JP2561753B2 - Immunological assay kit for human tissue plasminogen activator / human plasminogen activator complex and assay method - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunological measurement of human tissue plasminogen activator / human plasminogen activator inhibitor complex in a human sample. More specifically, it relates to an immunoassay method for measuring the amount of the complex in a human sample with high sensitivity and stability, and a kit therefor.

The symbols used in the present specification have the following meanings unless otherwise specified.

tPA: Human tissue plasminogen activator PAI: Human plasminogen activator inhibitor (tP
Inhibitor for A) tPA-PAI complex; human tissue plasminogen activator / human plasminogen activator inhibitor complex b. Prior Art Plasmin, which is an enzyme that lyses fibrin, is a tissue plasminogen activator of plasminogen. Generated after being converted by beta (tPA). In recent years, an inhibitor of this human tissue plasminogen activator (human plasminogen activator inhibitor (PAI) has been present in vascular endothelial cells, skin cells, platelets, placenta, etc., and human tissue plasminogen activator has been rapidly detected. It was found to form a complex with beta and suppress human tissue plasminogen activator activity (M.Phil
ips et al, Blochem Biophys, Acta, 802 , 99-110, 1984, S.
Thorsen, Biochem Biophys Acta, 802 , 111-118,1984, TW
un et al, J, Biol Chem. 262 , 3646−3653,1987, MASanzo
et al Biochem, 26 , 7443-7449, 1987, Y. Sakata et al, J.
Biol Chem. 263 , 1960-1969, 1988). Furthermore, the relationship between blood plasminogen activator inhibitor (PAI) activity and disease is becoming clear (B. Wiman et al.
Scand.j.Clin Lab Invest 45 , 43−43, 1985, P.Vague e
t al, Metabolism 35 , 250−253,1986, A. Hamsten, Lancet
8549, 3-8, 1987), plasminogen activator inhibitor (PAI) has been suggested to be an important regulator of the initiation mechanism of the blood coagulation line system. Therefore, PAI, tPA
If it is possible to know the blood level of the tPA / PAI complex and tPA / PAI complex, it is highly possible to monitor the above-mentioned fibrinolytic system and the pathological condition.

Currently, as a method for measuring tPA, a sandwich enzyme immunoassay method (JP-A-59-174759) or a commercially available kit (Biopool IMULYSE t-PA) is used.
On the other hand, as a method for measuring PAI, a method using a radioactive substance (RRSchlef et al, J.Lab Clin Med 10
6 , 408, 1985) and sandwich enzyme immunoassay kit using a monoclonal antibody (Biopool IMULYSE PAI-I).

In addition, it is said that the tPA / PAI complex is present in a trace amount in plasma. Takada et al. (Thro
mbosis Research 55, pp. 285-289, 1989), using a monoclonal antibody against PAI as an immobilized antibody, a polyclonal antibody against tPA as a labeling antibody, and a fragment thereof labeled with β-galactosidase, fluorescent. There is an encime immunoassay method.

The system for measuring the tPA / PAI complex is as follows. Immobilized monoclonal anti-PAI-1 antibody is reacted with a sample for 3 hours at 30 ° C., followed by washing, and β′-galactosidase-labeled polyclonal anti-tPA Fab ′. After the reaction with the fragment overnight at 4 ° C, the immune reaction was completed, 4-methyl-umbelliferylgalactoside was decomposed, and the produced 4-methylumbellieferon was measured by a fluorescence method to quantify the tPA / PAI complex. Is what you are doing. This method requires different reaction temperatures in the two immune reactions, requires a reaction time overnight, is extremely long, and uses a fluorometer, which is a special measuring device, so that it can be efficiently measured industrially and clinically. It was difficult to do.

On the other hand, as a method for measuring the tPA / PAI complex, Amiral et al. Have proposed a method for measuring the tPA / PAI complex by using a monoclonal antibody against PAI and a monoclonal antibody against tPA (Thrombosis Research, Supple.
ment VIII; 99-113, 1988). This method uses two kinds of monoclonal antibodies, and the tPA / PAI complex can be measured with relatively good sensitivity. However, according to the ELISA method described in this document, it takes about 5 hours or more in total. I need. Therefore, in order to actually apply the above-mentioned measurement system using these two types of monoclonal antibodies in clinical practice, it is indispensable to further increase the measurement sensitivity, simplify the operation, and shorten the time.

c. Problems to be Solved by the Invention Therefore, the first object of the present invention is to provide tPA / PAI in human samples.
An object of the present invention is to provide an immunoassay method and a kit capable of measuring a complex with high sensitivity.

A second object of the present invention is to provide a method and kit for measuring the tPA / PAI complex in a human sample by a convenient means that can be used clinically.

Another object of the present invention is to stably and highly sensitively measure the tPA / PAI complex in a human sample without being affected by the person who measures it and the measurement conditions (time, temperature, etc.) as much as possible. It is to provide an immunological method and a kit.

Yet another object of the present invention is the active PAI in human specimens.
To provide a measuring method of.

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 object and advantages of the present invention are that a kit for immunologically measuring the tPA / PAI complex in a human sample. (I) a monoclonal antibody (first antibody) to a human plasminogen activator inhibitor bound on an insoluble solid support having a mirrored surface (ii) an enzyme-labeled human tissue plasminogen activator Polyclonal antibody against (second
Antibody) (iii) Substrate and reaction terminator for measuring enzyme activity (iv) Diluent and (v) Detergent containing nonionic surfactant having HLB (Hydrophile Lipophile Balance) value of at least 16 It was found to be achieved by an immunoassay kit characterized in that

Further, according to the study by the present inventors, the following immunological assay method for tPA / PAI and assay method for active PAI are provided.

