling substance, the lysing agent, the detergent, and the serum of the bird are the same as those described in the immunoassay system for the thrombin / antithrombin III complex (TAT). be able to.

According to the study of the present inventors, in the immunological assay system for a trace amount of protein in a human sample, in particular, a coagulated fibrinolytic protein in the present invention, the target protein can be highly sensitive by using avian serum. Although it became possible to measure, it was found that more excellent sensitivity and stable sensitivity could be achieved by appropriately combining the following technical aspects.

(1) Use of a carrier having excellent surface smoothness as an insoluble carrier, (2) Use of an amphoteric surfactant or a surfactant-containing liquid having an LHB value of 16 or more as a detergent, (3) Use of a specific protein in an immune reaction Addition By further combining at least one of these embodiments (1) to (3) with the immunological assay system of the present invention, non-specific reactions can be further suppressed and humans can be stably and extremely highly sensitive. A trace amount of protein in a specimen can be measured. Hereinafter, these technical aspects (1) to (3) will be described.

(1) Use of an insoluble carrier having excellent surface smoothness as the insoluble carrier;
The use of an insoluble carrier having a mirror-finished and smooth surface with a non-specific adsorption reaction of a protein or a labeled antibody in a specimen with respect to the carrier as compared with a carrier having a rough surface improves measurement sensitivity. It was also found that the stability increased.

Heretofore, in order to increase the measurement sensitivity in an immunological assay system, an insoluble carrier whose surface has been polished and roughened to increase its surface area has been used. Indeed, if the target protein contains a relatively large amount of the target protein or does not contain other proteins having a similar structure to the protein, the use of an insoluble carrier having a rough surface is reasonable and effective. was there. However, when very little is contained in a sample such as TAT, and when other proteins having a similar structure coexist, non-specific adsorption is suppressed as the surface smoothness increases, The measurement sensitivity increases.

Thus, the insoluble carrier has a center line average roughness (R
Advantageously, a) has a mirror-finished smooth surface of 1.5 μm or less.

The center line average roughness (Ra) is obtained by extracting a portion of the measurement length 1 from the roughness curve in the direction of the center line, setting the center line of the extracted portion as the X axis and the direction of the vertical magnification as the Y axis. To y
= F (x) means that the value of Ra given by the following equation is expressed in micron units.

About this center line average roughness (Ra), JIS B 0601-1
982 (Japan), ANSI B 46.1-1979 (USA) and R 468-1966
(ISO).

 In the following examples of the present invention, the insoluble carrier is
SURFCOM, a surface roughness meter manufactured by Mikko Co., Ltd. Surface roughness using
Was measured.

The material and shape of the insoluble carrier having a smooth surface are not particularly limited, and those described above are shown. Particularly preferred examples include polystyrene beads.

(2) Use of an amphoteric surfactant or a surfactant-containing liquid having an HLB value of 16 or more as a detergent; generally, in immunological measurement, non-specifically remaining or adsorbed to a solid phase or a test tube after an immune reaction. An operation of washing and removing an antigen or a labeled antibody using a detergent is performed. As such a detergent, water, physiological saline or phosphate buffer is usually used.

In order to reduce the non-specific adsorption and increase the measurement sensitivity, it is proposed to increase the number of washings or the amount of detergent, or to specify the washing method and conditions in detail, in order to perform effective washing. Have been. However, even with these measures, a satisfactory cleaning effect may not always be achieved. In particular, in the case of the measurement of a very small amount of an antigen or the measurement of a specific protein in a sample in which a protein having a similar structure coexists, the type of the washing means and the type of the washing solution greatly affects the measurement sensitivity.

Thus, according to the study of the present inventors, an amphoteric surfactant and / or a surfactant having an HLB value of 16 or more contained 1 w / w% or less as a detergent for a solid phase or a container after performing an immune reaction. By using a detergent having a concentration of 6 or less and a pH of 6 or less, the washing effect can be enhanced, and non-specific adsorption can be reduced with a small number of washing times and without requiring special conditions or operations. Was found.

