JP2610808B2 - Immunoassay reagent using monoclonal antibody against tissue-type plasminogen activator derived from human normal cells - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a tissue-type plasminogen activator (hereinafter, referred to as a "plasminogen activator") collected from a cell culture of human normal tissue-derived cells.
(abbreviated as t-PA)).

(Prior art) Lymphocyte cells and mammals (eg, mice and rats)
Fusions with myeloma cells derived from E. coli produce hybrid cells capable of growing and replicating in vitro (Kohler
and Milstein, Nature 256, 495-497, 1975). Since such hybrid cells have the property of secreting a uniform antibody (monoclonal antibody) having a predetermined specificity, hybrid cells producing monoclonal antibodies against various hormones, proteins, and the like are produced. Attempts have been made to use the monoclonal antibodies produced by them for various studies. (E. Dale Servier et a
l., Clinical Chemistry Vol.27, No.11,1797-1806,198
1). However, there was no finding about a monoclonal antibody against t-PA derived from human normal cells.

As plasminogen activators, urokinase separated and purified from urine or cultured kidney cells, and streptokinase collected from Streptococcus are now practically used as thrombolytic agents.

However, since they are inferior in affinity for fibrin, they are often administered in large amounts in order to obtain the effect required for treatment, and it is known that side effects such as internal bleeding occur. That is, by these, plasmin produced in the circulating blood binds to the plasmin inhibitor in the blood and is quickly inactivated. It is necessary to produce plasmin in excess of the amount of plasmin inhibitor. However, the production of large amounts of plasmin breaks down fibrinogen, causing the side effect of bleeding tendency. On the other hand, it has high affinity for fibrin,
If plasmin can be produced on fibrin,
The fibrin can be decomposed in a small amount without being affected by the plasmin inhibitor in the circulating blood, and the action of decomposing fibrinogen in the circulating blood is weakened.
Under such circumstances, a thrombolytic agent having a high fibrin affinity, a small amount, a high thrombolytic activity, and few side effects is desired.

On the other hand, in recent years, a plasminogen activator having high fibrin affinity has been separated and purified from a human melanoma cell culture (see Japanese Patent Application Laid-Open No. 57-28009). However, since this is made from tumor cells, it has problems in antigenicity and carcinogenicity and cannot be put to practical use.

On the other hand, recently, t-PA having the following properties was found from a cell culture solution derived from a human normal tissue, and was isolated and purified (JP-A-59-51220).

a) molecular weight: 63,000 ± 10,000 b) isoelectric point: 7.0-8.5 c) affinity for fibrin: yes d) affinity for concanavalin A: yes e) optimal pH: 7-9.5 f) reaction with anti-urokinase specific antibody do not do.

A major advantage of t-PA is that it has a very high affinity for fibrin and is degraded from a human normal cell culture solution. Therefore, thrombolysis with few side effects that does not have the various drawbacks of the various plasminogen activators described above. It can be an agent.

In addition, plasminogen activator derived from human melanoma cells has no stabilizing effect by adding human serum albumin (Thromb Haemostas, 48: 3, 294-296, 1982).
On the other hand, t-PA has a stabilizing effect and has various different properties from human melanoma cell-derived plasminogen activator, such as long-term storage and no inactivation by lyophilization. is there.

t-PA plays an important role in the mechanism of fibrinolysis. Therefore, if the amount of human t-PA in blood can be measured accurately and easily, its prevention against various diseases,
Very useful for diagnosis.

Conventionally known methods for measuring human t-PA include a fibrin plate method, a synthetic substrate method and a fibrin solid phase method. However, in each case, the activity of plasmin generated by the action of t-PA on plasminogen is measured, and the enzyme activity of t-PA is measured indirectly. Also, the presence of plasminogen inhibitor (PAI) in blood is known, and even if the enzyme activity of t-PA is directly measured, it cannot be said that the amount of t-PA itself is necessarily reflected. , There was a problem with accuracy.