Immunological assay method for tPA / PAI; (1) contacting a human sample with a first antibody bound to an insoluble solid carrier, and then contacting with a labeled second antibody, or (2) using an insoluble solid carrier By simultaneously contacting the bound first antibody, the labeled second antibody, and the human specimen, the human tissue plasminogen activator / human plasminogen activator-inhibitor complex in the human specimen is immunologically analyzed. (A) a monoclonal antibody against a human plasminogen activator inhibitor bound on an insoluble solid support having a mirrored surface is used as a first antibody, and (b) is labeled with an enzyme. Second polyclonal antibody against humanized tissue plasminogen activator
An immunological measurement method, comprising: (c) a detergent containing a nonionic surfactant having an HLB (Hydrophile Lipophile Bakance) value of at least 16 as an antibody.

Method for measuring active PAI; (A) Human tissue plasminogen activator / human plasminogen inhibitor complex present in human specimen and (B) Present in human specimen to which human tissue plasminogen activator is added Human tissue plasminogen activator and human plasminogen inhibitor complex were measured by immunoassay by sandwich method, respectively, and the active human plasminogen activator in human specimen was analyzed based on the difference between the measured values. A method for determining the amount of an inhibitor comprising: (a) using as a first antibody a monoclonal antibody against a human plasminogen activator inhibitor bound on an insoluble solid support having a mirrored surface; Labeled human tissue plasminogen activator A polyclonal antibody against the Tar second
An active human plasminogen, which is used as an antibody, and (c) is a detergent containing a nonionic surfactant having an HLB (Hydrophile Lipophile Bakance) value of at least 16. Activator-on-hibitor measurement method.

In the present invention described above, (i) a monoclonal antibody against PAI is used as the first antibody (solidified antibody) and enzyme-labeled as the second antibody.
using a polyclonal antibody against tPA in combination, (ii) using a solid support having a mirror-finished surface, that is, having a very smooth surface, as an insoluble solid support for immobilizing the first antibody, And (iii) the use of a cleaning agent containing a nonionic surfactant having an HLB value equal to or higher than a certain value, and (i), (ii) and (ii)
The object of the present invention is achieved by the synergistic effect of i). That is, the above (i), (ii) and (iii)
As a synergistic effect of, the nonspecific adsorption of tPA, PAI and other solid carriers to a large number of proteins present in human specimens is suppressed as much as possible, and the nonspecific adsorption of the second antibody is also suppressed and specific Adsorption is selectively promoted, and components unnecessary for immune reaction can be effectively washed away.

Thus, according to the present invention, tPA / PAI contained in an extremely small amount in a human sample can be measured with high sensitivity, simply, accurately and stably, and the time required for the measurement is much wider than that of the conventional method. In addition, the practical effect that the amount of the cleaning agent used is also reduced is achieved.

Furthermore, according to the present invention, when performing an immune reaction in two steps, it is not necessary to set different temperatures in each step, and it is possible to easily carry out the reaction at the same temperature. The process can be performed in a short time of 2 hours or less.

Therefore, the immunoassay system according to the present invention can efficiently and rapidly measure a human specimen having or suspected to have an abnormality in coagulation / fibrinolytic activity, and its clinical significance is extremely great.

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

[A] Preparation and purification of antigen; The monoclonal antibody against PAI and the polyclonal antibody against tPA used in the present invention can be obtained by using the antigen described below.

That is, tPA as an antigen for obtaining these antibodies
Alternatively, as PAI, in principle, both a natural type extracted from a natural material and a recombinant type produced by gene recombination technology are used, but PAI has immunological properties equivalent to those of tPA or PAI. If it is available, it can be obtained by a genetic engineering method. A cell culture medium is preferably used as a material for obtaining natural tPA or PAI. Separation,
Purification can be carried out by combining commonly used protein separation techniques, for example, salting out, pouring out, centrifugation, ultrafiltration, various chromatographies and the like.

Using tPA or PAI thus obtained as an antigen, a polyclonal antibody or a monoclonal antibody can be prepared.

[A]-(1) Preparation of polyclonal antibody against tPA; The polyclonal antibody against tPA used in the present invention can be obtained according to a method known per se by using the early stage tPA as an antigen. For example, as described in "The Biochemical Society of Japan, Sequel Biochemistry Laboratory, Vol. 5, 1-10, Tokyo Kagaku Dojin, 1986", immunized animals such as guinea pig, rabbit, rat, mouse, goat, etc. After immunization with tPA by a usual method using an animal capable of producing antibodies, blood is collected to obtain antiserum. A purified antibody can be obtained from antiserum by combining a method usually used, for example, salting out, extraction, centrifugation, ultrafiltration, various chromatography and the like.

[A]-(2) Preparation of Monoclonal Antibody Against PAI; The monoclonal antibody against PAI used in the present invention uses PAI as an antigen, and the cell fusion method (K.
r and Mulstein, Nature (Lind on), 256, 495−497 (197
It can be obtained by culturing and secreting the hybridoma prepared in 5) and separating it from the culture solution. That is, after immunizing a mouse with PAI, pancreatic cells of this mouse are fused with mouse myeloma cells to prepare a hybridoma. The hybridoma thus obtained produces various monoclonal antibodies in response to various fused lymphocytes, so that the hybridoma producing the desired monoclonal solid is isolated as a cloned hybridoma by cloning. The cloned hybridoma is cultured in vitro or intraperitoneally in the mouse to secrete the monoclonal antibody. Monoclonal antibodies against PAI are separated from this culture medium by a conventional method.