The present inventors have studied the case where a pH 7 detergent containing a surfactant commonly used for an immune reaction is used. As a result, the background value (absorbance when the antigen concentration was 0) could be reduced to some extent, but the effect of increasing the measurement sensitivity up to a very low concentration range such as ng / ml or pg / ml was confirmed. It was difficult to obtain, and was not sufficient for the purpose of reducing nonspecific adsorption and measuring with high sensitivity. In order to improve the measurement sensitivity, it is important to suppress non-specific adsorption and lower the background value as much as possible. In this case, increasing the concentration of the surfactant was examined, but the specific reaction was often suppressed, and the sensitivity could not be improved after all. Therefore, the present inventors focused on the pH of the detergent, and when the pH was set to 6 or less instead of the conventional pH 7, surprisingly, the background value was suppressed without suppressing the specific reaction by adding a small amount of surfactant. It has been found that it can be lowered and the sensitivity can be improved.

The surfactant used for the cleaning agent is an amphoteric surfactant or a surfactant having an HLB value of 16 or more.

 As the amphoteric surfactant, for example, carboxylate type
(Betaine type), sulfonate type (sulfobetaine type)
And a phosphate type. Sulfobeta
Carcinogenicity may be a problem for in-type, and its use is
It is not necessarily favorable from an industrial point of view. Preferably
Tyne type amphoteric surfactant with general formula R₁R_TwoR_ThreeNR_Four[formula
Medium, R₁Is C₈~ C_{twenty two}Alkyl group, R_TwoAnd R_ThreeIs C₁~ C₆Al
Kill group, R_FourIs COO C having a substituent₁~ C₆An alkyl group
A betaine type represented by the following: More preferred
Is R_Two, R_ThreeIs a methyl group, and R_FourIs CH_TwoCOO so
is there. Suitable surfactants of this type include R₁But Lauri
Kao shares “Amphitol 24B” (trade name)
Sold by the company.

As a surfactant having an HLB value of 16 or more, for example, as a nonionic surfactant, polyoxyalkylene alkylaryl ether type, polyoxyalkylene alkyl ether type, polyoxyalkylene polyhydric alcohol fatty acid ester type, polyoxyethylene polyoxy It is sufficient if the HLB value of a propylene polyol type or the like is 16 or more,
For example, Triton X-305 (HLB value = 17.3), Triton X-405
(HLB value = 17.9), Emulgen 950 (HLB value = 18.2), Emulgen 985 (HLB value = 18.9), Tween20 (HLB value = 16.
7), Pluronic F68 (HLB value = 29), Tetronic 707
(HLB value => 20).

Examples of the anionic surfactant include carboxylate, sulfonate, sulfate, and phosphate.
It is sufficient that the HLB value is 16 or more, and examples thereof include sodium n-dodecyl sulfate (HLB value = 40) and sodium tetradecyl sulfate (HLB value = 39).

These surfactants can be used alone,
Two or more surfactants can be used in combination, as in the case of using an amphoteric surfactant and a nonionic surfactant in combination.

The concentration of the surfactant in the detergent is 1 w / w% or less, preferably 0.001 w / w% or more and 0.05 w / w% or less. 0.05w / w
% Of the surfactant, the cleaning effect is excellent, and the foaming during the cleaning is reduced. As a result, the foaming is severe, and the removal of the foam becomes complicated or the removal is insufficient. This is preferable because problems such as insufficientness are reduced.

The detergent contains the surfactant and has a pH of 6
It is adjusted to be as follows. Preferably, its pH is 4 or higher.

The solvent used for the detergent may be any solvent that does not adversely affect the measurement, such as water, physiological saline, or a buffer such as a phosphate-citrate buffer.

(3) Addition of specific protein in immune reaction; molecular weight in immunoreaction solution in the immunoassay system of the present invention
When a protein having a molecular weight of 16-50,000 and an isoelectric point of 1.0-5.0 or a mixture containing the same is present and adjusted so that the final concentration in these immunoreaction solutions is 0.02-0.9% by weight, non-specific adsorption is reduced. It was found that this was suppressed, so that the background was lowered and high sensitivity was easily obtained, which was preferable. Such a protein or a mixture containing the protein may be contained in an immunoassay reagent constituting the immunoassay method of the present invention and an immunoassay reagent constituting another part of the kit so as to have the predetermined amount in an immunoreaction solution. Can also. Such proteins include, for example, casein, pepsin, ovoglycoprotein, and orosomucoid. As such a mixture, for example, 10 to 60% by weight of the protein as a main component, preferably 20 to 50% by weight, 30 to 80% by weight of sugar (eg, lactose), preferably 40 to 60% by weight, and other fats (eg, 0.5 to 2% by weight), ash (eg, 5 to 12% by weight), moisture (eg, 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 compared to the case where casein is used alone, skim milk has better dispersibility in the immune reaction solution, has a higher effect of reducing non-specific adsorption, and is stored at a temperature of 4 ° C. It has the characteristic that the property is good (precipitation hardly occurs). The skim milk may be milk of any origin as long as it is defatted milk, and one of the most typical ones is commercially available Difco
There is skim milk made by the company.