(Problems to be Solved by the Invention) In the treatment of thrombosis and the like, studies on the mechanism of action of t-PA,
Development of a highly sensitive detection method for t-PA has been desired as a means for confirming blood levels and the like. At present, as a method for measuring t-PA, a method using a monoclonal antibody prepared using a plasminogen activator derived from melanoma as an antigen has been reported (see JP-A-59-5121). A kit by a sandwich enzyme immunoassay (ELISA) using a polyclonal antibody is commercially available (Biopool t-PA ELISA kit, manufactured by Biopool). However, these methods have poor sensitivity to t-PA,
It is not satisfactory in terms of accuracy, and improvement is desired.

(Means for Solving the Problems) The present inventors have conducted intensive studies to overcome these problems, and as a result, obtained a monoclonal antibody having specificity for t-PA by cell fusion. Succeeded.
This monoclonal antibody is industrially very useful, for example, it can be used for immunoassay and the like, and if a preserved hybrid cell is cultured, a large amount of the monoclonal antibody can be obtained at any time.

That is, the present invention relates to the use of a monoclonal antibody having specificity for t-PA collected from a cell culture of human normal tissue-derived cells.

Generally, the monoclonal antibody used for the immunoassay of t-PA includes (1) a monoclonal antibody that specifically binds to the site where the fibrinolytic activity is expressed (the fibrinolytic activity of plasminogen activator when the antibody binds). Disappears, but retains fibrin affinity), (2) a monoclonal antibody that specifically binds to the fibrin affinity site (the fibrin affinity of the plasminogen activator is lost when the antibody binds, (3) retains fibrinolytic activity), (3) Monoclonal antibody that binds to an antigenic determinant other than (1) and (2) above (even if the antibody binds, it has no effect on fibrinolytic activity and fibrin affinity) Is not shown).

Monoclonal antibodies used in the present invention include those having the property of not binding to the fibrinolytic activity expression site and the fibrin affinity site of t-PA and those having specific reactivity to the fibrin affinity site.

The use of a combination of monoclonal antibodies that specifically bind to different antigenic determinants as described above provides a more sensitive method for measuring t-PA.

In general, a method of producing an antibody bound to two different positions of an antigen and measuring the presence or absence or the amount of the antigen is called a sandwich method. For example, a wide-area "radioimmunoassay method (Radioimunoas say Methods)" 199-206
(1970).

The immunological assay reagent of the present invention uses two types of monoclonal antibodies that recognize different antigen-determining sites of t-PA, respectively. Thus, according to the present invention, t-PA in a solution state (for example, in plasma) can be constantly and accurately measured without a difference in the quality of the reagent. In addition, since human t-PA is directly measured, it can be measured accurately and in a short time without being affected by other contaminants at all. Therefore, according to the present invention, there is provided a reagent capable of accurately and rapidly measuring human t-PA which has not existed conventionally.

Next, a method for measuring the contents of the measurement reagent and human t-PA according to the present invention will be specifically described.

A monoclonal antibody (first antibody) against human t-PA is immobilized on a suitable insoluble carrier (for example, a plastic container) (hereinafter, this is referred to as “immobilized antibody”). Next, the surface of the insoluble carrier is coated with a suitable substance (for example, bovine serum albumin) in order to avoid non-specific binding between the insoluble carrier and the reagent or sample material to be measured.

The insoluble carrier on which the first antibody thus obtained is immobilized is brought into contact with the specimen sample for a certain period of time and at a temperature for reaction. During this time, the immobilized antibody (first antibody) and human t-PA in the specimen sample bind. Then, after washing with an appropriate washing solution, a solution (for example, an aqueous solution) of a monoclonal antibody (second antibody) against human t-PA labeled with an appropriate labeling substance is added to the human t-PA bound to the immobilized antibody on the insoluble carrier. -Contact with PA for a certain time and at a temperature, and react with the second antibody. This is washed with an appropriate washing solution, and then the amount of the labeling substance labeled on the second antibody present on the insoluble carrier is measured. Thus, the amount of t-PA in the sample can be calculated from the value.

