JP3036545B2 - Monoclonal antibody, hybridoma cell line producing the same, and immunoassay using the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a monoclonal antibody, a hybridoma cell line producing the same, and an immunoassay using the same.

More specifically, the present invention relates to a monoclonal antibody of diadenosine tetraphosphate (Ap ₄ A), a hybridoma cell line that specifically produces the monoclonal antibody, and a medical field, particularly, a diagnosis and treatment of cardiovascular diseases and the like. it relates immunoassay of Ap ₄ a in biological fluids using useful this monoclonal antibody.

(Prior art) Living body adenosine tetraphosphate (Ap ₄ A)
Is released mainly from activated platelets and appears in the blood, inhibiting platelet aggregation (MJ Harrison et al., FEBS Let
ters, 54 , 57, 1975), promoting histamine release from mast cells (BSGomperts, Secretary Process Butterwor
ths, London. 18-37, 1984), vasoconstrictor action (R. Busse
Am. J. Phyiol, 254 H828-H832, 1988).

Conventionally, two methods are known as a method for quantifying this Ap ₄ A in a biological fluid. One is to use a high-performance liquid chromatography (HPLC) to separate the sample containing Ap ₄ A on a reversed-phase column and measure the absorbance at 260 nm to determine the amount of Ap ₄ A in the sample. (The above R.
Busse et al.).

The other is called the enzymatic method, in which Ap ₄ A is split into adenosine monophosphate and adenosine triphosphate (ATP) by an enzyme (phosphodiesterase), and ATP is essential, and depending on the amount, is that quantify the Ap ₄ a by light emission using the luciferase activity changes. (BKKim, Blo
od 66, 735-737,1985).

(Problems to be Solved by the Invention) However, in these conventional measurement methods, for example, in the case of a method using HPLC, the sample is previously prepared with a hole diameter of 0.45 μm in order to prevent the column from being significantly deteriorated by biological components.
A complicated operation of processing with the following filters is required. Furthermore, in the case of this method, a problem of the hard surface of HPLC, stable it is difficult to obtain a baseline value, also including Ap ₄ A elution time becomes uneven by a slight temperature change, solvent change, etc. Some have disadvantages.

Also, in the case of the enzymatic method using phosphodiesterase and luciferase, since ATP is ultimately measured with luciferase, it is necessary to eliminate ATP mixed in biological components with phosphomonoesterase in advance, and this phosphomonoesterase Requires a complicated operation of inactivating it by heat treatment (90 ° C., 5 minutes) in order not to erase ATP generated in the next step.

Thus, in the conventional Ap ₄ A measurement method, the
Separation of Ap ₄ A using HPLC or separation with an enzyme makes the operation complicated, and the measurement accuracy was not always good.

In general, when a specific physiologically active substance in a living body is quantified, an antibody of the substance, in particular, if a monoclonal antibody is identified, for example, by adding the labeled antibody to the sample, the target substance can be determined. Direct quantitative immunological measurements are possible. However, monoclonal antibodies against Ap ₄ A far did not exist.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an Ap ₄ A monoclonal antibody that has not existed so far. The present invention also uses a hybridoma cell line that specifically produces the monoclonal antibody and the monoclonal antibody.
Some for the purpose of providing an immunological assay of Ap ₄ A.

(Means for Solving the Problems) The present invention provides a monoclonal antibody specifically recognizing diadenosine tetraphosphate to solve the above problems.

Also, the present invention provides a lymphocyte prepared from a mammal immunized with a diadenosine tetraphosphate antigen, and a diadenosine phosphate-specific recognition monoclonal antibody characterized by being produced from a hybridoma with myeloma cells; Provided are a hybridoma cell line capable of producing and an immunoassay for diadenosine phosphate using the monoclonal antibody.

 Hereinafter, the present invention will be described in detail.

Monoclonal antibodies of the present invention, as described above, Ap ₄ A
And lymphocytes prepared from mammals immunized with antigens, hybridomas are selected with myeloma cells, it can be obtained by culturing, in terms of production efficiency, of the above hybridomas, a monoclonal antibody against Ap ₄ A sorted cells that specifically produced, in terms of which was cloned and cell lines, preferably to obtain a monoclonal antibody of Ap ₄ a by culturing this cell line.