As described above, according to the immunoassay system of the present invention, a coagulated fibrinolytic protein in a human sample can be measured with high sensitivity and stably.

According to the study of the present inventors, in the case of a measurement system of human-thrombin-antithrombin III complex (TAT), which is one of extremely important proteins as a coagulation-fibrinolytic protein, an insoluble carrier is used as a solid phase antibody. For higher sensitivity by selecting an anti-human thrombin antibody or an equivalent fragment immobilized on it, and selecting a labeled antibody with a labeled substance bound to a human antithrombin III antibody or an equivalent fragment. A small amount of TAT
Can be measured.

Thus, according to the present invention, there are provided the following methods (I) to (III) for measuring TAT in human samples, reagents and kits.

(I) Immunoreaction using a solid-phase antibody in which an anti-human thrombin antibody or an equivalent fragment is immobilized on an insoluble carrier, and an anti-human anti-thrombin III antibody or a fragment equivalent thereto bound to a labeling substance as a labeling antibody A method for measuring human thrombin / antithrombin III complex (TAT) in a human sample, wherein the immune reaction is carried out in the presence of avian serum. TAT) immunoassay method.

(II) (a-1) a solid phase antibody in which an anti-human thrombin antibody or a fragment equivalent thereto is immobilized on an insoluble carrier; and (a-2) an anti-human antithrombin III antibody or a fragment equivalent thereto, with a labeling substance. A reagent for immunologically measuring a human thrombin / antithrombin III complex (TAT) in a human specimen comprising a bound labeled antibody, wherein the reagent is the solid-phase antibody or (a-
An immunoassay reagent characterized by combining bird serum or a liquid containing the same with the labeled antibody or both of the above 2).

(III) (a-1) a solid phase antibody in which an anti-human thrombin antibody or a fragment equivalent thereto is immobilized on an insoluble carrier; (a-2) a labeling substance is applied to the anti-human antithrombin III antibody or a fragment equivalent thereto When the bound labeled antibody, (b) lysing agent, (c) detergent, and (d) labeling substance are enzymes, human and human in a human sample combined with a substrate and a reaction terminator for measuring enzyme activity. A kit for immunologically measuring a thrombin-antithrombin III complex (TAT), comprising the solid-phase antibody of (a-1),
An immunoassay kit, wherein at least one of the labeled antibody and the lysing agent (c) is a combination of avian serum or a solution containing the same.

In the immunoassay system for TAT described in the above (I) to (III), the anti-human thrombin antibody used as the solid phase antibody includes an anti-human thrombin polyclonal antibody, an anti-human thrombin monoclonal antibody, An antibody fragment having a function equivalent thereto, for example, F
Each fragment such as (ab ') ₂ , Fab' or Facb is mentioned. Among them, anti-human thrombin polyclonal antibodies are preferable, and such polyclonal antibodies can be prepared by a conventionally known method (for example, Pelzer et al, Thrombosis and Haem).
ostasis 59 (1), 101-106, 1988), which is obtained by purifying an antiserum obtained by immunizing an animal with human thrombin as an antigen using a thrombin-fixed column. Among these, rabbit anti-human thrombin polyclonal antibody or goat anti-human thrombin
Polyclonal antibodies and the like are preferably used.

As a solid phase for immobilizing such anti-human thrombin antibody, as described above, the center line average roughness (Ra) of the surface is 1.5.
It is preferable to use an insoluble carrier having a size of μm or less. By using such an insoluble carrier having a smooth surface, non-specific adsorption by free human antithrombin III in a human sample is remarkably reduced, and TAT can be measured with higher sensitivity.