As such a monoclonal antibody against PAI, JTI-1, JTI-2, JTI-3 or JTI-4 previously proposed by the present applicant and described in European Patent Publication No. 339302 can be used. Of these, JTI-3 and JTI-4 are preferable in the present invention, and JTI-4 is particularly excellent.

The two JTI-3 and JTI-4 monoclonal antibodies have been internationally deposited under the Budapest Treaty as described below.

Monoclonal antibody JTI-3 against PAI; this monoclonal antibody JTI-3 against PAI has a hybridoma producing it, which is an international deposit period under the Budapest Treaty.
nstaitute) on November 22, 1988 as FERMp-10405 and subsequently converted to an international deposit on March 2, 1989 (international deposit; No. BP-2317).

The monoclonal antibody JTI-3 against this PAI is not inhibited from binding to PAI even when the subclass is IgG ₁ and tPA binds to PAI (that is, to the tPA-PAI complex), but to PAI. When bound, tPA has the property of recognizing an antigenic determinant site that inhibits binding.

Monoclonal antibody JTI-4 against PAI; this monoclonal antibody JTI-4 against PAI is a hybridoma producing the same, which was reported to the above-mentioned Micro Engineering Institute in November 1988.
It was deposited as FERMp-10406 on the 22nd and was subsequently converted to the international deposit on March 2, 1989 (International Deposit No. BP-231.
8).

The subclass of this monoclonal antibody JTI-4 against PAI is IgG ₁ , and even if tPA binds to PAI, the binding to PAI is not inhibited.

[B] Preparation of tPA-PAI complex measurement kit; [B]-(1) Preparation of first antibody As the first antibody of the present invention, PAI bound to an insoluble solid carrier having a mirrored surface is used. It is necessary to use a monoclonal antibody against.

By using a solid support having a mirror surface, that is, an extremely smooth surface, non-specific adsorption is reduced and the measurement sensitivity of the tPA-PAI complex is improved.

Conventionally, as an insoluble solid carrier for highly sensitive measurement, a carrier whose surface has been polished to roughen it to increase its surface area has been used. Certainly, increasing the surface area has the advantage of increasing the amount of immobilized antibody, but has the disadvantage of increasing non-specific adsorption. According to the research conducted by the present inventors, in the measurement of tPA-PAI complex, a mirror-finished solid support having a center line average roughness (Ra) of 1.5 μm or less was
It was found that the amount of immobilized antibody was almost the same as that of the roughened solid support, but non-specific adsorption was drastically reduced, which was advantageous for highly sensitive measurement of tPA-PAI complex. 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 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.

Regarding this center line average roughness (Ra), JIS B 060
1-1982 (Japan), ANSI B46.1-1979 (USA) and R468
-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.

As a monoclonal antibody against PAI immobilized on an insoluble carrier that becomes harder, not only the above-mentioned monoclonal antibody molecule but also its fragment such as (ab ′) ₂ , Fab, Fab ′, Facb or the like which binds to the antigen It is a derivative of an antibody or fragment thereof that does not lose its ability. Methods for immobilizing these antibodies on insoluble carriers include physical adsorption methods such as immersing a polystyrene carrier in a solution of the antibodies; ionic bonding methods such as ion exchange resins or amine groups, carboxylic acid groups, sulfonic acid groups. , A method using a carrier having an ionizable functional group such as a phosphate group; or 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 activity Polysaccharide method, diazo method, active ester method, carrier binding method using a cross-linking reagent (glutaraldehyde, hexamethylene isocyanate, succinimide / maleimide compounds etc. as cross-linking reagents), and PAI
A method of binding via a substance which has no binding ability to PAI but can bind to a monoclonal antibody against PAI by a biological reaction, for example, a method using a protein A binding carrier.

[B]-(2) Preparation of second antibody (labeled antibody); In the present invention, a polyclonal antibody against tPA labeled with an enzyme is used as the second antibody.
The poclonal antibody against tPA described in [A]-(1) above or a fragment equivalent thereto is labeled with an enzyme used in an immunoassay.

Examples of the enzyme used for this labeling include lysozyme malate dehydrogenase, glucose-
6-phosphate, dehydrogenase, peroxidase, glucose oxidase, alkaline phosphatase, luciferase, β-galactosidase and the like can be mentioned. As a method for binding these enzymes to the polyclonal antibody, a usual method such as a glutaraldenide method, a periodate acid method, and a maleimide method can be used, but the maleimide method is preferably used.

Maleimidation of antibodies and enzymes is a method known per se (Eiji Ishikawa, “Enzyme Immunoassay”, Medical Institute), for example, succinimidyl 4- (N-maleimidomethyl) cyclohexane carbonate (SMCC), sulfosuccinimidyl, 4-. (N-maleimidomethyl) cyclohexane carbonate (sulfo SMCC), succinimidyl-metamaleimidobenzoate (MBS), succinimidyl 6
It can be done using a cross-linking reagent such as maleimidohexanoate (EMCS).

The reaction of a polyclonal antibody against tPA or a fragment thereof with a labeling substance is carried out by using the above-mentioned cross-linking reagent for maleimide-ized polyclonal antibody or a fragment thereof and, for example, an enzyme introduced with an SH group by δ-acetylmercaptosuccinic anhydride, for example, 4 It is carried out at a temperature of -30 ° C and a molar ratio of the antibody or fragment to the SH group-introduced enzyme of 1: 1 to 1:80 and for 1 to 48 hours.