Thus, the measurement reagent of the present invention mainly comprises an immobilized antibody in which the first antibody is bound to an insoluble carrier and a labeled second antibody. In order to use this reagent efficiently and conveniently, a kit can be formed containing various auxiliary agents in addition to these antibodies. As such an auxiliary, for example, a dissolving agent for dissolving a solid reagent,
When an enzyme is used as a detergent for washing an insolubilized carrier or a labeling substance of an antibody, it is usually used as a kit for immunological measurement reagents such as a substrate for measuring enzyme activity and a reaction terminator. Things.

Examples of the insoluble carrier used in the measurement reagent of the present invention include:
For example, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin,
Examples thereof include polymers such as cross-linked dextran and polysaccharide, as well as paper, glass, metal, agarose, and combinations thereof.

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

Further, it is advantageous to use a radioactive substance, an enzyme or a fluorescent substance as the labeling substance. As a radioactive material
¹²⁵ I, ¹³¹ I, ¹⁴ C, ³ H, etc., enzymes such as alkaline phosphatase, peroxidase, β-D-galactosidase, etc., and fluorescein isothiocyanate, tetramethyl rhodamine isothiocyanate, etc. as fluorescent substances However, these are not limited to those exemplified, and any other ones can be used as long as they are used in the immunological measurement method.

Hereinafter, a method for producing a monoclonal antibody will be specifically described.

(A) Preparation of antibody-producing cells Preparation of antibody-producing cells may be performed according to a conventional method. That is, a method of immunizing an animal with t-PA as an antigen and obtaining antibody-producing cells of the animal may be used.

Examples of animals include mice, rats, rabbits, guinea pigs, sheep, horses, cows, and the like. Examples of antibody-producing cells include splenocytes, lymph node cells, and peripheral blood cells.

(B) Preparation of myeloma cells Myeloma cells used in the cell fusion method are not particularly limited, and cell lines of many animals such as mouse, rat, rabbit, and human can be applied. The cell line used should preferably be drug resistant so that unfused myeloma cells cannot survive in the selection medium and only hybrid cells will proliferate. The most commonly used cell line is 8-azaguanine resistant, which is hypoxanthine.
Guanine phosphoribosyltransferase is deficient and hypoxanthine-aminopterin-thymidine (HA
T) It cannot grow in a medium. The cell line used is preferably a so-called “non-secretory” cell line. For example, P ₃ / X63-Ag8U1 (P ₃ U1), P ₃ / from mouse myeloma strain SP-1 or mouse myeloma strain MOPC-21
X63-Ag ・ 6 ・ 5 ・ 3, P ₃ / NS1-I-Ag4-1, Sp2 / O-A
g14, rat myeloma cells 210, RCY3, Ag1, 2.3, etc. can be preferably used.

(C) Cell fusion Normally, Eagle's minimum basic medium (MEM), Roswell
Park Memorial Institute (RPMI) 1640
1-5 × 10 ⁷ myeloid cells and 1-5 × 10 ⁸ antibody-producing cells were mixed in a medium such as a medium (the mixing ratio was 1: 4 to 1: 1).
0), cell fusion is performed. As a fusion promoter, polyethylene glycol having an average molecular weight of 1,000 to 6,000 (PE
G) is preferable, but a virus or the like can also be used. PEG
Is usually 30 to 50%.

(D) Selective growth of hybrid cells Cells that have completed cell fusion contain 10-20% fetal bovine serum.
Dilute appropriately with RPMI 1640 medium and inoculate about 10 ⁵ to 10 ^{6 in a} microtiter plate. A selection medium (for example, HAT medium) is added to each well, and after that, the selection medium is appropriately replaced and cultured. If an 8-azaguanine-resistant strain is used as the myeloid cell, the unfused myeloid cell will
In the medium, all the cells died by about 10 days, and the antibody-producing cells were normal cells.
o) cannot grow for a long time. Therefore, culture 10-14
Everything that grows from day to day is hybrid cells.