On the other hand, in the TAT measurement system, as the anti-human antithrombin III antibody as a labeled antibody, anti-human anti-thrombin III polyclonal antibody, anti-human anti-thrombin III monoclonal antibody and those having the same function as those Fragments of antibodies, for example F
Each fragment such as (ab ′) ₂ , Fab ′, and Facb is mentioned. Among them, anti-human antithrombin III polyclonal antibody is preferable in terms of sensitivity. Such anti-human
Antithrombin III polyclonal antibody, a rabbit using human antithrombin III as an antigen by a conventionally known method,
Examples include polyclonal antibodies obtained by immunizing goats and the like. Particularly, such anti-human antithrombin
III The polyclonal antibody was prepared by the method of Nisonoff et al. (A. Nisonof
f et al., Arch. Biochem. Biophys. 89 , 230, 1960), using Fab 'obtained by subjecting F (ab') ₂ obtained by subjecting the obtained F (ab ') This is preferable because a measurement system with higher sensitivity can be obtained.

The binding between the labeling substance and the anti-human antithrombin III antibody or a fragment thereof can be performed according to a conventional method such as a glutaraldehyde method or a maleimide method. A bonded one is preferred. In such a case, the labeling substance can be converted to maleimid in advance to increase the reactivity with the sulfur atom.

In the present invention described above, by allowing bird serum to be present in the immune reaction, it was possible to measure trace proteins with high sensitivity.However, according to another finding of the present inventors, bird serum was labeled antibody. Was found to be very effective in the preparation of

That is, generally, labeled antibodies are lyophilized and used as reagents.Dried labeled antibodies containing avian sera obtained by adding avian sera during lyophilization of the labeled antibodies are added with avian sera It was found that the preparation could be performed more stably than in the case where no preparation was performed, and that a labeled antibody of stable quality could be obtained.

Thus, according to the present invention, there is provided a stabilized labeled antibody or fragment thereof containing avian serum.
In particular, when the antibody in the labeled antibody is an antibody against a coagulation / fibrinolytic protein or its Fab ′ or F (ab ′) ₂ fragment, the above-mentioned stabilizing effect is excellent.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the results of examining the effect of addition of serum from various animals on TAT measurement in an immune reaction.

FIG. 2 shows the results obtained by examining the serum concentration of the added chicken and the effect on the TAT measurement in the immune reaction.

FIG. 3 shows the results of examining the effect of adding chickens on freeze-drying of antibodies.

(F) Examples Hereinafter, the present invention will be described in detail with reference to examples.

In Examples, anti-human thrombin antibody, anti-human ATIII
All antibodies used were polyclonal antibodies obtained by a conventionally known method.

Reference Example 1. Construction of thrombin / antithrombin III complex measuring system (1) Labeling of anti-human / ATIII antibody Fab 'with horseradish peroxylase (HRP): Anti-human / ATIII antibody 2 mg / ml in PBS solution Then, 100 μl of 1M acetate buffer (pH 4.2) and 40 μg of pepsin were dissolved in 20 μl of the same buffer and reacted at 37 ° C. for 16 hours. After the reaction was completed, a Sephadex G-25 column (φ2 cm × 45 cm) equilibrated with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA
And F (ab ') ₂ was collected. Then F
(Ab ') ₂ is reduced using mercaptoethylamine,
Fab 'was purified by gel permeation HPLC using a Tosoh G3000SW column.

On the other hand, as for HRP, maleimidated HRP was isolated using M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Finally, Fab 'and maleimidated HRP
Was mixed and reacted using a Filtron (Ultrafiltration Unit). The reaction was carried out at 4 ° C. overnight, and an HRP-labeled anti-human ATIII antibody Fab ′ was isolated and purified using Tosoh G3000SW.

(2) Measurement of human / thrombin / ATIII complex (TAT) by San Deutsch enzyme immunoassay; one polystyrene bead (Ra = 0.8 μm) immobilized with anti-human / thrombin antibody and purified human / TAT 0n
350 μl of 0.5% BSA-containing PBS solution (pH 7.4) containing g / ml to 10 ng / ml was added to each test tube and incubated at 37 ° C. for 20 minutes. Next, the solution in the test tube is removed by suction, and then washed with PBS.
Add the HRP-labeled antibody prepared in
Incubated for 20 minutes at a temperature of ° C. Next, after aspirating and removing the solution in the test tube, 3,3 ′,
0.02% 5,5'-tetramethylbenzidine hydrochloride, H ₂ O ₂ 0.
0.1 M phosphate-citrate buffer containing 005% (pH 4.0)
Was added to each test tube at a temperature of 37 ° C for 20 minutes, and then a 1N aqueous sulfuric acid solution was used as a reaction terminator.
The enzyme reaction was stopped by adding 1 ml each.

Next, the solution was measured for absorbance at a wavelength of 450 nm using a spectrophotometer to prepare a calibration curve.