In this case, it is preferable to obtain mainly one molecule of the labeling substance per molecule of the antibody or its fragment through the sulfur atom of the antibody or its fragment.

[B]-(3) Detergent In the immunological assay method by the sandwich method, non-specific adsorption occurs with high sensitivity by simply combining the immobilized antibody (first antibody) and the labeled antibody (second antibody). I cannot assemble the measurement system. Therefore, suppressing non-specific reactions is the key to high-sensitivity measurement. For example, when a surfactant is used in the measurement system, non-specific adsorption is reduced, but not all surfactants are effective in the measurement system of tPA-PAI complex.

According to the present inventors, a substance which does not suppress the immune reaction in the assay system of tPA-PAI complex and which does not participate in the immune reaction, in which the first antibody and the second antibody are combined as described above 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 has an HLB value (Hydraphile
Lipophile Balance) is 16 or more nonionic surfactants.

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

Examples of the non-ionic surfactant having an HLB value in the 16th position include polyoxyalkylene alkylaryl ether type, polyoxyalkylene alkyl ether type, polyoxyalkylene polyhydric alcohol fatty acid ester type, polyoxyethylene polyoxypropylene polyol. The HLB value of the system or the like may be 16th or higher, for example, Triton X-305 (HLB17.3), Triton X-405 (HLB17.9), Emulgen 950 (HLB18.2), Emulgen 985 (HLB18. 9),
Examples include Tween20 (HLB16.7), Bluronic F68 (HLB29), and Tetranic 707 (HLB> 20).

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

The odor of the surfactant in the cleaning solution is 1 w / w% or less, preferably 0.001% or more and 0.1 w / w% or less.

In the present invention, even with a surfactant amount of 0.1 w / w% or less, excellent cleaning effect, less foaming during cleaning, resulting in severe foaming, complicated removal of foam, or its removal. This is preferable because it causes less problems such as insufficient cleaning and conversely insufficient cleaning.

As a solvent for the nonionic surfactant, water, physiological saline,
Alternatively, any buffer such as a phosphate buffer may be used as long as it does not adversely affect the measurement.

[B]-(4) Diluent: The diluent used in the measurement kit and the measurement method of the present invention may be any diluent usually used in immunological measurement. 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.

[B]-(5) Substrate and reaction terminator for measuring enzyme activity: The substrate and reaction terminator used in the assay kit and assay method of the present invention correspond to the type of enzyme as a labeling substance. Then, those generally known in immunological measurement can be used. As an example thereof, 2,2'-azino-di- [3ethylbenzthiazolinesulfonic acid] diammonium salt (AB
TS), orthophenylenediamine (OPD), 3,3 ', 5,5'
-There is tetramethylbenzidine (TMB), etc., and H ₂ SO ₄ , HCl, acetic acid, glycine buffer (pH 10.3),
Sodium fluoride solution and the like.

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

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

[B]-(6) Use of protein According to the studies conducted by the present inventors, in the measurement of tPA-PAI complex, the molecular weight of the immunoreaction solution was 16,000 to 50,000, preferably 20 to 46,000 and isoelectric. Point 1.0-5.0, preferably 1.2-
When the protein of 4.8 is present and adjusted so that the final concentration of this protein in the immunoreaction solution is 0.02 to 0.9% by weight, the nonspecific adsorption is further suppressed, and therefore the background is remarkably lowered and the high sensitivity is further enhanced. It turned out that it was easy to obtain. Examples of such substances include casein, β-casein, α-casein, pepsin, ovoglycoprotein, orosomucoid and the like. Alternatively, such proteins can be used as a mixture. Examples of such a mixture include, for example, 10 to 60% by weight, preferably 20 to 50% by weight, of the above-mentioned protein as a main component, 30 to 80% by weight of sugar (e.g., lactose), preferably 40 to 60% by weight, and other fats (for example, 0.5 to 2% by weight), ash (eg 5 to 12% by weight), water (eg 2 to 8% by weight) and the like can be contained. A typical example of such a mixture is skim milk. Skim milk contains casein as a protein, but skim milk has better dispersibility in an immunoreaction solution, a higher effect of suppressing non-specific adsorption per unit weight of protein, and a higher temperature than the case of using casein alone. It has the characteristic of good storage stability at ℃ (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. That is, it is possible to allow skimmed milk to exist in the immune reaction solution and adjust its final concentration in the immune reaction solution to 0.002 to 0.8% by weight.
Similarly, high sensitivity is obtained, which is preferable. If the concentration is lower than 0.002% by weight, sufficient inhibitory effect is not obtained, and if the concentration is higher than 0.8% by weight for skim milk and 0.9% by weight for the above proteins, the specific reaction is also suppressed. Not preferable.

The above-mentioned protein may be used so as to have the above-mentioned concentration in the immune reaction solution as described above, and may be contained in the diluent or contained in the secondary antibody.

[B]-(7) Measuring method As the human sample in the measuring method of the present invention, an ordinary clinical sample, for example, blood in the form of serum or plasma, synovial fluid, lymph, thymus fluid, ascites fluid, amniotic fluid, cell tissue fluid, bone marrow. Any liquid such as liquid or urine may be used as long as it contains the tPA-PAI complex. Preferred is blood in serum or plasma form.