(E) Search for antibody-producing hybrid cells Hybrid cells may be screened by a conventional method, and there is no particular limitation. For example, a part of the supernatant of the well in which the hybrid cells have grown is collected and reacted with t-PA, and then the amount of the label is reduced by reaction with a second antibody labeled with an enzyme, a radioisotope, a fluorescent substance, or a luminescent substance. It can be measured and assayed for the presence of anti-human tissue-type plasminogen activator antibody.

(F) Cloning Since there is a possibility that two or more hybrid cells may grow in each well, cloning is performed by a limiting dilution method or the like to obtain a monoclonal antibody-producing hybrid cell.

(G) Obtaining antibodies The purest monoclonal antibody can be used to generate 10
% Of fetal bovine serum, and cultured in a suitable culture medium such as RPMI 1640 medium, and can be obtained from the culture supernatant.

On the other hand, in order to obtain a larger amount of antibodies, pristane (2, 6, 10, 14
-Hydrogen oil such as (tetramethylpentadecane) may be administered intraperitoneally, and then the hybrid cells may be administered to allow the hybrid cells to grow in large quantities in vivo.
In this case, an ascites tumor is formed in about 10 to 18 days, and a high concentration of antibody is generated in serum and ascites.

(H) Purification of Monoclonal Antibody Purification of anti-human tissue-type plasminogen activator monoclonal antibody from ascites can be performed by a method similar to a conventional method for recovering an antibody from serum, for example, Hudson et al. (Practica
l Immunology, Blackwell Sci. Pub., 1976) can be applied.

The titration of t-PA purified as an antigenic substance was performed by the following method (the same applies to the following experiments).

95% coagulated fibrinogen (containing plasminogen
Using fibrin plates with agar prepared using 50 casein units / g coagulated protein) as a raw material, the measurement was performed by a plate method using urokinase as a standard. t-PA solution with 1% gelatin,
Contains 0.1M sodium chloride and 0.1% sodium nitride
After dilution with 0.067 M Tris-HCl buffer (pH 8.0), the concentration of the plasminogen activator solution used in the present invention, which shows the same dissolution window as 101 U / ml urokinase on the fibrin plate, was 10 U / ml.

According to the present invention, a monoclonal antibody characterized by having specificity for t-PA derived from human normal cells is provided by a hybrid cell line.

(Effect of the Invention) The anti-human tissue-type plasminogen activator monoclonal antibody of the present invention is used for immunoassay of t-PA in clinical and the like. By introducing the monoclonal antibody into the analysis of the coagulation / fibrinolysis system, such as the study of the mechanism of action of t-PA in blood, highly sensitive microquantification becomes possible.

Further, the measurement system of the present invention is extremely useful in the following points.

(1) Since an antibody that recognizes the active site is not used,
T-PA bound to an inhibitor such as PAI-I can also be measured. Measurement of the total amount of t-PA is an important index for diagnosing disease and determining prognosis.

(2) t-PA is characterized by being a substance having high fibrin affinity. In recent years, a new t-
PA development is underway. Each of the novel t-PAs has a structure that determines fibrin affinity. Since this measurement system uses an antibody that recognizes a fibrin-affinity site, it is necessary to measure all t-PAs of biological origin and fibrin-saturated t-PAs present in blood that are present. Can be.

(Example) Example 1 Purification of antigen Ammonium sulfate was added to human embryonic kidney tissue culture solution 4 at a rate of 300 g / day, and the mixture was allowed to stand at 4 ° C overnight. The resulting precipitate was collected by filtration and 1 M
Dissolve in 1M rhodamon ammonium solution containing sodium chloride. The obtained lysate has a volume of 400 ml and the activity of t-PA is 21 U.
/ ml, specific activity was 10 U / A _280. After adsorbing this on a phenyl sepharose column (1 × 10 cm), ethylene glycol was increased to about 50% by a concentration gradient method, and t
-Elute PA. The eluate has a volume of 150 ml and the activity of t-PA is 5
The specific activity was 2 U / ml and 250 U / A ₂₈₀ .