Sampling method of creating monoclonal antibodies of such cell lines, and immunological assay of Ap ₄ A using the monoclonal antibodies can be performed as follows.

(1) Ap ₄ create hybridoma cell lines which specifically produces monoclonal antibodies of A: First, to create a hybridoma, Ap ₄ lymphocytes of immunized mammal A antigen, myeloma to be fused thereto (bone marrow Tumor cells). Among them, to collect the above lymphocytes, first, a complex in which Ap ₄ A and a carrier protein are covalently bonded (hereinafter referred to as Ap ₄ A-carrier)
Create an Ap ₄ A antigen consisting of, this mammal, preferably immunizing mice or rats.

The method of immunization, such as the amount of antigen used, the site of administration, and the use of an adjuvant, may be in accordance with the conventional method for obtaining antisera. For example, if a mouse is used, 0.001 to 10 mg per dose per mouse, preferably Ap ₄ A- carrier 0.01 to 1 mg, initial adjuvant (e.g., Freund's complete adjuvant) is mixed with well, subcutaneous, The composition is administered intraperitoneally or the like. After two weeks or more, an adjuvant (for example, Freund's incomplete adjuvant) is mixed well and administered subcutaneously or intraperitoneally. After more than two weeks,
Ap ₄ A- carrier only intravenous, subcutaneous, and administered to the abdominal vagina etc., sufficiently immune. The animals immunized in this way are
Preferably, the cells are killed 2 to 4 days after the final immunization, and the lymphocytes are collected. For the preparation of lymphocytes, spleen, lymph nodes, peripheral blood and the like are used. The lymphocytes are loosened in a culture solution.

On the other hand, it is preferable to use myeloma derived from the same species as the animal to be immunized. Further, the myeloma is preferably a mutant strain with low drug resistance, and it is preferable that the unfused myeloma does not grow on the hybridoma selection medium. Most commonly, 8-azaguanine resistant cell lines are used. This is a hypoxanthine-guanine-phosphoribosyltransferase (Hypoxantin g
uanine phosphoribosyl transferase), a type of selective medium called hypoxanthine-aminopterin-
Cannot grow on thymidine (HAT) medium. It is also desirable that the myeloma used does not itself secrete antibodies. From the above points, for example, commercially available mouse myeloma P3 X63
・ Ag8 ・ 6 ・ 5.3 ・ (X63 ・ 6.5 ・ 3); P3 ・ X63 ・ Ag8
・ U1 (P3U1), rat myeloma 210 ・ RCY3 ・ Ag1 / 2
It is preferable to use 3 or the like.

This myeloma was added to serum, preferably fetal bovine serum, Eagle's minimal medium (MEM), RPMI1640 medium (RPMI1
640).

Next, the lymphocytes and myeloma obtained above are each suspended in a medium such as MEM and RPMI1640 and mixed. The mixing ratio at this time can be arbitrarily selected, but it is preferable to use a ratio of lymphocyte: myeloma of 1: 1 to 20: 1, preferably 5: 1 to 10: 1 in terms of the number of cells. The mixed cells are fused using a fusion promoter. As a fusion method, for example, Immunological Methods Volume 2, p. 285 (Immunological Methods V
ol. II, 1981, Academic Press). As the fusion promoter, various polymer substances, viruses and the like can be used, but polyethylene glycol (PEG) and Sendai virus are preferably used. PEG is
Those having an average molecular weight of 400 to 20,000 can be used, but those having an average molecular weight of 1,000 to 7,500 are preferably used. The use concentration is preferably 40 to 60 vol%.

The fused cells were washed to remove the fusion promoter,
The cells are suspended in MEM or RPMI1640 medium containing 15% by volume of serum and dispensed into 96-well culture dishes at a rate of 0.5 to 5 × 10 ⁶ / well. Furthermore, if a selection medium (for example, HAT medium) is added to each well and the selection medium is replaced as appropriate, unfused myeloma will die after 10 to 14 days, and only hybridomas will grow.
By the way, lymphocytes cannot grow for long periods of time in vitro and also die after 10-14 days.

