JPH01112157A - Method, means and kit for measuring activity of tissue plasminogen activator - Google Patents

Abstract

PURPOSE:To measure the t-PA activity in blood as it is by using a solid conjugated with an antibody fragment having affinity to the part of the tissue plasminogen activator t-PA except the active part thereof. CONSTITUTION:The total blood 8 is taken in a vacuum blood taking tube 7 and a stick-shaped measuring means 1 is immediately inserted therein and is brought into reaction with the total blood. The tube is thereafter cleaned and the measuring means 1 is taken out. This measuring means 1 is inserted into a vessel 9 which ought to contain a substrate soln. The means is then bent and broken at the aperture edge 10 thereof as a fulcrum and only the broken piece 11 contg. an anti-t-PA monoclonal antibody-coated part 3 is charged into the vessel 9. The substrate soln. is put into the vessel 9 and is incubated; thereafter, the stick piece is taken out and is measured by a measur ing instrument, by which the quantity of the color forming or light emitting material liberated from the substrate is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention is a novel method for measuring the true activator activity of tissue plasminogen activator (hereinafter referred to as t-PA) in blood.

The present invention relates to a measurement kit and a measurement tool used for the measurement. More specifically, 9 the present invention uses a solid phase bound to a t-PA antibody IgG fragment that has affinity for sites other than the active site of t-PA and is specific for t-PA, and plasma or serum as a specimen. An attempt to measure the true activity of t-PA in blood by measuring the activator activity of a composite of the solid phase and t-PA in the sample using unseparated whole blood or a diluted product thereof. It is.

(Prior Art) t-PA is a type of serine protease involved in the fibrinolytic mechanism. It is known that its action is to prevent the formation of thrombus by dissolving and removing fibrin formed in blood vessels and tissues. Furthermore, t-PA is synthesized by vascular endothelial cells and secreted and released as necessary, so it is an essential factor for preventing thrombus formation and maintaining blood flow. For this reason, a high level of t-PA activity in the blood indicates a tendency to bleed.

It is easily assumed that low levels cause a thrombotic tendency, but due to the presence of an inhibitor (FAI) for t-PA in the blood and its short half-life, it is also easily inactivated in plasma separated from blood under water cooling. Therefore, no suitable measurement method has been developed so far. however,
The results of conventional measurement methods showed that clinical cases with high values were associated with liver damage [J, Cl1n-Invest, 43.681
(] 964), J-Cl1n, Pathol.

? 1. 772 (1983)], ovarian cancer [Eur-J,
CancerClin, 0nco1. , 20.577
(1984)], cerebral hemorrhage [Blood.

61.267 (1983)] and deep vein thrombosis [Br, J, Surg. ,7
0.369 (1983), Br1tish Med
, J-, 29 (1, 1453 (1985)) Atro
phic Branche [Arch, Patho]
l, Lab, Med, +110 +517 (19
86)], Child metrial tumor Cancer+ 48
+1484 (+982)], myocardial infarction and hyperlipidemia [T
he New England Journal o
f Medicine, 313. No.25,
1557 (1985)]. Furthermore, there are reports of families with repeated venous thrombosis due to abnormalities in the t-PA production mechanism [Acta, Chir, 5cand,
+ -ias, 313 (1972), Acta
, Med, 5cand, , 203, 477 (
1978)] From the above, t-P fi in blood,
Knowing the concentration is clinically very important.

- Until now, the concentration of t-PA in plasma has been measured by the amount of antigen and activity value for boat fishing.
-RIA (radioimmunoassay) using PA antibody
and IRMA (immuno radiometric
assay ) + E L I S A (Enzym
e 1 inked-immunosorbent a
sSay) etc. are used (Japanese Patent Application Laid-Open No. 1747-1989)
No. 59.

61-1.48200).

On the other hand, the measurement of activity is broadly classified into fibrinolytic method and synthetic substrate method, and the following literatures are cited as the fibrinolytic method.

Thromb-Haemostas・, 52(1),
19 (1984) Thromb, Haemosta
s, 45(2), 107107 (1981) Thr
o Diathes, Haemostas, 32.
356 (1974) and the following literature regarding the synthetic substrate method.

Thrombosis Res,, 43.129(1
986) Thrombosis Haemostasis
, 53(3), 356 (1985) Thrombos
isRes, 46.213 (1987) C1ini
ca, Chimica, Acta, +127+27
9 (1983) Plasma is commonly used as a material for examining t-PA activity in blood, but various treatments are performed to remove inhibitors to t-PA. For example, after centrifuging blood for 5 minutes, separate plasma and immediately adjust the pH to 5.9 with acetic acid water to
A method for precipitating and centrifuging the lobulin fraction [Thromb, H
amostas, +dan (3), 266 (1982)
]', or a method in which plasma is adjusted to pH 3.9 and immediately frozen at -70°C (dry ice) (dissolved by heating and adjusted to pH 7.4 before use); t-PA was added to Lysine Sepharose (Lysine 5e).
A method of removing t-PA inhibitor by adsorbing it on t-PA inhibitor (Throm-bosis) and washing it [Throm-bosis
Res, + 4←413 (1,987)].