Example 1. Measurement of cross-reactive substances in various animal sera: Each of the animal sera shown in FIG. 1 was added to a human / TAT-free system to a final concentration of 10%. In accordance with the above, an immunoreaction was carried out, the absorbance was measured, and the human / TAT conversion concentration (ng / ml) in each animal serum was determined from the value and displayed in FIG. As a result, it was found that substances having a cross-reactivity with human / TAT were not contained in chicken serum of chicken goose, turkey, quail, and pigeon. Furthermore, it was found that the serum of cattle, pigs, sheep and horses contained substances having cross-reactivity with human / TAT.

Example 2. Effect of chicken serum addition on TAT measurement (1) Chicken serum was added to primary immune reaction at 0, 10, 20, 30 v / v%
And TAT = 0, 10, 20 ng / according to the measurement method of Reference Example 1.
An immune reaction was carried out using a normal human sample diluted 4 times as the sample and the sample, and the results were shown in FIG. As shown in FIG. 2, especially when a human specimen was used, it was found that non-specific adsorption was reduced by adding chicken serum, and the specific reaction was maintained.

Example 3. Effect of adding chicken serum in TAT measurement (2) In the TAT measurement described in Example 1, the effect of adding chicken serum to the secondary immune reaction was examined. That is, in Example 2, an immunological reaction was performed by adding TAT = 2.5 ng / ml, chicken serum 10 v / v% to the primary immune reaction, and then an enzyme-labeled antibody (a ), An enzyme-labeled antibody (b) subjected to lyophilization treatment, and an enzyme-labeled antibody (c) subjected to freeze-drying treatment by adding 10 v / v% chicken serum, were used to carry out an immunoreaction. The result is shown in FIG. In these cases, the measurement sensitivity was set to a CV value of 0% at 0 ° C., and the [OD value + 3SD] value at the 0 point obtained from this was determined as the TAT concentration of 0 ng / ml and 2.
The TAT concentration at the point of intersection with the straight line connecting 5 ng / ml was defined as the measurement sensitivity, and this value is also shown in FIG. As shown in FIG. 3, by adding chicken serum (c), the non-specific adsorption of the enzyme-labeled antibody was effectively prevented by lyophilization, and the specific immune reaction was maintained. I understand.

Example 4 [Study of Sensitivity by Each Fractionation of Enzyme-Labeled Antibody] (1) Preparation of Each Enzyme-Labeled Antibody (IgG, F (ab ') ₂ , Fab') (a) HRP Labeling of Anti-Human ATIII Antibody IgG Was added to a 1.0 mg / ml PBS solution of anti-human / ATIII antibody IgG.
50 ml of a 10 mg / ml dimethylformamide solution of (m-maleimidobenzoic acid) -N-succinimide ester (MBS) was added and reacted at a temperature of 25 ° C. for 30 minutes. Then, using a column packed with Sephadex G-25, 0.1M
Gel filtration was performed with a phosphate buffer (pH 6.0) to separate the maleimidated antibody from unreacted MBS.

On the other hand, N-succinimidyl-3- (2-pyridylthio) propionate (SPD) was added to a HRP 1.0 mg / ml PBS solution.
P) Add a 10 mg / ml ethanol solution at 25 ° C
The reaction was performed for 30 minutes. Then, using a column packed with SEPHADEXS G-25, purification was carried out by gel filtration with a 0.01 M acetate buffer (pH 4.5), and a fraction containing pyridyl disulfide-modified HRP was collected. The solution was concentrated about 10 times under ice-cooling. Next, add 0.85% NaCl
0.1 M acetate buffer containing 0.1 M dithiothreitol (pH
4.5) Add 1 ml, stir at 25 ° C. for 30 minutes to reduce the pyridyl disulphide group introduced into the HRP molecule, then run the gel on a Sephadex G-25 column, and fractionate the fraction containing thiolated HRP. I got

Next, the maleimidated antibody and the thiolated HRP were mixed, concentrated to a protein concentration of 4 mg / ml under ice cooling using a collodion back, and allowed to stand at 4 ° C for 24 hours a day. Thereafter, the gel was passed through a column packed with Ultrogel Ac A44 (LKB) to obtain an HRP-labeled anti-human / ATIII antibody IgG fraction.