Next, find the hybridomas producing antibodies against Ap ₄ A, sorted. As a method for that, an ELISA method can be used. However, the hybridomas in that case, to the Ap ₄ A- carrier and antigen, in addition to those that produce antibodies against Ap ₄ A, since there are also those that produce antibodies against a carrier protein, antibodies against Ap ₄ A Only the hybridomas that need to be produced need to be selected.
For this purpose, for example, complexes covalently coupling the Ap ₄ A to a protein other than a carrier protein to create a (hereinafter, Ap ₄ A- to as carrier 2), which was adsorbed to the ELISA plate, this hybridoma After washing and washing, a labeled antibody against a commercially available immunized animal immunoglobulin (for example, horseradish peroxidase (HRP) -labeled antibody or ¹²⁵ I-labeled antibody) is added. As a result, when there antibodies against Ap ₄ A in hybridoma supernatants, it is a solid phase Ap ₄ A- attached to the carrier 2, further bound to the labeled antibody which for immunoglobulin, with labeled A signal is obtained. On the other hand, if the antibody to Ap ₄ A is not present in the supernatant, the solid phase Ap ₄ A- carrier 2
Does not bind anything and therefore no signal is obtained. As described above, hybridomas can be selected based on the presence or absence of a signal due to the label.

Then, by selectively culturing a hybridoma which specifically produces monoclonal antibodies against Ap ₄ A thus obtained, it is possible to create a hybridoma cell line.

Incidentally, the pre-Ap ₄ A- 1 hybridomas cell lines were generated by fusing spleen lymphocytes and mouse myeloma cells from mice immunized with the carrier in accordance with this method was designated hybridoma UNH3H10. On July 1, 1990,
The procedure for depositing the microorganism with the Research Institute of Microorganisms of the Ministry of International Trade and Industry was carried out on the 1st, and was accepted as Fungus No. 11600 (FERM P-11600). This UNH3H10 can be cryopreserved almost permanently at −120 ° C. or lower, and is constantly placed in a state where it can be distributed.

This hybridoma UNH3H10 can grow in a commonly used medium. For example, RPMI1640 or MEM containing 5 to 20% fetal bovine serum is used as a medium, and grows well at 37 ° C. in air containing 5 vol. Further, since also has a tumorigenicity myeloma, in vivo (e.g., syngeneic animals, nude mouse) grown in, it is possible to produce monoclonal antibodies against Ap ₄ A.

(2) Ap ₄ A large amount harvesting of monoclonal antibodies: by culturing a hybridoma cell line that was created as described above, can be mass collected monoclonal antibodies to Ap ₄ A. There are roughly two methods for collecting this monoclonal antibody. One is
This is a method of culturing in a culture vessel such as a flask using a medium and collecting the antibody from the supernatant. For example, 5-10vol.
0.5-5 × 10 ^{5 in} MEM or RPMI1640 medium containing
Planting two hybridoma cell lines, 10-
This is a method of growing 20 times and collecting the antibody from the supernatant after the culture. Another method is to inoculate a syngeneic animal with the hybridoma cell line cultured in the culture vessel in this manner. That is, the hybridoma cell line 10
Administer ⁵ to 10 ⁷ animals subcutaneously or intraperitoneally, etc.
This method collects serum and ascites when the hybridoma cell line grows 7 to 20 days later and the tumor grows. When administering intraperitoneally, beforehand (3-7 days before)
Administration of mineral oils such as 2,6,10,14-tetramethylpentadecane results in greater ascites.

The antibody thus obtained can be purified and used as necessary. For example, the protein can be purified using a means usually applicable to proteins, such as ammonium sulfate fractionation, ion exchange chromatography, and affinity chromatography with protein A immobilized thereon.

In this way, easily monoclonal antibodies to Ap ₄ A, and can be obtained in a large amount.

(3) Immunological measurement of Ap ₄ A using a monoclonal antibody: First, an Ap ₄ A-carrier is immobilized on a solid phase. As the solid phase, a plastic tester, a microtiter plate, glass beads, plastic beads, a membrane, or the like can be used. A general physical adsorption method can be used to immobilize the Ap ₄ A-carrier, but a covalent bonding method can also be used to immobilize the Ap ₄ A-carrier on a solid phase having a functional group. At this time, the concentration of Ap ₄ A-carrier is
The concentration is 0.001 mg / ml or more, preferably 0.05 to 0.2 mg / ml, which may be brought into contact with a solid phase.