(Problem to be Solved by the Invention) However, all methods for measuring the amount of t-PA antigen also measure the reaction product of t-FA and its inhibitor as the amount of antigen, and thus the true amount of t-PA cannot be measured. There is a drawback that PA activity cannot be measured.

Moreover, the above-mentioned conventional methods for measuring t-PA antigen amount and t-PA activity.

(1) The time required for centrifugation to separate plasma from blood is at least 10 minutes from the start to the end of centrifugation, during which time t-PA activity in plasma is lost.

(2) It takes a long time (approximately 20 hours) to judge the results, and it is not only inconvenient.

Depending on the method, the true t-PA activity cannot be measured because the effect of the t-PA inhibitor continues during the reaction time.

There were some problems. In addition, conventional methods require dilution of tens to thousands of times to avoid the effects of t-PA inhibitors, and unless the measurement method is highly sensitive and accurate,
There was a problem in that even t,-PA activity could not be detected.

For the above reasons, methods for measuring t-PA activity in blood as it is have not been successful so far.

(Means for Solving the Problems) Therefore, in order to establish a method for measuring true t-PA activity, the present inventors used whole blood that had not been subjected to centrifugation that would cause the loss of t-PA activity, or This is the result of intensive research into the method of measuring diluted products.

A t-PA-specific synthetic substrate is directly applied to the reaction product produced by reacting a solid phase on which an anti-t-PA monoclonal antibody is immobilized with t-PA in a sample in a short period of time, or a t-PA-specific synthetic substrate is directly applied to the reaction product. According to this method, the true t-PA activity in the specimen can be detected in an extremely simple and short processing time by applying a specific enzyme substrate to the t-PA and reacting the produced enzyme with a synthetic substrate specific to the enzyme. Measure scales with high accuracy and sensitivity 9
The present invention has now been completed.

That is, 1 the present invention involves reacting a solid phase bound with a t-PA antibody IgG fragment having affinity for a site other than the active site of t-PA with t-PA in whole yuzu or a diluted solution thereof. The present invention is characterized in that the activity of the product is then measured, and the object of the present invention is to provide a method for this measurement. In addition, 1 the present invention provides a t-PA antibody IgG fragment having affinity for at least a site other than the active site of t-PA÷-Yaichi←÷←
a solid phase to which the fragment is bound; and a measurement reagent for measuring the activity of the reaction product of the solid phase to which the fragment is bound and t-PA in whole blood or a diluted product thereof. The object of the present invention is to provide a kit for the measurement.

Furthermore, the present invention is characterized in that a coating layer of a t-PA antibody IgG fragment having an affinity for a site other than the active site of t-PA is fixed to one end of a plastic stick, and the measuring device The purpose is to provide.

The present invention will be explained in more detail below.

(Measurement method) The measurement method of the present invention uses a solid phase bound to an anti-t''-PA monoclonal antibody.

(1) No secondary antibody is used. (2) t-PA activity is measured, not the amount of anti-W.

In this respect, it is completely different from the conventional ELISA method using monoclonal antibodies. The traditional ELISA method is
In the presence of an inhibitor, part of the -PA becomes t-P.
Although the t-PA activity is lost due to binding to the A inhibitor, the composite of the t-PA and t-PA inhibitor is also measured as the amount of t-PA antigen. The present invention is a method for measuring whole blood containing inhibitors or its diluted product in a short period of time, and is
Since the reaction product with the inhibitor is not measured, the true t-PA activity in the blood can be determined.

The t-PA antibody IgG fragment used in the present invention may be any anti-t-PA monoclonal antibody that does not bind to t-PA at its activator active site and has specificity for t-PA, and commercially available ESP -2 (manufactured by Bioscotto, t-PA monoclonal antibody), JP-A-59-5121.

JP-A-61-148200, JP-A-61-181
All of the anti-t-PA monoclonal antibodies described in Publications No. 964 (and No. 61-183299) that do not bind to the t-PA active site can be used;
Anti-t-PA that is exceptional in terms of enhanced PA activity
Monoclonal antibody 5P-322 (separately filed by the applicant, Japanese Patent Application No. 178073/1986, 328004/1982)
No.) is particularly preferred.

In addition, as the solid phase used in the present invention, the above-mentioned t-PA
Any material capable of binding antibodies can be used, such as microplates, beads, stains, test tubes, etc. made of polymers such as polystyrene, polycarbonate, polypropylene, and polyvinyl. Among them, the t-PA monoclonal antibody-bonded stain described below is preferred because of its ease of use.

The measuring method of the present invention aims at short processing time, and therefore, the whole blood as a sample is preferably whole blood that has just been collected from a test subject in the presence of an anticoagulant. In addition, the whole surface dilution is preferably one obtained by diluting whole blood 4 to 16 times with a diluting medium such as physiological saline.9 In the measurement method of the present invention, a dilution of about 4 times reduces the influence of the t-PA inhibitor. It is possible to do so.

t-PA antibody IgG fragment-bound solid phase and t-
To measure the activity of the reaction product with PA.

A synthetic substrate specific for t-PA is immediately applied to the composite, and the amount of a chromogenic or luminescent substance released from the synthetic substrate is optically measured.