This eluate was dialyzed against a physiological saline containing 0.1% twin 80, passed through an anti-urokinase 1g-G Sepharose column, and then passed through an arginine Sepharose column (1.5 × 10 cm).
Adsorb continuously. After thoroughly washing with 0.5M sodium chloride solution containing 0.1% twin 80, 0.5% containing 0.1% twin 80
Elute t-PA with M arginine solution. The solution, liquid volume 52 ml, the activity of t-PA is 98U / ml, specific activity was 3200U / A _280.

After concentration by lyophilization, 1.5M sodium chloride, 0.1M
0.0 with arginine, 0.1M EDTA and 0.1% Twin 80
The active portion is collected by gel filtration on a column (1.5 × 100 cm) of Sephadex G-150 equilibrated with 1 M phosphate buffer (pH 7.0). The resulting solution liquid volume 15 ml, the activity of t-PA is 270 U / ml, specific activity was 12500U / A _280.

Immunization of mice 100 μg of t-PA purified as described above was emulsified in Freund's complete adjuvant, and mouse BALB / c
♂ group was subcutaneously administered three times at two-week intervals for immunization. Among these mice, mice with the highest serum antibody titer to t-PA were injected intraperitoneally with 100 μg of t-PA to complete the immunization.

Cell fusion Three days after the final immunization, the mice are killed, the spleens are removed,
After shredding, pressing and filtering with stainless steel mesh, Eagle's Minimum Essential Medium (ME
M) to give a spleen cell suspension. These spleen cells and mouse myeloma cells SP-1 each do not contain serum
After washing three times with MEM, spleen cells and SP-1 were mixed at a ratio of 10: 1 and centrifuged (800 rpm, 5 minutes), the precipitate was loosened gently, and 1 ml of a 44% polyethylene glycol 2000 / MEM solution was gradually added. , 37
The cell fusion was carried out by slowly rotating the centrifuge tube for 1 minute in hot water at ℃. After 1 minute, add 1 ml of MEM and rotate slowly,
Further, MEM was added at a rate of 2 ml per minute to make a total of 10 ml, and the supernatant was removed by centrifugation at 1,000 rpm for 5 minutes. The cell pellet was purified from Roswell Park® containing 10% fetal calf serum.
SP on Memorial Institute (RPMI) 1640 medium
-1 was suspended at 1 × 10 ⁶ cells / ml, and 0.1 ml was inoculated in a 96-well microplate (manufactured by Nunc).

One day later, RPMI 1 containing HAT (hypoxanthine 1 × 10 ⁻⁴ M, aminopterin 4 × 10 ⁻⁷ M, thymidine 1.6 × 10 ⁻⁵ M)
A 640-10% FCS medium (HAT medium) was added to each well in an amount of 0.1 ml, and thereafter, the HAT medium was replaced every half day with a HAT medium every 3 to 4 days to proceed with HAT selection. The hybrid cells grew in some wells from about day 7 and hybrid cells grew in almost all wells after 10 to 14 days.

Screening for antibody-producing cells
Hybrid antibody-producing hybrid cells against t-PA were examined by ked immnosorbent assay (ELISA). ELISA is Jean-Luc Guesd
On (J. Histochem. cytochem. 27, 1131 , 1979), a biotin-avidin system was used. T-PA in 96-well microplate
Was dispensed at 0.05 μg / 100 μ / well, and allowed to stand at 25 ° C. for 18 hours to adsorb t-PA to the solid phase. Phosphate buffered saline (PB
S) After washing three times with 200μ, 200μ of PBS containing 0.5% bovine serum albumin was added, and the mixture was allowed to stand at 4 ° C overnight to block unadsorbed portions of each well.