Next, on the solid phase on which the Ap ₄ A-carrier was immobilized in this manner,
Add several liquids with known Ap ₄ A concentrations or samples with unknown Ap ₄ A concentrations. Furthermore, this enzyme, RI, either added antibodies to Ap ₄ A labeled fluorescent substance or the like, or after addition of antibody to Ap ₄ A, adding the enzyme, RI, a secondary antibody labeled with a fluorescent substance or the like. In addition, HRP, bovine intestinal alkaline phosphatase, and the like can be used as the enzyme. Also, RI
¹²⁵ I or the like, and fluorescein isothiocyanate, tetramethyl rhodamine isothiocyanate or the like as the fluorescent substance.

Then each measured signal for several Ap ₄ A known concentration of labeled antibody liquid to prepare a calibration curve, applying the signal obtained from the Ap ₄ A unknown concentration of the analyte in the calibration curve, Ap of the specimen ₄ Quantify A concentration.

Hereinafter, the present invention will be described specifically with reference to Examples.

Example 1 (Screening of immunogen and hybridoma cell line) Keyhole Lympet Hemocyanin was used as a carrier protein.
(KLH) and bovine serum albumin (BSA) were used to screen immunogens and hybridoma cell lines.

The Ap ₄ A10mg dissolved in 0.1M NaIO ₄ 0.2ml, 2 at a temperature of 25 ° C.
Oxidized for 0 minutes. Ethylene glycol was added thereto to decompose excess NaIO ₄ . Then added 28mgKLH or BSA, adjusted to pH9.0~9.5 with aqueous sodium carbonate, Ap ₄ A
We have created a -KLH or Ap ₄ A-BSA. From the absorbance at 260 nm and the amount of protein, Ap ₄ A bound per KLH molecule was about 1000 molecules, and Ap bound per BSA molecule.
₄ A was determined to be about 0.5 molecule.

Example 2 (Preparation of hybridoma) Ap ₄ A-KLH prepared in Example 1 was completely mixed with 8-week-old mouse Balb / c (obtained from Oriental Bioservice) with complete Freund's adjuvant (obtained from Hanoi Chemical). mixture is emulsified in intraperitoneally, of 50μg after two weeks Ap ₄ A-
After booster injection with KLH intravenously, the spleen was removed 3 days later, and M
The suspension was loosened and washed with an EM medium (obtained from Hanoi Chemical).
On the other hand, mouse myeloma X63.6.5.3 (obtained from Kyoto University) was cultured for two days, and cells in the logarithmic growth phase were collected by centrifugation. 10 ⁸ splenocytes were used for myeloma X63-6
・ Mix with 5.310 ⁷ and pellet by centrifugation.
1 ml of 50% PEG4000-RPMI1640 (obtained from Gibco) was gradually added in a water bath at 1 ° C. over 1 minute, and after gentle stirring for 1 minute, 9 ml of RPMI1640 medium was gradually added, and PEG40 was added.
00 was diluted. The PEG solution was removed by centrifugation, 20 ml of HAT medium containing 10% fetal calf serum was added to the pellet, and
0.1 ml was dispensed into each well of a 96-well culture dish (manufactured by Corning). Half of the culture medium was discarded a total of three times on days 4, 8, and 11, and fresh HAT medium was added.

As a result, growth of hybridoma was observed in 24 of 384 wells 10 days later.

The lines producing antibodies against Ap ₄ A of the hybridomas obtained in Example 3 (preparation of hybridoma cell lines) Example 2 was searched using the ELISA method.