Alternatively, an enzyme substrate specific for t-PA is applied to the composite in the presence or absence of an active stimulant.

This is carried out by reacting the enzyme produced with a synthetic substrate specific to the enzyme and optically measuring the amount of the liberated chromogenic 9 luminescent substance.

Here, the synthetic substrate specific for t-PA is t-P
By reacting with A, it decomposes and liberates a substance with 1 color and 9 luminescent properties, and the amount of the 2 luminescent substances.

Any substance that correlates with t-PA activity may be used. Commercially available synthetic substrates include S-2288 (H
-D -11e -Pro-Arg-NH-C)1-N
o, 2HC1; manufactured by Kabi Co., Ltd.), S-2444 (H
-L-pGlu -Gly-L-Arg-NH-CyN
O2/HCI; manufactured by Kabi Co., Ltd.) etc. [JP-A-52-245
No. 81 (Special Publication No. 15.238 of 1982), Special Publication No. 15.238 of 1983
No. 500076], all of which are chromogenic substrates and liberate p-nitroaniline. p-Nitroaniline has a characteristic wavelength of 405% in visible light in the yellow band.
nm. Although not commercially available, if the chromogenic residue in the above substrate is 9, for example, a 4-methoxy-β-naphthylamide residue or a 7-amino-4-methylcoumarin residue, it can be used as a 2-fluorescent substrate. Become.

The amounts of liberated 4-methoxy-β-naphthylamine and 7-amino-4-methylcoumarin can be detected by measuring light.

On the other hand, the enzyme substrate specific for t-PA is typically plasminogen, preferably human lysine plasminogen, glutamyl plasminogen, and the like.

Active stimulants that may be used in the assay of the present invention include, for example, bromcyan-treated fibrinogen.

Soluble fibrinogen monomer, polylysine.

Examples include fibrin.

When plasminogen is used as the enzyme substrate, the enzyme produced by the reaction between the complex and the t-PA-specific enzyme substrate becomes plasmin.

A synthetic substrate specific to the producing enzyme decomposes by reacting with the producing enzyme, liberating a substance with 1 color and 1 luminescence, and the amount of the luminescent substance with 1 color and 9 luminescence.

Any substance may be used as long as it correlates with the generated enzyme activity and, ultimately, with the t-PA activity. For example, if the enzyme produced is plasmin, the above-mentioned Japanese Patent Application Publication No. 52-24581
No. 38), Special Publication No. 500076 of 1982, and Japanese Patent Publication No. 1983-500076
No. 3494 (Special Publication No. 56-22519), JP-A-57
-2253 (Japanese Patent Publication No. 62-571.97), JP 58-63399 (Japanese Patent Publication No. 61-9840), JP 58-172354, JP 62-294.695
No. 62-294696, etc.

Examples include those that serve as substrates for plasmin.

Commercial product S-225, which is particularly specific for plasmin
1, (H-D-Val-L-Leu-L-Lys-NH
-D-Pro-Phe-Arg-NH+NOz・2HC
1: Kabisha), Nr, 51-32 (HD-V
an, 5HT-Lys-pNA・2AcOH;
(manufactured by Pentafirm).

Chromozym PL
;Tos-Gly-Pro-Lys-pNA;
(manufactured by Boehringer Mannheim) is preferably used. When these are used as synthetic substrates, p-nitroaniline is liberated as a dichromogenic substance. The measurement and the luminescent substrate are the same as described above.

Specifically, the measurement was performed by (1) coating a solid phase with a t-PA antibody IgG fragment;

(2) This was washed to remove unbound t-PA antibody IgG fragments.

(3) Whole blood or its dilution is added to this and reacted with t-PA in the specimen within a short period of time to cause it to bind.

(4) This is washed to remove unbound components and inhibitors).

(5) In this, t-PA is directly incubated with a specific Fe synthetic substrate such as S-2288, or (4) is further incubated with a t-PA specific enzyme substrate such as lysine plus nogun. If the activity is low, an activity stimulant such as bromocyanated fibrinogen and a synthetic substrate specific for the producing enzyme such as plasmin such as S-225 may be added.
1. Incubate 1-9 by adding Nr-51-32 or Chromozyme PL (6) Detect and quantify the liberated substance.

(7) Conversion to t-PA activity is performed if necessary.

Processes such as washing and incubation are carried out at specified pH,
Performed using ionic strength buffers.

Such buffers include phosphoric acid, carbonic acid, boric acid, citric acid, barbituric acid buffers, Tris-HCl buffer,
Examples include amino acid buffer. Although it depends on the processing means, Tris-HCl buffer or a mixture thereof with sodium chloride or Tween (polyoxyethylene sorbitan fatty acid ester) is advantageously used. The reaction temperature is preferably room temperature, which is sufficient for the formation of the bulk, and about 37° C. for incubation for detection and quantification.

Measurements using synthetic substrates are performed with or without deactivation of t-PA or the enzyme produced, depending on the type of free substance.2 Spectrophotometers, fluorescence detectors, etc. are used.

Preferably, this is carried out using conventional measuring equipment.