Next, a culture medium as a specimen was put in a 100 μ / well, and 37
The reaction was carried out at 2 ° C. for 2 hours. P containing 0.05% Triton X-100
After washing three times with BS, 100 µ / well of biotinylated-goat anti-mouse IgG (manufactured by EY Laboratories) was added, and reacted at 37 ° C for 1 hour. After washing, add horseradish peroxidase-avidin (manufactured by EY Laboratories) 100 μ / well, and after 1 hour, add P
After washing with BS-Triton, 0.001% hydrogen peroxide, 0.15m
g / ml Azino-bis (3-ethylbenzothiazoline-6,6-su
0.1M citric acid of lf onic acid (manufactured by Hanoi Chemical)
A sodium hydroxide buffer (pH 4.0) was added, and the absorbance at a wavelength of 414 nm was measured. In the sample, coloring is observed only in the holes where the antibody against t-PA was present.

Cloning of single antibody-producing cells against t-PA Hybrid cells in the wells of the microplate that showed a positive result in ELISA were taken out, and the number of cells was measured. Dilute with RPMI 1640 medium supplemented with 10% fetal bovine serum and add 0.5 /
It was inoculated into the hole. The microtray used was the one inoculated and cultured at 2 × 10 ⁵ cells / ml of mouse peritoneal cells as a feeder cell the day before. The culture was carefully continued for about 2 weeks while changing the medium, and the colony formation of the hybrid cells was awaited. The amount of antibody produced in the hole where the hybrid cells proliferated and colonies appeared was determined by EL
Hybrid cells that showed a positive result as determined by ISA were cloned again.

Clonal cells X-21 and X-23 having high antibody-producing ability were obtained.

Production of single antibody against t-PA in vivo 0.5 ml of Pristane (manufactured by Aldrich) was intraperitoneally administered to BALB / c mice 7 weeks or older, and after 1 week or more, culture and proliferation in vivo One to 9 × 10 ⁸ hybridized cells / mouse were inoculated intraperitoneally. One week after inoculation of the hybrid cells, the mice gained weight rapidly and peaked at 10-15 days. Mice were sacrificed under ether anesthesia before and after the peak body weight, and ascites was collected. This was centrifuged at 3,000 rpm for 10 minutes to obtain ascites containing a monoclonal antibody at 5 to 15 ml / animal.

Example 2 Purification of monoclonal antibody From 10 ml of ascites fluid obtained in Example 1, Hudson et al.
Cal immunology Blackwell Sci. Pub., 1976), and the monoclonal antibody was purified.

First, 10 ml of a saturated ammonium sulfate solution (10 ml
% Saturated) and left at 4 ° C. overnight. The resulting precipitate was centrifuged, dissolved in 5 ml of 0.01 M phosphate buffer (pH 8),
It was dialyzed overnight against the same volume of the same buffer. 10,000 rotations after dialysis, 5
After centrifugation for minutes, the supernatant was separated. This sample was equilibrated with a sufficient amount of 0.01 M phosphate buffer (pH 8).
DEAE Toyopearl 650M column (manufactured by Toyo Soda Co., Ltd.) subjected to 20 ml, 0.01 M Tris buffer until the A ₂₈₀ is 0.02 or less (pH
8) After washing in 0.01M, the solution was
The concentration was changed linearly to a Tris buffer (pH 8), and the monoclonal antibody was fractionated and eluted. Monoclonal antibody fractionation
Antibody activity by EIA was determined by SDS-polyacrylamide gel electrophoresis, followed by fractionation and elution using a Sephadex G-200 column (Pharmacia) equilibrated with phosphate buffered saline. 21 monoclonal antibodies
106 mg and 73 mg of X-23 monoclonal antibody were obtained.

Example 3 Physicochemical Properties of Monoclonal Antibody The following physical properties were measured using the monoclonal antibodies X-21 and X-23 purified in Example 2.

a) Molecular weight: 15,000 ± 5,000 (X-21) 153,000 ± 5,000 (X-23) SDS-polyacrylamide gel slab electrophoresis using a 5-10% gradient gel was performed using Laemmli's buffer system. The molecular weight of the antibody was estimated. The molecular weight marker is SDS-PAGE standard molecular weight marker (Hi
gh) (Myosin 200K, β-galactosidase 116.25K, phosphorylase B 92.5K, bovine serum albumin 66.2K, ovalbumin 45K).