In the measurement method of the present invention, the primary antibody and the secondary antibody are used in combination, and (i) a human sample is contacted with a first antibody bound to an insoluble solid carrier, washed, and then labeled with a second antibody. By contacting the antibody, or (ii) allowing the first antibody bound to the insoluble solid carrier, the labeled first antibody, the labeled second antibody and the human sample to exist in the same system, The amount of tPA-PAI complex in a sample can be measured with high sensitivity. The method (i) is superior to the method (ii) when tPA is present in a human sample at a high concentration, for example, even in a patient sample to which tPA is administered for the purpose of treatment, tPA is not affected.

The temperature of the immune reaction at the time of measurement is not limited as long as it does not denature the properties of the protein as a constituent element and significantly suppresses the immune reaction, but it is generally 50 ° C or lower, preferably about 4 to 45 ° C. The reaction may be performed under temperature conditions for about 5 minutes to 5 hours, preferably about 30 minutes to 3 hours.

For example, when the reaction temperature is close to the body temperature such as 37 ° C., the nonspecific adsorption reaction can be extremely reduced,
It is also one of the characteristics of the system of the present invention that it exerts great power in terms of high sensitivity of the measurement system.

[C] Method for measuring active PAI The method for measuring the tPA-PAI complex according to the present invention described above is
Since the tPA-PAI complex in a human sample can be measured with extremely high sensitivity, the use of this method makes it possible to easily and sensitively measure the amount of active PAI in a human sample. .

Conventionally, tPA or PAI was used to measure the concentration of active PAI.
Takada et al. Have reported a method that utilizes the reaction to generate a tPA-PAI complex. [Thrombosis Rese
aech55, 285-589 (1989)]. In this method, the reaction between PAI and tPA in the sample is carried out at 4 ° C overnight,
The amount of tPA-PAI complex is then measured for production.

And the method to measure this tPA-PAI complex is 30
After reacting for 3 hours at ℃, washed and then reacted with β-galactosidase-labeled polyclonal antibody tPA.Fab 'fragment overnight at 4 ℃, complete the immunoreaction, 4-
Methyl-umbelliferylgalactoside was decomposed, and the resulting 4-methylumbelliferone was measured by fluorescence,
It is a quantification of the tPA-PAI complex. Such a method requires different reaction temperatures in two immune reactions, requires two days for measurement, is very long, and uses a fluorometer, which is a special measuring device, so that it is industrially and clinically efficient. It was difficult to measure.

On the other hand, according to the present invention, tPA was added to a human sample to react with active PAI and tPA, the amount of the resulting tPA-PAI complex was measured, and the complex in the sample to which tPA was not added was measured. The amount of active PAI can be measured easily and in a short time from the difference with the amount of PAI.

e. Effect of the Invention Thus, according to the method of the present invention, a sample containing a trace amount of tPA-PAI complex such as a clinical sample is used as a sample, and the tPA-PAI complex in the sample is highly sensitive and highly accurate, and It can be quantified by a simple operation.

Further, similarly, the amount of active PAI in the sample can be easily measured in a short time with high sensitivity and high accuracy.

f. Examples The present invention will be described in detail below with reference to examples. In the examples, "%" indicates "% by weight".

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a calibration curve for immunological measurement of tPA-PAI complex, showing the relationship between the concentration of tPA-PAI complex and the absorbance.

FIG. 2 shows the relationship between the washing time and the absorbance in the immunological measurement of the tPA-PAI complex.

FIG. 3 shows the relationship between the degree of surface roughness of the insoluble solid support and the nonspecific adsorption rate in the immunological measurement of the tPA-PAI complex.

Reference Example [Preparation of peroxidase-labeled anti-tPA monoclonal antibody Fab 'fragment] Monoclonal antibody against human tissue plasminogen activator (JTA-l; European Patent Publication No. 33930)
Fab ′ fragment (see No. 2) was prepared according to the method of Nisonov, and bound to peroxidase by a conventional method to obtain a peroxidase-labeled Fab ′ (edited by the Biochemical Society of Japan, sequel to biochemistry laboratory, Vol. 5, 109- 112 pages, Tokyo Kagaku Dojin, 1986
Year). That is, 40 mL of 2% of a 1.0 mg / mL solution of the monoclonal antibody (0.01 M phosphate-0.15 M NaCl pH7.2 (PBS)) was used.
Add μg of pepsin and p with 1M citrate buffer (pH 3.5).
After adjusting to H3.7, it was digested and decomposed at 37 ° C. for 1 hour. 1N NaOH was added dropwise to the reaction solution to adjust the pH to 8 to stop the reaction. TSKGe
i G-3000SW column (Tosoh) -5 mM for eluate by HPLC
An F (ab ') ₂ fragment having a molecular weight of 100,000 was separated from this reaction solution using EDTA-0.1M phosphate buffer (pH 6.0).

It was concentrated by ultrafiltration. 0.1M 2-
200 μm of mercaptoethylamine was added and reduction treatment was carried out at 37 ° C. for 2 hours. The reaction solution was concentrated by an ultrafiltration filter and then concentrated by HPLC in the same manner as F (ab ′) ₂ by HPLC to obtain a Fa of 50,000.
b'is separated and 1.07 mg / ml of Fab 'fragment is added to 1.0 ml.
Obtained.