(Measurement kit) The present invention includes a kit applicable to the above measurement method, preferably a solid phase used in the above measurement method.

t-PA monoclonal antibody, t-PA activity measurement reagent, i.e., t-PA-specific synthetic substrate, or t-PA
From the viewpoint of ease of use, it is preferable to cover all of the specific enzyme substrates, (activation stimulators if necessary) synthetic substrates specific to the produced enzymes, and other standard solutions. It is not limited to what you have. That is, 2
The measurement kit of the present invention is characterized in that it contains a small amount of an anti-t-PA monoclonal antibody or a monoclonal antibody thereof bound to a solid phase, and a reagent for measuring t-PA activity in the form of a composite. 9 The present invention includes all kits for measuring t-PA activity of whole blood or diluted products thereof.

(Measurement device) Among the solid phases bound to anti-t-PA monoclonal antibodies, the present invention uses an anti-t-PA monoclonal antibody-binding stick that is particularly easy to use and most suitable for short-time measurement processing. included.

Specifically, the stain-like measuring device for measuring t-PA activity of the present invention is made of plastic, and one end thereof has t-
-t-PA that has affinity for sites other than the active site of PA
It is made by fixing a coated phase of an antibody IgG fragment. Among these, it is preferable to use a material that has an easy-to-cut part and can be easily cut by bending the opening edge of the container used for measurement as a fulcrum. Further, a measuring device in which this stick is previously housed in a vacuum blood collection tube is also mentioned as a preferred specific example of the present invention.

Below, two measuring instruments of the present invention will be explained in detail based on drawings which are examples. FIG. 1 is a perspective view of a stick-shaped measuring tool 1 of the present invention, and FIG. 2 is a process diagram of a method for measuring using the measuring tool 1.

The stain main body 2 is made of plastic to which an anti-t-PA monoclonal antibody is bound, and has a rectangular flat plate shape in Figure 9. Examples of the plastic material include those mentioned above as the solid phase material, and in particular, an easily cut portion is provided.

A suitable material is one that can be easily cut by bending the opening edge of the container as a fulcrum. In the embodiments of the present invention, any material may be used as long as the end of the polystyrene that is not coated with the stick-null antibody can be pinched with fingers.

It is not limited to the illustrated rectangular or flat shape.

However, in order to provide an easy-to-cut portion and to enable the container to be easily cut by bending the opening edge of the container as a fulcrum, a flat plate as shown in the drawing is preferable to a rod-like shape.

An anti-t-PA monoclonal antibody coating section 3 is provided at one end of the stick body 2, and an anti-t-PA monoclonal antibody is coated and fixed thereon. Covering and fixing are performed by conventional methods. That is, after washing and drying the stick, the end was immersed in a container containing a buffer solution of anti-t-PA monoclonal antibody.

Leave to coat for about 4 hours at room temperature, or overnight if the temperature is below 4°C, then put the coating stick into a container containing a buffer solution containing serum albumin etc. for about 1 hour at room temperature.

If the temperature is below 4°C, leave it for about one night to fix.

It is preferable to coat the stick until the base end 4 of the stick is completely immersed in the upper surface of the substrate solution to be placed in the measurement container (approximately 6 mm from the base end 4 in the drawing). Also.

The coating is usually applied to the stick body 2. Although the coating is applied to the entire front and back surfaces of the edges, it is not prohibited to coat only one side.

In the figure, 5 indicates the area blocked with serum albumin. The fixing region 5 may be any region that can achieve the fixing purpose, and may be larger than the covering portion.

It is preferable that the stain main body 2 is provided with an easy-to-cut portion 6 in advance, and as shown in FIG. (10 points from the proximal end in the drawing)
mrn) is suitable.

The easy-to-cut part 6 is not limited to the illustrated slit, but may be a perforation, etc., as long as it can be easily cut, especially by bending the opening edge of the measuring container as a fulcrum. .

The method of measuring using this stick-shaped measuring device 1 is explained with reference to FIG. , anticoagulant such as sodium citrate or heparin], put the stick-shaped measuring device 1 into this immediately after blood collection, and leave it for about 5 to 120 minutes at room temperature to collect whole blood. Bend and cut using the opening edge 10 as a fulcrum.

Cut piece 1 containing anti-t-PA monoclonal antibody coated area 3
Pour only 1 into container 9 [(a)]. A substrate solution is put into the container 9 containing the cut piece 11.

Incubate at 37°C for about 30 minutes to 20 hours) [(e)
] After that, the stick piece is taken out and attached to a measuring device (not shown) to measure the amount of color-forming or luminescent substance released from the substrate [(f)]. (Note that colorimetric measurements are possible even after the two reactions have stopped).

Also, if we explain the case of using diluted whole blood in (2),
A dilution medium 12 and a stick-shaped measuring tool 1 are prepared in advance. Collect blood into a vacuum blood collection tube 7 containing an anticoagulant if necessary [(ml
], Immediately mixed and left for 5 to 120 minutes to react with whole blood dilution 13 [fb'1], washed, processed in the same manner as above (cl to (e)), and measured.

Note that this is not limited to the case where whole blood dilution 13 is used.