b) IgG subclass: IgG1 (X-21, X-23) 1% agarose in 1/15 M phosphate buffered saline (pH 7.2)
In addition, after boiling and dissolving, 5 ml was put on a slide glass and solidified. Holes having a diameter of 3 mm were made at intervals of 3 mm, and each hole was filled with 15 μl of a culture solution of the fused cells that had been successfully ascited or an antiserum (anti-mouse Ig) against a mouse antibody produced by a goat. The slide glass was placed in a wet box, allowed to stand at room temperature for 18 hours, and the formation of a sedimentation line due to an antigen-antibody reaction was observed between the two wells containing the culture solution and anti-mouse Ig.

c) Isoelectric point: pI = 6.7-7.0 (X-21) pI = 6.6-6.9 (X-23) Using an LKB-column isoelectric focusing apparatus, add about 1 mg of partially purified reclonable antibody. Then, in the presence of an ampholine carrier having a pH of 3.5 to 9.5: ampholite (manufactured by LKB), current was applied at 4 ° C. and a constant voltage of 700 V for 40 to 60 hours. After pH measurement under ice cooling, 2
The absorbance at 80 nm and the detection of a specific antibody against t-PA were measured using a monoclonal antibody screening method.
The results are shown in FIG. 1 and FIG.

d) Binding site with antigen: Antigenic determinant other than fibrin affinity site and fibrinolytic activity expression site (X-21) Fibrin affinity site (X-23) X-produced in Examples 1 and 2 After reacting an antigen-antibody reaction with t-PA using a 21 monoclonal antibody and an X-23 monoclonal antibody, the remaining fibrinolytic activity was measured by the fibrin plate method. Table 1 shows the results.

Table 1 X-21 X-23 Residual activity (%) 100 25 A fibrin sepharose column [Thromb. Res.
2 137-154 (1973)] and eluted in a physiological saline solution containing 0.3 M arginine / hydrochloric acid. The fibrinolytic activity of the eluate was measured by the fibrin plate method. Table 2 shows the results.

Table 2 Fibrinolytic activity (%) Eluate t-PA alone t-PA-X-21 t-PA-X-23 Passage fraction 20 23 86 Eluted fraction 80 77 14 e) Amino acid sequence (less than N) Sequence Analysis was performed using Edman decomposition (Edman et al., Euro pean J. Bi
ochem., 1, 80, 1967). Samples were analyzed using a gas-phase protein sequencer (Applied Biosystems, M
odel 470 A) The cartridge is introduced into the cartridge, and the coupling, cleavage and co
An nversion reaction was performed. The obtained PTH (phenylthiohydantoin) -amino acid was dissolved in acetonitrile / H ₂ O / TFA (trifluoroacetic acid) = 33/67 / 0.02 (v / v / v), and reverse-phase high-pressure liquid chromatography (column: 4.6φ × 30cm, injected into Senshu Scientific SEQ-4), and the retention time is standard P
Each PTH by comparing with the retention time of the TH-amino acid
-The amino acids have been identified.

 The obtained amino acid sequence is as follows.

(1) X-21 (In the formula, parentheses indicate putative residues, and X indicates an unidentified residue.) (2) X-23 (In the formula, parentheses indicate putative residues, X indicates an unidentified residue, and Pca indicates pyrrolidone carboxylic acid.) F) Cross-reactivity with various plasminogen activators Human urinary urokinase and porcine heart extract plasminogen The reactivity with the activator was examined by ELISA (biotin-avidin system). X-21 and X-23 monoclonal antibodies bound only t-PA and did not react with other urokinase, porcine heart extract plasminogen activator.

Example 4 Quantification of t-PA by ELISA Using Monoclonal Antibody ELISA reagents were prepared using monoclonal antibodies X-21 and X-23 produced according to Examples 1 and 2.