First, 50 μl of Ap ₄ A-BSA dissolved in 50 mM sodium carbonate buffer, pH 9.6 was dispensed into an ELISA plate (manufactured by Corning) at a temperature of 25 ° C. for 2 hours.
After removing this solution, each well was filled with 20 mM sodium phosphate buffer (pH 7.2) containing 1.0% BSA and 0.15 M sodium chloride, and allowed to stand at a temperature of 25 ° C. for 1 hour. This solution is further removed, and a 20 mM sodium phosphate buffer (pH 7.2) containing 0.2% Tween 20, 0.2% BSA and 0.15 M sodium chloride is used.
It washed well each well with wash solution composed of, creating the Ap ₄ A-BSA immobilized plate.

Next, 50 μl of the supernatant of the hybridoma obtained in Example 2 was added to the plate, and the plate was allowed to stand at a temperature of 25 ° C. for 2 hours.
(Purchased from Funakoshi) Add 1 μg / ml to each well and add 50 μl at 25 ° C
For 2 hours. Further, after washing, 50 μl of a peroxidase measurement reagent (manufactured by Bio-Rad) was added, and 25
The mixture was reacted at a temperature of 20 ° C. for 20 minutes, the peroxidase activity was stopped by adding 50 μl of 10% SDS, and the absorbance at 415 nm (A ₄₁₅ ) was measured. As a control, a reaction in which only the HAT medium was reacted instead of the hybridoma supernatant was used.

As a result, a supernatant showing a higher signal than the signal without the supernatant used as a control was obtained. The strain in the well of the supernatant was selected and cloned by the limiting dilution method to obtain a monoclonal hybridoma cell line UNH3H10.

Example 4 (Collection of monoclonal antibodies to Ap ₄ A) culturing a hybridoma cell line UNH3H10 obtained in Example 3, was taken of the monoclonal antibodies to Ap ₄ A.

First, UNH3H10 was cultured in RPMI1640 medium containing 10% fetal calf serum, and a 300 m culture having a cell concentration of 2 × 10 ⁶ cells / ml was obtained.
The supernatant was separated by centrifugation, the crude antibody fraction was separated by 50% saturated ammonium sulfate fractionation, dialyzed, and then adsorbed on protein A-Sepharose (pH 9.0) to obtain a citrate buffer (pH 3.0). )
In elution to Ap ₄ antibodies against A (hereinafter referred to as UNA3H10) was obtained.

Next, we examined the specificity for Ap ₄ A in this UNA3H10.

First, ELISA plates were fixed Ap ₄ A-BSA, each well 10
50 μl of Ap ₄ A or adenosine (A), adenosine monophosphate (AM) to give ^-7 , 10 ^-6 , 10 ^-5 , 10 ^-4 mol /
P), adenosine diphosphate (ADP), adenosine triphosphate (ATP), adenosine tetraphosphate (Ap ₄ ) are each injected into each well of the plate,
To each well was added 1050 μl of UNA3H at a concentration of 4.0 μg / ml. After incubating the plate at a temperature of 25 ° C for 2 hours,
After thoroughly washing with a 20 mM sodium phosphate buffer containing 0.2% Tween 20, 0.2% BSA and 0.15 M sodium chloride, an HRP-labeled goat anti-mouse IgG antibody was added to each well. Next, this was washed, 50 μl of a peroxidase measurement reagent (manufactured by Bio-Rad) was added, and reacted at a temperature of 25 ° C. for 20 minutes.
The reaction was stopped by adding 50 μl of 10% SDS, and the absorbance at 415 nm was measured. The results are as shown in Table 1 and FIG.

These Table 1 and as is apparent from Figure 1, the monoclonal antibody UNA3H10 is, A, AMP, and not at all aware of ADP, also slightly discernible to the ATP and Ap ₄ thereto also Ap ₄ A It is about 1/100 of cognitive ability.

For these reasons, UNA3H10 is considered to be a specific monoclonal antibody to Ap ₄ A.

Example 5 (Immunological measurement of Ap ₄ A using monoclonal antibody) Using the monoclonal antibody UNA3H10 obtained in Example 4,
Ap ₄ A concentration in the sample was measured immunologically.

First, UNA3H10 and pepsin at a weight ratio of pH 3.5.
Mix 200: 1, digest at 37 ° C. for 6 hours, UNA3H10F
(Ab ′) ₂ was obtained and further reduced to obtain UNA3H10Fab ′.