Vacuum blood collection tube 7 is also used as a stain-like measuring device in the whole blood method.
It can be stored in advance.

That is, the immobilized anti-t-PA monoclonal antibody is stable enough to be commercially available in such a form.

Furthermore, it is not impossible to adopt a method in which the diluting medium 12 is not stored in advance in the diluting substance, and the diluting medium 12 is added and mixed immediately after blood collection.

However, in the present invention, which aims at short-time processing, the illustrated method is convenient.

(Effect of the invention) The method for measuring t-PA activity of the present invention can detect true t-PA in blood.
This method enables the measurement of activity for the first time, and has various advantages in terms of simplicity, sensitivity, accuracy, etc. as described below.

(1) Since there is no centrifugation operation, the influence of t-PA inhibitors is small and t-PA deactivation can be minimized.

(2) t-PA activity can be measured within 100 to 130 minutes after blood collection, which is much shorter than the conventional method (about 20 hours).

(3) Measurements can be made using plasma or whole blood dilutions that are lighter in dilution (approximately 4 to 16 times) than conventional dilutions (generally 50 to 3,200 times diluted), thereby minimizing the inactivation of t-PA-24=. It is possible to measure true t-PA activity with high sensitivity.

(4) There is no need to adjust to acidity (pH 3, 9) or freeze with dry ice (-70°C).

(5) It is extremely easy to operate.

(6) Sensitivity is further improved in systems containing active stimulants.

Therefore, the measurement method of the present invention includes: t-PA, t-PA
We not only screen derivatives and other fibrinolytic enhancers and confirm their clinical efficacy, but also diagnose specific diseases, monitor medical conditions, and perform therapeutic drug therapy (TDM).
rug Monitoring).

Furthermore, the measurement kit of the present invention has the advantage that it can be used for simple measurements. In particular, the stick-shaped measuring device coated with a t-PA antibody IgG fragment-bonded solid phase, especially the t-PA antibody IgG fragment, and the kit containing the same are completely covered with this.
It is only necessary to add the diluted product to the reaction volume measurement reagent, incubate, and detect, which is extremely simple.

Also, if you use a stick-shaped measuring tool.

■ After adsorbing blood t-PA to the stick, there is no loss of activity after washing with a buffer solution, and it can be stored for one week (
4°C), which facilitates measurement.

■ Washing is easy because monoclonal antibodies are already immobilized. For example, a buffer solution can be placed in a beaker, or it can be washed directly with ordinary water.

■ The coated and fixed stick-shaped measuring device is stable for more than 3 months if stored in a buffer solution at 4°C.

■ Stick-shaped measuring instruments also have excellent transport stability.

(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.

(Measurement method 1) Anti-t-PA monoclonal antibody 5P-322 at 50mM
Adjust the concentration to 1 μg/ml with carbonate buffer (pH 9,6), and transfer to a microplate (Linbro EIA).
plate Flow Laboratories + I
nc,) in 200 μt portions and incubate at room temperature for 4 hours or 4 hours.
Leave at ℃ overnight.

The contents of the wells were removed by suction, and 150 mM sodium chloride (Kanto Kagaku Co., Ltd.) and bovine serum albumin (S
igma, A-7030), 10mM)'J sous-acid buffer (p
Dispense 200 μl of H7,4) into wells and leave at room temperature for 1 hour or at 4°C overnight.

Aspirate the well contents and add 0.05% Tween 20.
(Hani Chemical Co., Ltd.) 10 mM Tris-HCl buffer (pH 7,4) (referred to as buffer ■) 30
Wash 4 times with 0 μL.

Specimen immediately containing t-PA (whole blood or its dilution)
Dispense 200 μt and allow to react at room temperature for 10 minutes.

Aspirate well contents and wash 6 times with Buffer I 300ILt.

Then 0.45mM S-2251 (
Kabi), 150mM sodium chloride, 0.
01% Tween 80 (Hakai Chemical Co., Ltd.), 0
．． 042μM lysine-plasminogen, 120μg/
2 ml bromocyanated fibrinogen added
After incubation for 90 min at 37°C, dispense 200 td L of 0 mM Tris-HCl buffer (+) H7,4) into the wells.

The reaction was stopped with 50 μL of 50% acetic acid aqueous solution, and measured using an absorption photometer (T1tertek' Multiskan Ty
pe 310 C) at 4 (measured by absorbance at 15 nm (the principle diagram is shown in Figure 3).
A diagram of the principle of measuring A activity using a synthetic substrate is shown in Figure 4).

In addition, lysine-plasminogen, bromcyan-treated fibrinogen, and anti-t-PA monoclonal antibody 5P
-322 preparation method is as follows.

Method for preparing lysine-plasminogen Lysine-plasminogen (Midori Juji).

Code No. 1089 , ) (2100CU
) in pure water.

Excess aprotinin (Sigma No.-A-601-2) 20 to completely inactivate the contaminated plasmin.
Add 000KI/inZ 20mZ and leave at 4°C overnight.