(1) Preparation of Horseradish Peroxidase (HRP) Labeled Monoclonal Antibody-Fab 'The method of S. Yoshitake et al. (J. Biochem. 92, 1413-1424 , 198)
According to 2), 30 mg of the monoclonal antibody X-23 IgG was digested with pepsin, and subjected to gel filtration using Sephadex G-100 to collect an F (ab ') ₂ fraction. The obtained F (ab ')
Β-mercaptoethylamine was added to the buffer solution of ₂ ,
After reduction treatment at 37 ° C. for 2 hours, fractionation was performed using Sephadex G-25, and Fab ′ was collected. Maleimidated HRP and Fa
b ′ was mixed and reacted at 4 ° C. for 20 hours, then purified using Sephacryl S-200 equilibrated with 0.1 M sodium phosphate buffer (pH 6.5), and the absorbance at 280 nm and 403 nm was measured. The fraction of the absorption maximum was collected.

(2) ELISA measurement system Monoclonal antibody X-2 dissolved in 0.05 M sodium carbonate (pH 9.6) was added to each well of a 96-well flat bottom microplate.
1 was added in 200 μl increments. Incubate in a humid box at 25 ° C for 3 hours, discard the solution, and add PB containing 0.05% Twin 20
The S solution was poured into each well, washed by shaking for several minutes, and the solution was discarded. t-PA was diluted with a PBS / twin solution containing 0.1% BSA, poured into each well at 200 μl, and reacted at 25 ° C. for 16 hours. After washing with PBS / twin solution as before, HRP
200 μl of a PBS / twin dilute solution of the labeled monoclonal antibody X-23-Fab ′ was added and reacted at 25 ° C. for 3 hours, and the solution was discarded. After washing, 200 μg of sodium citrate phosphate buffer (pH 5.0) containing 0.01% hydrogen peroxide and o-phenylenediamine (0.4 mg / ml) was added as an enzyme substrate,
The reaction was performed at 25 ° C. for 30 minutes. After stopping the reaction by adding 50 μM of 4.5 M sulfuric acid, the absorbance at 492 nm was measured.

(3) Sensitivity of the measurement system Using t-PA (1.6 to 0.05 ng / ml) at various concentrations, a calibration curve was created from an average value measured ten times. The results are shown in FIG.

From these results, using this measurement system, t-PA 0.1 ng / ml
Can be quantified.

Example 5 Quantification of t-PA in human blood t-PA in human blood was determined by ELISA prepared in Example 4.
Was determined. The sample was prepared by the method of Bergsdarf et al. (Tromb. Hae
most. 50, 740-744 , 1983). Citrate-added blood was centrifuged (3000 rpm, 15 minutes), and 25 µ of plasma was added to 25 µ of 1 M sodium acetate buffer (pH 3.9).
And leave at room temperature for 1 hour. After neutralizing by adding 50 μM of 0.1 M disodium hydrogen phosphate-0.4N sodium hydroxide aqueous solution, 400 μL of a PBS / twin solution containing 0.1% BSA was added, and 2
A 0-fold dilution was used as a sample. Blood was collected from 22 healthy people,
Quantification of t-PA was performed. The results are shown in FIG.

From these results, the average of t-PA in healthy human blood was 8.88 ±
It was 0.044 (SD) ng / ml.

[Brief description of the drawings]

FIG. 1 is a table showing the correlation between isoelectric focusing results of X-21 monoclonal antibody and antibody activity, and FIG. 2 is a graph showing the correlation between isoelectric focusing results of X-23 monoclonal antibody and antibody activity. The chart shown, FIG. 3 is a chart of a calibration curve by ELISA, and FIG. 4 is a chart showing the results of measuring the amount of t-PA in the blood of a healthy subject.

Claims (1)

(57) [Claims] 1. An immunological tissue type plasminogen activator assay reagent using a sandwich method, wherein an antibody bound to an insoluble carrier and a labeled antibody each have a specific fibrin affinity site of the tissue type plasminogen activator. Monoclonal antibody (X-23)
And a monoclonal antibody (X-21) that recognizes an antigenic determinant other than a fibrin affinity site and a fibrinolytic activity expression site of a tissue-type plasminogen activator. A tissue type plasminogen activator assay reagent using a monoclonal antibody against beta.