(B) HRP labeling of anti-human ATIII antibody F (ab ') ₂ The 2.0 mg / ml PBS solution of the anti-human ATIII antibody IgG fraction
100 μl of M acetate buffer (pH 3.7) and 40 μg of pepsin were dissolved in 20 μl of the same buffer and reacted at 37 ° C. for 3 hours. After the reaction, Sephadex G equilibrated with PBS
Separation using -25 column (φ2cm × 45cm) and F (ab ')
₂ were collected. HRP-labeled anti-human ATIII antibody F (ab ') ₂
Was adjusted according to Example 1 (1) (a).

(C) HRP labeling of anti-human ATIII antibody Fab 'in a 2 mg / ml PBS solution of anti-human ATIII antibody, 100 μl of 1 M acetate buffer (pH 4.2) and 20 μl of 40 μg pepsin in the same buffer And reacted at 37 ° C. for 16 hours. After completion of the reaction, a Sephadex G-25 column (φ2 cm × 45 cm) equilibrated with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA.
m), and F (ab ') ₂ was collected. Next, the F (ab ') ₂ was reduced using mercaptoethylamine, and Fa
b 'was purified.

On the other hand, HRP isolated maleimidated HRP using MBS. Finally, while mixing Fab 'and maleimidated HRP, the mixture was concentrated and reacted using a Filtron (Ultra-Period Unit). Incubate overnight at 4 ° C, HRP-labeled anti-human / ATIII antibody
Fab 'was isolated and purified using Tosoh G3000SW.

(2) Measurement of human / thrombin / ATIII complex (TAT) by sandwich enzyme immunoassay One polystyrene bead (Ra = 0.8 μm) on which anti-human / thrombin antibody is immobilized and purified human / TAT
400 μl of a PBS solution (pH 7.4) containing 0.5% BSA and 30% chicken serum containing g / ml to 10 ng / ml was added to each test tube, and incubated at a temperature of 37 ° C. for 20 minutes. Next, the solution in the test tube was removed by suction, washed with PBS, and then the three types of HRP-labeled antibodies prepared in (1), (a), (b) and (c) were added to the test tube, respectively. And incubated at 37 ° C. for 20 minutes. Next, after aspirating and removing the solution in the test tube, while washing with PBS, 3,
3 ', 5,5'-tetramethylbenzidine hydrochloride 0.0225%, H
0.1 M phosphate-citrate buffer containing 0.009% ₂ O ₂ (p
H4.0) was added to each test tube in a volume of 0.4 ml.
After incubating for 1 minute, the enzyme reaction was stopped by adding 1 ml of a 1N aqueous sulfuric acid solution as a reaction terminator.

Next, the absorbance of this solution was measured at a wavelength of 450 nm using a spectrophotometer, and the N / S ratio (%) by the combination of various labeled antibodies, where N is the OD value at TAT 0 ng / ml and S is TAT
The OD value at 10 ng / ml is shown. The results are shown in Table 1.

From Table 1, Fab 'and F (ab') _{2 were used} as labeled antibodies.
It can be seen that when is used, nonspecific adsorption is reduced and sensitivity is particularly excellent.

Example 5 [Study of Sensitivity Based on Roughness of Antibody Solid-Phase Surface] Anti-human thrombin antibody was immobilized and fixed using polystyrene beads for EIA having various center line average roughness shown in Table 2 below. A phase antibody was obtained. On the other hand, a PB solution containing 20% of chicken serum and 25% of TAT-deficient plasma having TAT concentrations of 0 ng / ml and 20 ng / ml was prepared.

Example 4 (2) using these solid phase antibodies and solutions, and using an anti-human ATIII antibody Fab 'as an enzyme-labeled antibody
The TAT was measured in accordance with

Table 2 shows the sensitivity of the measurement of the center line average roughness (Ra) of each surface using polystyrene beads.

From the results in Table 2 above, it can be seen that the use of an insoluble carrier having a smooth surface with Ra of 1.5 μm or less makes it possible to perform TAT measurement with even higher sensitivity.

Example 6 Thrombin / antithrombin by San Deutsch method
In the measurement system of III complex (TAT), the effect of the surfactant used for the washing solution was examined.