Biogel P100 gel (BIORAD) at 140mM
Salt 28 20mM) Lis-HCl buffer (p
After equilibration with H7,5) and elution with the same buffer, the lysine-plasminogen peak was fractionated. The amount of protein obtained in this experiment was 1100 rlI. The resulting gin-plasminogen is aliquoted, stored frozen at -20°C, and dissolved before use.

Preparation method of bromocyanated fibrinogen C, Zam
marron [J, Biol, Chem-, 259
No. 4.2080-2083 (1984)]. human fibrinogen 0
．． 8g of protein was dissolved in 70% formic acid containing 1.3g of bromocyanide.

Treat for 17 hours at room temperature in nitrogen gas. after that.

It is prepared by dialysis against 301 pure water for one day and night, and then by adding it to 51 pure water and dialysis.

The amount of protein obtained in this experiment was 700 mg.

The obtained bromcyan-treated fibrinogen is aliquoted, stored frozen at -20°C, and dissolved before use.

Preparation method of anti-t-PA monoclonal antibody ■ Preparation of hybridoma 5P-322 a) Preparation of immunized tile cells T derived from human melanoma cells purified from BALB/C female mice (8 weeks old at the start of immunization) - pA (manufactured by Pius Conte) at a dose of 30 μg/mouse or 7.5 μg/mouse,
After mixing with the same volume of Freund's complete adjuvant to emulsify, the mixture was administered intraperitoneally (first immunization). From then on 3~
T-PA 30 μg/mouse or 7.0 μg/mouse twice at 4-week intervals.
5 μg/mouse was mixed and emulsified with the same volume of Freund's incomplete adjuvant and injected intraperitoneally. Six weeks after the third immunization, t-PA was administered at 30 μg/[ffi or 7.5 μg
0.4 ml of physiological saline solution containing 50% of each animal was administered intraperitoneally (
final immunization). Three days after the final immunization, spleen cells were collected from two mice and suspended in DMEM medium. Red blood cells were disrupted by treatment with 0.17M ammonium chloride for 10 minutes at 0°C and removed by centrifugation.

b) Preparation of hybridoma 5P-322 The tile cells (5 x 108 cells) prepared above were added to mouse myeloma cells P3X63.
Ag8, Ul (P3Ul) (108 IX) and Köhler and Milstein's method [Nature + 25th
6, p. 495 (1975)].

That is, after washing the tile cells and P3Ul cells several times with DMEM, they were placed in a 50 mZ plastic test tube.
Mix thoroughly.

Next, the medium was removed by centrifugation, and 1 ml of DMEM containing so% (w/v) polyethylene glycol (manufactured by Sigma, average molecular weight 3640) kept at 37°C was added.
was added to the bodies under stirring over a period of 1 minute. Next, 10 ml of DMEM kept at 37° C. was added dropwise to terminate the cell fusion reaction.

After centrifuging the reaction solution and removing the supernatant.

HAT medium [hyboxanthin (], X10-' M)
.

Aminopterin (4X10-7 M), Thymidine (
DMEM medium containing 100% fetal bovine serum supplemented with 1.6 x 10-5 M);
The number of cells was adjusted to 10-5 times/ml.

This suspension was dispensed into a 24-well plastic plate in an amount of 2 ml per hole (106 tile cells). 4~
Half of the medium was removed by suction every 5 days, and the above HAT medium was added. Seven to ten days after cell fusion, hybridoma growth was observed in all the wells, so anti-t-PA activity in the culture supernatant was measured by the immunoprecipitation method described below.

C) Measurement of anti-t-PA activity in culture supernatant by immunoprecipitation method t-PA labeled with radioactive iodine (+25I) (by chloramine T method) [ioooμZ, 0.7'
Z bovine serum albumin, 0.3M sodium chloride, 0.
05 (w/v) dissolved in 25mM) Lys buffer pH 7.4 containing Tween 8o. Hereinafter abbreviated as 1machi-t-PA] and 20 µL of the hyperidoma culture supernatant were mixed, left overnight at room temperature in a plastic box, and then 1 µL of rabbit anti-mouse IgG antiserum was added.

The mixture was left at room temperature for an additional 3 hours.

To this was added 10 μt of Pansorbin (trade name, manufactured by Calbiochem) suspension, mixed, and after 3°-32 minutes, 2 ml of Tris buffer was added and immediately centrifuged (3000 rpm + 15 minutes). The supernatant was removed, and the radioactivity of 125I-t-PA in the precipitate was measured using an Autowell gamma counter ARC300 manufactured by Aloka. For hybridoma enrichment wells showing significant +25I-t-PA immunoprecipitation.

The hybridoma clone obtained was named SP-322. Also, hybridoma 5P
The anti-t-PA monoclonal antibody produced by -322 was also named 5P-322.

■ Production and purification of monoclonal antibodies BALB/C male mice, 2°6.1 at 6-8 weeks old
After intraperitoneal injection of 0.5 ml of 0.14-tetramethylpentadecane (Pristan, manufactured by Aldrich),
107 hybridomas 5P-322 after 7-14 days
was suspended in physiological saline O05mZ and inoculated intraperitoneally.