On the other hand, N-succinimidyl-maleimide carboxylate was reacted with HRP to introduce a maleimide group.

By mixing this maleimide group-introduced HRP and UNA3H10,
HRP-labeled UNA3H10 Fab 'was prepared.

Next, ELI on which Ap ₄ A-BSA prepared in Example 1 was immobilized
Ap ₄ A concentration known (0,10 ^-7 , 3 × 10 ^-7 , 10 ^-6 , 3
50 μl of each standard solution (× 10 ⁻⁶ , 10 ⁻⁵ mol /) and a serum sample of unknown Ap ₄ A concentration were injected, and 50 μl of 1 μg / ml HRP-labeled UNA3H10 Fab ′ was added thereto. After reacting at the temperature for 1 hour, wash well and use HRP color reagent 50
μl was added and allowed to react for 20 minutes was measured A ₄₁₅ to stop the reaction by adding 10% SDS50μl.

The measurement results are shown in Table 2. FIG. 2 shows a calibration curve obtained from A415 of each standard solution having a known Ap ₄ A concentration.

From Table 2 and FIG. 2, the concentration of Ap ₄ A contained in the serum sample used as the specimen was 5.6 × 10 ⁻⁷ mol /.

Comparative Example (Measurement of Ap ₄ A by Conventional Method) Ap of Serum Sample Used as Sample in Example 5
The ₄ A concentration was measured using the HPLC method.

That is, each standard solution and serum sample having a known Ap ₄ A concentration as in Example 4 were subjected to a reverse-phase _C18 column (Waters).
Then, 500 μl of each was injected using a phosphate buffer solution (pH 7.0) containing 0.1% tetra-n-butylammonium as a solvent, and
Absorbance at 0nm the (A ₂₆₀₎ was measured. The concentration of Ap ₄ A was represented by the peak area of A ₂₆₀ . The results are as shown in Table 3 and FIG.
_{The 4} A concentration is 5.6 × 10 ⁻⁷ mol /.

Thus, for the same serum samples, and Ap ₄ A concentrations measured by HPLC method is a conventional method, Ap ₄ A concentration by enzyme immunoassay using monoclonal antibodies of this invention shown in Example 5 Thus, it can be said that the Ap ₄ A measurement method of the present invention is a sufficiently reliable measurement method.

Of course, the present invention is not limited by the above examples,
It goes without saying that various aspects are possible.

As described (Effect INVENTION) above in detail, the present invention by enabling the Ap ₄ A immunoassay of using monoclonal antibodies, the Ap ₄ A in a biological fluid easily and quickly, yet quantified with high precision can do.

Moreover, the establishment of a hybridoma cell line that specifically produces the monoclonal antibody against Ap ₄ A makes it possible to easily and easily obtain a large amount of Ap ₄ A monoclonal antibody useful for diagnosis and measurement.

[Brief description of the drawings]

FIG. 1 shows the degree of binding between monoclonal antibody UNA3H10, Ap ₄ A and its competitor as a correlation diagram between the concentration of each sample and the absorbance. FIG. 2 is a correlation diagram of Ap ₄ A concentration and absorbance showing an example of a calibration curve of Ap ₄ A used in the immunoassay of the present invention. FIG. 3 is a correlation diagram showing an example of a calibration curve of Ap ₄ A by a conventional HPLC method.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12P 21/08 C12R 1:91) (72) Inventor Hiroshi Nakajima 23 Uji Kozakura, Uji City, Kyoto Prefecture Unitika Research Center (56) ) References JP-A-61-93199 (JP, A) Am. J. Physiol. Vol. 254, (1988), PH828-832 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 21/00-21/08 C12N 5/00-5/28 G01N 33/53 G01N 33 / 577 C12N 15/00-15/90 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A fusion cell of lymphocytes prepared from a mammal immunized with a diadenosine tetraphosphate antigen and myeloma cells, wherein the hybridoma cell line UNH3H10 (FERM P -11600). 2. The hybridoma cell line according to claim 1,
Diadenosine tetraphosphate specific recognition monoclonal antibody produced by UNH3H10 (FERM P-11600). 3. An immunoassay for diadenosine tetraphosphate using the monoclonal antibody according to (2).