(1) Anti-human antithrombin III (ATIII) antibody Fa
b ′ HRP labeling 20 ml of 7 mg / ml PBS solution of anti-human ATIII antibody (pH 7.2)
Was added with 2 ml of 1 M acetate buffer (pH 4.2), and then pepsin
540 μl of the same buffer containing 2.7 mg was added and reacted at 37 ° C. for 5 hours. After completion of the reaction, Sephadex G-25 equilibrated with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA.
The column (φ2 cm × 45 cm) was separated, and F (ab ′) ₂ was collected. Next, the F (ab ') ₂ was reduced using mercaptoethylamine, and Fab' was purified by gel permeation HPLC using a Tosoh G3000SW column.

On the other hand, HRP was maleimidated using SMCC (succinimide 4- (N-maleimidomethyl) cyclohexane-1-carboxylate) to isolate maleimidated HRP. Finally, while mixing Fab 'and maleimidated HRP, the mixture was concentrated and reacted using a Filtron (Ultra-Period Unit). Incubate overnight at 4 ° C, HRP-labeled anti-human / ATIII antibody
Fab 'was isolated and purified using Tosoh G3000SW. The obtained Fab 'was lyophilized by adding 10% of chicken serum thereto.

(2) Measurement of human / thrombin / ATIII complex (TAT) by San Deutsch enzyme immunoassay One polystyrene bead (Ra = 0.8 μm) on which anti-human / thrombin antibody is immobilized and 0n of purified human / TAT
0.1% skim milk, 0.5% BSA at g / ml and 20ng / ml concentration
And PBS solution containing 30% chicken serum (pH 7.4) 400μl
Was added to each test tube and incubated at a temperature of 37 ° C. for 20 minutes. Next, the solution in the test tube was removed by suction, and then washed twice with 3 ml of a physiological saline solution containing 0.0025% of a surfactant of the type shown in Table 3 and adjusted to pH 5.5 and 7.0. Next, Fab 'of the HRP-labeled antibody obtained in (1)
Add 400 μl of 0.1% skim milk and 0.5% BSA-PBS solution adjusted to a concentration of 1 μg / ml to the test tube, and add 37 ° C.
At 20 ° C. for 20 minutes. Next, the solution in the test tube was removed by suction, and then washed three times with 3 ml of the same washing solution as in the washing after the primary reaction. After washing, 400 μl of a 0.1 M phosphate-citrate buffer (pH 4.0) containing 3,225,3,5,5′-tetramethylbenzidine hydrochloride 0.0225% and H ₂ O ₂ 0.009% was placed in each test tube. After incubation for 20 minutes at a temperature of 37 ° C., the enzyme reaction was terminated by adding 1 ml of a 1N aqueous sulfuric acid solution as a reaction terminator.

Next, the absorbance of this solution was measured at a wavelength of 450 nm using a spectrophotometer. The N / S ratio when each cleaning solution was used was compared in the same manner as in Example 4.

The results are shown in Table 3. As is clear from Table 3, when the pH of the washing solution containing the surfactant is 7.0, the absorbance at 0 ng / ml and the N / S ratio are higher when the pH is 7.0 than when the pH is 5.5. It turns out that .1 or less is more advantageous.

Example 7 As a washing solution, a physiological saline solution (pH 5.5) of betaine lauryldimethylaminoacetate at each concentration shown in Table 4 was used, and the effect of the used concentration was examined in the same manner as in Example 6. Table 4 shows the results.

As can be seen from Table 4, the N / S ratio is small over the range up to 0.001%, and the measurement sensitivity in the low concentration region is improved.

At 2%, bubbling was severe and the absorbance of TAT 20 ng / ml was significantly reduced.

Example 8 human plasmin-.alpha. ₂ · plasmin inhibitor complex (PIC) addition effect of chicken serum in the measurement.

(1) Preparation of solid phase antibody An IgG component was separated and purified from an antiserum against human plasminogen obtained by immunization with rabbit and human plasminogen. The obtained anti-plasminogen antibody was adjusted to PBS to a protein concentration of 20 μg / ml, and polystyrene beads (Ra = 3.9 μm) were added thereto to obtain anti-plasminogen antibody-immobilized polystyrene beads by a conventional method. Was.

(2) with anti-alpha ₂ · plasmin inhibitor monoclonal antibody obtained in has been Example 3 The method described in Japanese Patent Publication Sho 60-222426 preparation of labeled antibody of Example 4 (a) to obtain a HRP-labeled anti-alpha ₂ · plasmin inhibitor antibodies in a similar way. The obtained labeled antibody was diluted with PBS containing 0.5% BSA (pH 7.2) to a concentration of 0.4 μg / ml to prepare a labeled antibody solution (A). On the other hand, similarly, the labeled antibody solution (B) was prepared by diluting with PBS containing 0.5% BSA supplemented with 10% chicken serum.