After 10 to 20 days, the produced ascitic fluid was collected from the sacrificed mice that underwent laparotomy. Ascitic fluid containing monoclonal antibodies of 5 to 10 mZ was obtained from one mouse. This ascites was centrifuged to remove insoluble matter, and then 1 equivalent volume of saturated ammonium sulfate solution was added, and the mixture was left at 4° C. for 1 hour to overnight. The resulting precipitate was centrifuged and diluted with 0.1 M containing a small amount of 0.9% sodium chloride.
It was dissolved in phosphate buffer (pH 8) and then dialyzed against 100 times the volume of the same buffer overnight (gamma globulin fraction). From this fraction, 9MAPS-■Mouse monoclonal antibody purification kit (trade name, manufactured by Bioland Laboratories)
IgG was purified using Namely.

Add the same volume of binding banophur to the gamma globulin fraction, mix, and then add Affigel Protein A (manufactured by Bioland Laboratories) or Protein A Sepharose CT, 4B, which has been sufficiently equilibrated with the same binding vanofur. (manufactured by Pharmacia Fine Chemicals) (Gelpet volume 5 or 1 omZ) and washed with Binding Mesophor 10 column volume. The IgG was then eluted with approximately 3 column volumes of the elution buffer included in the kit. Eluted Ig
G was concentrated to 0.9% by salting out with ammonium sulfate.
The sample was dialyzed against a 0.01-0.1M IJ acid buffer containing 5P-322 and used as an IgG standard of 5P-322. Normally, 11 to 18 Ir1 g of IgG was obtained per 1 ml of ascites.

(Standardization) t-PA (-mercury) standard specimen 0.02-40 ngAl
A calibration curve was created using Dilution was performed in 025-chi bovine serum albumin supplemented buffer ■.

According to the results, 0.03 ng/i'nZ ~20
It was revealed that the calibration curve for ng/Fnl was correct.

(Measurement of t-PA activity in the blood of healthy subjects) After the subject was kept at rest for 10 minutes and restrained at a pressure of 100 mmHg for 5 minutes, the counterpressure mesothelial vein was punctured, and the syringe used to collect blood in advance was filled with dilute solution 9. Add 1 part of Sodium Citrate (Midori Juji) in Section 38. After blood collection, remove the needle and
Place it in a test tube that has been prepared in water beforehand. Immediately, 200 μl of whole blood is dispensed into a prepared antibody-coated microtube and used for assay.

The results are shown in Table 1.

Table 1 + 16 ] ~ 5 100 mrnHg Blood collection after 5 minutes loading ■ 46 ~ 10 Normal blood collection Note that t-PA activity of 1 ng/ml is 0.55 IU/mt when expressed in international units.

(Measurement method 2) Creation of stain-like measuring tool Polystyrene plastic stick (manufactured by Nippon Soda,
100mm long, 5.5mm wide, 1mm thick).

Extrane M.A. 01■ (Product name, Ext
ranMAOI (manufactured by Merck) was soaked overnight in a 20-fold diluted detergent in a water bath at a 60°C rice grain temperature. After washing the stick in an ultrasonic cleaner for 3 hours and in regular water for 3 hours, soak it in purified water overnight.

The stain ink is dried in a dryer at 40° C., and slits, which are easily cut, are provided on both sides of the stain ink at a distance of 10 mm from the proximal end.

Anti-t-PA monoclonal antibody 5P-322 at 50mM
Carbonate buffer < T) + 1 μgA in 4(9,6)
Adjust to nl, dispense 200 μl into PPN Chee-B0 (trade name, Dainippon Pharmaceutical Co., Ltd.), add the stick, and leave it at room temperature for 4 hours or overnight at 4°C to coat the stick with the antibody. do. coating stick,
150mM sodium chloride, 10mM containing bovine serum albumin (manufactured by Sigma), 0.02% sodium azide
) Place 500 μt of Lis-hydrochloric acid buffer (pH 7.4) into a PPN tube and let stand at room temperature for 1 hour or at 4° C. overnight to block the surface.

It was washed with the buffer I and stored at 4°C in the buffer II.

Note that this coating-blocked stick was stable for more than 3 months when stored in a buffer at 4°C.

measurement 1) Measurement of t-PA activity using whole blood From the counterpressure mesothelial vein of a subject (healthy individual).

3.1: In a vacuum collection tube containing 3% sodium citrate,
Blood was collected at a ratio of 1 part sodium citrate to 9 parts blood, and immediately after blood collection, the anti-t-PA monoclonal antibody prepared in advance was coated and the blocked stick was placed in the blood collection tube for 10 minutes at room temperature. Make it react.

Take out the stick, wash it with buffer solution ■, insert it into a microplate, bend and cut using the opening edge of the microplate as a fulcrum, and place only the stick piece containing the anti-t-PA monoclonal antibody coated part into the microplate. put in.

Then, 0.45mMS-2251 (
(manufactured by Kabi Co., Ltd.), 150mM sodium chloride, 0.01
%Tween 80 (Gyudon Chemical Co., Ltd. L O, 042μM
IJ resin plasminogen, 120μg/ml +
20mM supplemented with bromcyanized fibrinogen
) Dispense 200 μL of Lis-HCl buffer (pH 7.4) into the wells of the microplate containing the stick,
After incubation for 17 hours at 37°C, the sticks were removed and placed in a spectrophotometer 1 titer tick 0 Multiskan model 310C (Titertek' Multiskan 310C).
t-PA activity was measured by absorbance at 405 nm.