On the other hand, horseradish peroxidase (Toyobo IC)
6.0mg of 0.1M sodium phosphate buffer (pH 7.0) 0.9ml
Dissolved in N-succinimidyl-3-maleimidomethyl-cyclohexane carbonate (Pierce, SMCC)
Dimethylformamide solution of 60μ (2.9mg / 80μ)
It was dropped and reacted at 30 ° C. for 1 hour. This reaction solution was added to a Sephadex G-25 column, and 0.1 M sodium phosphate buffer (pH 6.0) was passed to separate the maleimidated peroxidase. 3.2 mg of the thus obtained maleimidated peroxidase and 1.0 mg of Fab ′ fragment were reacted at 25 ° C. for 24 hours, and TSK Gel G-3000 SW column-HPLC (eluent: 0.
After separation and purification with (01M PBS), 0.35 mg of peroxidase-labeled mouse anti-tPA antibody Fab ′ fragment with a molecular weight of about 130,000 was obtained. The binding molar ratio of Fab ′ fragment to peroxidase determined from the absorbance at 280 nm and 403 nm of the obtained labeled antibody was 1: 2.

Example 1 [Preparation of peroxidase-labeled anti-tPA polyclonal antibody] Goat anti-human tPA antibody (Biopool) in PBS (1 m
g / ml) 5.0 ml to N- (m-maleimidobenzoic acid) -N-
250 μm of succinimide ester (MBS, Pierce) dimethylformamide solution (10 mg / ml) was added and reacted at a temperature of 25 ° C. for 30 minutes. Then, using a column packed with Sephatex G-25, 0.1 M phosphoric acid was added. Gel filtration was performed with a buffer solution (pH 6.0) to separate the maleimidated polyclonal antibody and unreacted MBS.

On the other hand, 5.4 ml of a 0.1M phosphate buffer solution (pH 6.5) containing horseradish peroxidase (HRP) (10 mg / ml) was added to dimethylformamide solution of S-acetylsuccinic anhydride (60 mg).
/ ml), and react at 25 ° C for 30 minutes, then
Add 2.2 ml of 0.1M-Tris buffer (pH 7.0), 4.3 ml of 1M-hydroxylamine aqueous solution, 0.4 ml of 0.1M EDTA aqueous solution, and add 4.
The mixture was stirred for 1 minute, and gel filtration was performed with 0.1 M phosphate buffer (pH 6.0) using a column packed with Sephatex G-25 to separate SH-modified peroxidase.

The MBS antibody (2.4 mg) and the SH-oxidized peroxidase (5.3 mg) were reacted at 25 ° C for 20 hours and then subjected to HPLC (TSK Gel
G-3000 SW) was used for gel filtration to obtain 1.9 mg of peroxidase-labeled anti-tPA polyclonal antibody.

[Preparation of Immobilized Antibody] Polystyrene beads having a center line average roughness (Ra) of 1.35 μm (D-7, Immunochemical Co., Ltd.) were added with 0.1 M phosphoric acid citrate of mouse anti-PAI monoclonal anti-PAI antibody (JTI-4). Immerse in a buffer solution (pH 3.0) (20 μg / ml) at 4 ℃ for 2
Left for 0 hours. After washing the beads with PBS solution, 1%
After standing still in the BSA-PBS solution at room temperature for 2 hours, the beads were washed again with the PBS solution to obtain mouse anti-PAI antibody-immobilized beads.

[Preparation of calibration curve] Human tPA / PAI complex containing 0.25% skim milk, 0.
Diluted with 01M phosphate-0.5M NaCl-2% BSA buffer (pH 7.2) to prepare 25, 12.5, 6.25, 3.125, 1.56 and 0 ng / ml solutions. 0.4 ml of each concentration solution and mouse anti-PAI antibody-immobilized beads were placed in a small test tube and incubated at 37 ° C. for 1 hour. Then, the plate was washed twice with a washing solution containing 0.05% Tween 20 in PBS.

1 μg of peroxidase-labeled tPA polyclonal antibody
/ ml with PBS-0.05% Tween 20 solution,
0.4 ml each was added to a small test tube and incubated at 37 ° C. for 30 minutes. After washing three times with a washing solution, 0.4 ml of a substrate solution for peroxidase, 2.5 mM H ₂ O ₂ -0.025%, 3,3 ′, 5,5 ′
-Tetramethylbenzidine 0.1M phosphoric acid citric acid (pH 4.
0) The solution was added and color was developed at 37 ° C for 30 minutes, the color development was stopped with 1N sulfuric acid, and the absorbance at 450 nm was measured.

 The calibration curve is shown in FIG.

Example 2 [Comparison of peroxidase-labeled anti-tPA monoclonal antibody and anti-tPA polyclonal antibody] 0.01 containing 0.25% skim milk of human tPA / PAI complex.
M Phosphate-0.5M NaC Rittor-2% BSA buffer (pH7.2)
Was diluted with to prepare solutions of 25, 12.5, 6.25 and 0 ng / ml.
0.4 ml of each concentration and the mouse anti-PAI antibody-immobilized beads prepared in Example 1 [Preparation of immobilized antibody] were put in a small test tube.
Incubation was carried out at ℃ for 1 hour. Then 0.05 in PBS
After washing twice with a washing solution containing 10% Tween 20, the peroxidase-labeled anti-tPA polyclonal antibody obtained in Example 1 or the peroxidase-labeled mouse anti-tPA antibody Fab ′ fragment obtained in Reference Example was adjusted to 1 μg / ml with PBS-0.05.
Adjust with% Tween20 solution and add 0.4 ml to each small test tube.
Incubated for 30 minutes at ℃. After washing twice with the washing solution, add 3 ml of the washing solution to a small test tube, and add 0, 15, 1
After standing for 0, 20 and 30 minutes, it was removed by suction with an aspirator.
Next, 0.4 ml of peroxidase substrate (the same as in Example 1) was added, color was developed for 30 minutes, and the color development was stopped with 1N sulfuric acid.
Absorbance at 0 nm was measured. FIG. 2 shows the relationship between the absorbance and the time when the cleaning solution was added and allowed to stand. From this figure, it can be seen that when the peroxidase-labeled anti-tPA polyclonal antibody is used, the decrease in absorbance is small and the effect of the time required for the washing operation is small.