(3) Sandoitsuchi enzyme immunoassay 0.5% BSA containing 0 ng / ml and 50 ng / ml of the measuring method by PIC measured purified human plasmin-.alpha. ₂ · plasmin inhibitor (PIC)
200 µl of the PBS solution (pH 7.2) was collected in a test tube, and 200 µl of the labeled antibody (A) or the labeled antibody (B) obtained in the above (2) was further added thereto. Next, one anti-plasminogen antibody-immobilized polystyrene bead obtained in the above (1) was added to each test tube, and the mixture was incubated at a temperature of 37 ° C. for 60 minutes. Next, after aspirating and removing the solution in the test tube, washing with a physiological saline solution, 1.5% A
BTS (2,2' Ajinoji - [3-ethyl-Benz thiazoline sulfonate (6)]) and 0.1M containing 1 mM H ₂ O ₂
400 μl of a phosphate-citrate buffer (pH 4.5) was added to each test tube and incubated at 37 ° C. for 30 minutes. Next, 0.25 M oxalic acid was added in 1 ml portions to stop the enzyme reaction. Next, the absorbance of this solution was measured at a wavelength of 420 nm using a spectrophotometer. The results are shown in Table 5.

Table 5 shows that when chicken serum was added to the immune reaction system, non-specific adsorption was reduced and the sensitivity was excellent.

 ────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kazuhiko Ito 3-1-2-2, Yamate-cho, Iwakuni-shi, Yamaguchi (56) References JP-A-59-102161 (JP, A) JP-A-52-41222 ( JP, A) JP-A-60-36964 (JP, A) JP-A-62-238463 (JP, A)

Claims (8)

(57) [Claims] 1. A solid phase carrier comprising an anti-human thrombin antibody or a fragment equivalent thereto immobilized on an insoluble carrier,
A method for measuring a human thrombin-antithrombin III complex in a human sample by an immune reaction using an anti-human antithrombin III antibody or a fragment equivalent thereto labeled with a labeling substance as a labeling antibody, A method for immunologically measuring the complex in a human specimen, which is performed in the presence of avian serum that has not been subjected to a specific treatment. 2. The method according to claim 1, wherein the serum of the bird is 5 to 50 v / v in an immune reaction system.
2. The method according to claim 1, wherein the amount is in the range of%. 3. The method according to claim 1, wherein the avian serum is chicken, gachiyo or turkey serum. 4. The insoluble carrier has a surface smoothness with a center line average roughness (Ra) of 1.5 μm or less.
The measurement method described. 5. In the measurement, an amphoteric surfactant and / or a surfactant having an HLB value of 16 or more is used as a detergent at 1 w / w.
The method according to claim 1, wherein a detergent having a concentration of not more than 6% and a pH of not more than 6 is used. 6. The method according to claim 1, wherein the anti-human thrombin antibody or a fragment equivalent thereto is an anti-human thrombin polyclonal antibody or Fab ′, F (ab ′) ₂ or Facb fragment thereof. 7. A method for labeling (a-1) a solid phase carrier in which an anti-human thrombin antibody or a fragment equivalent thereto is immobilized on an insoluble carrier and (a-2) an anti-human antithrombin III antibody or a fragment equivalent thereto. A reagent for immunologically measuring a human thrombin-antithrombin III complex in a human specimen comprising a labeled antibody to which a substance is bound, wherein the reagent is the solid phase carrier of (a-1) or (a-
2) An immunoassay reagent characterized by combining the labeled antibody or both thereof with avian serum not subjected to a specific treatment or a solution containing the same. 8. A solid phase carrier comprising (a-1) an anti-human thrombin antibody or a fragment equivalent thereto immobilized on an insoluble carrier, and (a-2) a labeling on an anti-human antithrombin III antibody or a fragment equivalent thereto. A labeled antibody to which a substance is bound; (b) a lysing agent; (c) a detergent; and (d) if the labeled substance is an enzyme, a human sample in which a substrate for measuring enzyme activity and a reaction terminator are combined. A kit for immunologically measuring a human thrombin-antithrombin III complex, wherein at least one of the solid support (a-1), the labeled antibody (a-2) and the lytic agent (c) One is an immunoassay kit characterized by combining avian serum which has not been subjected to a specific treatment or a solution containing the serum.