(Incidentally, 50μ of a 50% acetic acid aqueous solution is used as a reaction stop solution.)
can also be used, but it was not used in this measurement experiment).

2) Measurement of t-PA activity using diluted whole blood Immediately after blood collection in the same manner as in 1), prepare the anti-t-PA monoclonal antibody coated, blocked stick and buffer I 1.5. ml (including anticoagulant)
Immediately dispense 0.5 ml of blood into the test tube containing the test tube and leave it at room temperature for 60 minutes.

Blood 1-PA activity was measured in the same manner as in 1) except that the reaction time with the substrate buffer was changed to 4 hours. .

In addition, +1.5 mZ buffer I was added to the vacuum blood collection tube.

Store 0.05 ml of anticoagulant and the above-mentioned stick in advance, collect 0.45 ml of blood with the blood collection tube, and measure blood t-I) A activity using the same procedure. I could do that. This method is.

The ability to react immediately after blood collection is excellent (5 points); however, the data obtained from the test tubes are shown below.

(Standardization) Similar to measurement method 1, the stick method]) and 2) were also standardized.

Figure 5 shows the semi-logarithmic calibration curve obtained by the whole blood method using Stain Ink; Figure 7 shows the calibration curve.

The calibration curves of linear functions obtained by method 2) are shown.

According to these calibration curves, it is clear that both of the measurement methods 1) and 2) will result in a calibration curve of Q, OO8~'l IU/mL.

(Measurement results of t-PA activity in the blood of healthy subjects) By the whole blood method using the above-mentioned stick.

The measured results are shown in FIG. 8, and the results measured by the dilution method are shown in FIG. 9.

According to this measurement result, the true blood t-PA
It is clear that the activity can be measured, and this shows that the measuring method of the present invention, especially this stick method, is far superior to the conventional method.

[Brief explanation of the drawing]

Figure 1 is a perspective view of an anti-t-PA monoclonal antibody coated stick, Figure 2 is a process diagram of the measurement method using the stick, Figure 3 is a diagram of the principle of the measurement method of the example, and Figure 4 is a diagram of the measurement method using the stick. A diagram of the principle of the method for directly measuring the t-PA activity of the product using a synthetic substrate. Figures 5 and 6 show semi-logarithmic calibration curves of whole blood and diluted standard samples using sticks, and are shown by Seiya Nono, patent attorney and representative of Yamanouchi Pharmaceutical Co., Ltd., the seventh patent applicant.

Claims (1)

[Scope of Claims] 1. A solid phase bound to a tissue plasminogen activator antibody IgG fragment having affinity for a site other than the active site of tissue plasminogen activator;
Method 2 for measuring tissue plasminogen activator activity, characterized by reacting tissue plasminogen activator in whole blood or a diluted product thereof, and then measuring the activity of the tissue plasminogen, The activity is measured by reacting the composite with a synthetic substrate specific for tissue plasminogen activator and detecting the amount of the substance liberated from the synthetic substrate. Measurement method 3 described in Section 1, the activity of the assembled product is determined by measuring the assembled product and an enzyme substrate specific for tissue plasminogen activator in the presence or absence of a tissue plasminogen activator activity stimulator. The scope of the claim is that measurement is performed by reacting an enzyme produced thereby with a synthetic substrate specific to the enzyme and detecting the amount of the substance liberated from the synthetic substrate. Assay method 4 described in item 1 includes at least a solid phase bound to a tissue plasminogen activator antibody IgG fragment having affinity for a site other than the active site of tissue plasminogen activator, and a solid phase bound to the fragment. For measuring tissue plasminogen activator activity, the method comprises a measuring reagent for measuring the activity of a reaction product between a phase and a tissue plasminogen activator in whole blood or a diluted product thereof. Kit 5: Kit 6 for measuring reaction product activity according to claim 4, wherein the reagent for measuring reaction product activity is only a synthetic substrate specific for tissue plasminogen activator. The measuring kit 7 according to claim 4, which contains at least a synthetic substrate specific for the enzyme produced by the reaction of the composite with a substrate specific for tissue plasminogen activator, is made of plastic. Measurement of tissue plasminogen activator activity by fixing a coating layer of a tissue plasminogen activator antibody IgG fragment having affinity for sites other than the active site of tissue plasminogen activator on one end of a stick. The stick-shaped measuring device 8 for use in the present invention has a stick portion that includes at least a portion fixed to the coating layer at the edge of the coating layer or its near edge on the non-coated side, and can be easily cut by bending the stick portion using the opening edge of the container as a fulcrum. The stick-shaped measuring device 9 for measuring tissue plasminogen activator activity according to claim 7 is provided with an easy-to-cut part, and the vacuum sampling device may contain an anticoagulant and/or a diluting medium. Claim 7 in a form pre-stored in a blood vessel
Stick-shaped measuring device for measuring tissue plasminogen activity according to item 1 or 8