Example 3 Made of polystyrene having a direct diameter of 6.35 mm and different surface roughness
The beads were treated in the same manner as in [Preparation of immobilized antibody] in Example 1.
Thus, mouse antibody PAI antibody-immobilized beads were obtained. Human tPA / PA
A solution containing 0.25 mg / ml of I complex was added to 0.25% skim milk-
Prepared with 0.01 M phosphate-0.5 M NaCl-2% BSA buffer,
Concentration solution 0.4ml and mouse anti-PAI antibody with different surface roughness
Put in a fixed bead and a small test tube and incubate at 37 ℃ for 1 hour.
I did it Next, wash twice with PBS containing 0.05% Tween 20.
Washed. The peroxidase-labeled anti-antibody obtained in Example 1
PBS-0.05% Tween20 solution of body tPA polyclonal antibody
Add 0.4 ml of (1 μg / ml) to a small test tube at 37 ℃ for 30 minutes.
Ink used. Washed three times with the cleaning solution
And 0.4 ml of peroxidase substrate flow at 37 ° C for 3
Color for 0 minutes, stop coloring with 1N sulfuric acid, and absorb at 450 mm
It was measured. From the obtained absorbance, the non-specific adsorption rate (0ng / ml
Absorbance of ÷ absorbance of 25 ng / ml x 100) was determined. Polisti
The surface roughness of renbeads is that of Tokyo Seimitsu Co., Ltd.
Elf It was measured at. Non-specific adsorption rate on the vertical axis, surface
The centerline average roughness (Ra), which indicates the roughness, is plotted on the horizontal axis in Fig. 3.
The smaller the surface roughness, the more non-specific absorption
It turns out that the arrival rate is small. Non-specific adsorption rate is 2.5% or less
At that time, the center line average roughness was 1.5 μm or less.

Example 4 As the washing solution in Example 1 [Creation of calibration curve], various surfactants described in Table 1 were dissolved in physiological water and used, and the tPA / PAI concentration was 0 ng / ml (N), 25 ng / The absorbance of ml (S) and its ratio (S / N) are shown. When the S / N ratio is set to 40 as the allowable limit for nonspecific adsorption, as shown in Table 1, the use of a nonionic surfactant having an HLB of 16 or more is effective in suppressing nonspecific adsorption. However, nonionic surfactants having an HLB of 16 or less, such as P-8P and Tween 40, suppressed nonspecific adsorption but also suppressed specific reaction (S), and were not effective for highly sensitive analysis.

Example 5 [Suppression of non-specific adsorption by adding skim milk] (a) Effect of adding skim milk to primary reaction solution 0, 0.01, 0.125, 0.25, 0.5, 0.8
Contains 1.0%, 0.01M PB-0.5M NaCl-2% BSA (pH
7.2) Dilute human tPA / PAI complex with buffer solution, and add 0,25ng /
A ml solution was made. 0.4 ml of each solution and mouse anti-PAI antibody-immobilized beads were placed in a small test tube and incubated at 37 ° C for 1 hour. Next, wash with PBS containing 0.05% Tween20
Washed twice. The peroxidase-labeled anti-tPA polyclonal antibody was adjusted to 1 μg / ml with BPS-0.05% Tween20 solution, 0.4 ml each was added to a small test tube, and incubated at 37 ° C. for 30 minutes. After washing three times with a washing solution, 0.4 ml of a peroxidase substrate solution was added to develop color for 30 minutes at 37 ° C., color development was stopped with 1N sulfuric acid, and absorbance at 450 nm was measured. The results are shown in Table 2a. It can be seen that when the S / N ratio of non-specific adsorption (N) and specific reaction (S) is 40.0 as an allowable limit, it is effective when the skim milk concentration is 0.01 to 0.8%.

(B) Effect of adding skim milk to the secondary reaction solution 0.01M-PB-0.5M NaC containing 0.25% skim milk
The human tPA / PAI complex was diluted with 1-2% BSA (pH 7.2) buffer to prepare a solution of 0, 25 ng / ml. 0.4 ml of each solution and mouse anti-PAI antibody solid beads were placed in a small test tube and incubated at 37 ° C. for 1 hour. Then add 0.05% Tween to PBS.
It was washed twice with a washing solution containing 20. Prepare a solution of PBS-0.05% Tween20 containing peroxidase-labeled anti-tPA polyclonal antibody at a concentration of 1 μg / ml and 0, 0.01, 0.25, 0.5, 0.8, 1.0% skimmed milk, and add 0.4 ml to each small test tube. Incubated at 37 ° C for 30 minutes. After washing three times with the washing solution, 0.4 ml of the substrate for peroxidase was added and color was developed for 30 minutes at 37 ° C, and the color development was stopped with 1N sulfuric acid.
The absorbance at 45 nm was measured. The results are shown in Table 2b.
The S / N ratio due to non-specific adsorption (N) and specific reaction (S) is increased by adding 0.01-0.8% skim milk,
It turns out that skim milk is effective.

Example 6 [Measurement of normal humans and patient samples] In Example 1 [Creation of calibration curve], plasma obtained from 5 normal humans and 3 thrombotic patients was diluted 4-fold and measured as the third sample. Shown in the table.

The concentration of tPA / PAI complex in patients with thrombosis was clearly high, and it was found that the concentration of tPA / PAI complex could be a marker for the diagnosis of thrombosis.

Example 7 [Measurement of Active PAI Concentration] Human tPA solution (0.5 μg / ml, 50 μ) was added to normal human plasma and thrombotic patient plasma (50 μ), and incubated at 37 ° C. for 30 minutes. After that, the concentration of the tPA / PAI complex was determined according to the preparation of the calibration curve of Example 1 (Result A). At the same time, the concentration of human tPA / PAI complex was determined for normal human plasma to which tPA was not added and plasma of patients with thrombosis according to the preparation of the calibration curve of Example 1 (Result B). The concentration of active PAI was obtained by calculating the difference between the measured values and converting the molecular weight of PAI (result C). The results are shown in Table 4.

The active PAI level of thrombosis was significantly higher than that of normal subjects, and it was found that the active PAI level could be a diagnostic marker for thrombosis.

Front page continued (72) Inventor Toshinori Murakami 1-7-9-202 East, Kokubunjicho Station, Shimotsuga-gun, Tochigi Prefecture (72) Yoichi Sakata 253-77 Yamamotocho, Utsunomiya City, Tochigi Prefecture (56) Reference JP-A-59 -68674 (JP, A) JP-A-53-32114 (JP, A) JP-A-60-36964 (JP, A) International Publication 89/1624 (WO, A) THROMBOSIS RESEARCH CH, VOL. 55, (1989), p. 285 ~ 289

Claims (12)

(57) [Claims] 1. A kit for immunologically measuring a human tissue plasminogen activator-human plasminogen activator inhibitor complex in a human specimen, comprising: (i) center line average roughness (Ra). ) Is a monoclonal antibody against human plasminogen activator inhibitor bound to a polystyrene carrier having a surface of 1.5 μm or less (first antibody) (ii) Enzyme-labeled polyclonal antibody against human tissue plasminogen activator (Second antibody) (iii) Substrate and reaction terminator for measuring enzyme activity (iv) Diluent, and (v) Nonionic surfactant having HLB (Hydrophile Lipophile Balance) value of at least 16 were contained. An immunoassay kit comprising a detergent. 2. A molecular weight of about 16,000 to about 50,000 and an isoelectric point of 1.0.
The immunoassay kit according to claim 1, wherein a protein of -5.0 is further contained in the secondary antibody and / or the diluent. 3. The nonionic surfactant is selected from the group consisting of polyoxyalkylene alkylaryl ether type, polyoxyalkylene alkyl ether type, polyoxyalkylene polyhydric alcohol fatty acid ester type and polyoxyethylene polyoxypropylene polyol type. The immunological measurement kit according to claim 1 or 2, which is at least one of the following: 4. The immunoassay kit according to claim 1, wherein the monoclonal antibody is JTI-4. 5. The method according to claim 2, wherein the protein is skim milk.
4. The immunological measurement kit according to any one of 4 above. 6. A human specimen is brought into contact with a first antibody bound to an insoluble solid carrier, and then a labeled second antibody is brought into contact therewith, or (2) a first antibody bound to an insoluble solid carrier. A method for immunologically measuring a human tissue plasminogen activator-human plasminogen activator inhibitor complex in a human specimen by simultaneously contacting the labeled second antibody and the human specimen. (A) using a monoclonal antibody against human plasminogen activator inhibitor bound on a polystyrene carrier having a surface with a centerline average roughness (Ra) of 1.5 μm or less as the first antibody, (b) using an enzyme A second polyclonal antibody against a labeled human tissue plasminogen activator was used.
An immunological assay method, comprising: (c) a detergent containing a nonionic surfactant having an HLB (Hydrophile Lipophile Balance) value of at least 16 is used as a detergent. 7. The immunoassay method according to claim 6, wherein a human sample is brought into contact with a first antibody bound to an insoluble solid carrier, and then a labeled second antibody is brought into contact therewith. 8. A molecular weight of about 16,000 to about 50,000 and an isoelectric point of 1.0.
The immunological assay method according to claim 6 or 7, wherein a protein of -5.0 is further contained in the secondary antibody and / or the diluent. 9. The nonionic surfactant is selected from the group consisting of polyoxyalkylene alkylaryl ether type, polyoxyalkylene alkyl ether type, polyoxyalkylene polyhydric alcohol fatty acid ester type and polyoxyethylene polyoxypropylene polyol type. The immunological measurement method according to any one of claims 6 to 8, which is at least one of the following: 10. The immunological assay method according to any one of claims 6 to 9, wherein the monoclonal antibody is JTI-4. 11. The protein is skim milk.
11. The immunological measurement method according to any one of to 10. 12. (A) Human tissue plasminogen activator-human plasminogen inhibitor complex present in human specimen and (B) human tissue present in human specimen to which human tissue plasminogen activator is added. Tissue plasminogen activator-human plasminogen inhibitor complex was measured by an immunological measurement method using the Sangerti method, and based on the difference in the measured values, active human plasminogen activator in human specimens was measured. In the method for measuring the amount of an inhibitor, (a) a monoclonal antibody against a human plasminogen activator inhibitor bound on a polystyrene carrier having a surface with a center line average roughness (Ra) of 1.5 μm or less is used as a first antibody. (B) human tissue labeled with an enzyme Polyclonal antibodies against Las plasminogen activator second
An active human plasminogen activator characterized in that it is used as an antibody and (c) a detergent containing a nonionic surfactant having an HLB (Hydrophile Lipophile Balance) value of at least 16 is used as a detergent. Method for measuring beta inhibitor.