JPH0488997A - Monoclonal antibody, hybridoma cell strain producing the same antibody and labeling using the same cell strain - Google Patents

Abstract

NEW MATERIAL:A Bacillus stearothermophilus derived diaphorase specific reactivity monoclonal antibody obtained from a hybridoma made by fusion of lymphocyte prepared from an animal immunized against Bacillus stearothermophilus derived diaphorase and myeloma cell. USE:A reagent for immobilizing enzymes useful for enzyme immunity measurement and tissue dyeing by using Bacillus stearothermophilus derived diaphorase as a labeled enzyme. PREPARATION:For example, Balb/c mouse is immunized against Bacillus stearothermophilus derived diaphorase by administration, after additionally immunized, splenocyte is collected, fused with mouse myeloma cell and cultured in HAT medium to give a hybridoma. The hybridoma is screened, anti- diaphorase antibody-forming strain is selected and cloned by limiting dilution to give a monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a monoclonal antibody, a hybridoma cell line that produces the same, and a labeling method using the same. More specifically, the present invention provides Bacillus
Stearothermophilus (hereinafter abbreviated as B, S, bacteria)
The present invention relates to a monoclonal antibody against diaphorase derived from B, S, and bacteria, a hybridoma cell line that specifically produces the same, and a method for labeling diaphorase derived from B, S, and bacteria using this monoclonal antibody.

(Prior art) Conventionally, in order to create an enzyme-labeled antibody by binding an enzyme and an antibody, a trifunctional chemical reagent is used to bind the amino group of the enzyme and the antibody, such as glutaraldehyde, to the amino group of each of the enzyme and the antibody. Method of crosslinking (S, ^Vralie
aS, I11+1unOCelliStr111.6
．． 43-52, 1969), or the amino group of the enzyme and the thiol group produced by reducing the antibody.
A method of crosslinking using a chemical reagent (for example, N-succinimidyl maleimidocarboxylate) that has a group that reacts with an amino group and a group that reacts with a thiol group in the molecule (Noshi Ishikawa, Enzyme immunoassay, 3rd edition, Medical It is also known that the thiol group of an enzyme and the thiol group of an antibody are crosslinked using a trifunctional reagent (for example, ortho-phenylene dimaleimide) for the thiol group. .

Furthermore, in recent years, enzyme-labeled antibodies using bispecific antibodies have been created. For example, an anti-urease antibody and an anti-choriogonadotropin antibody are made into a bispecific antibody by cell fusion, and this anti-urease antibody is produced as a bispecific antibody. urease-labeled antibody (H, Takaha)
shi, Cl1n, Chen, 34°1693-169
8.1988) or a horseradish peroxidase (HRP)-labeled antibody using a similar bispecific antibody (
H,R,5uresh,Proc,Natl.

Acad, Sci, LISA, 837989-799
3.1986) etc. are known.

By the way, diaphorase is an enzyme that catalyzes the reduction reaction of pigments with nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate.Diaphorase causes a change in color as the pigment is reduced, so it is useful for example in immunohistometry and tissue analysis. It has the excellent feature of being extremely easy to detect when used as a label for staining.

Among them, diaphorase obtained from thermophilic bacteria B, S, and Bacteria has the advantage of excellent thermostability in addition to the above characteristics, and is expected to be widely used as a labeling enzyme. There is.

(Problems to be Solved by the Invention) However, when this B, S, and fungus-derived diaphorase is chemically combined with an antibody against a given substance to be measured to create an enzyme-labeled antibody, its enzymatic activity is approximately 30% The problem is that sufficient sensitivity cannot be obtained. Therefore, despite their excellent characteristics, B, S, and bacterial-derived diaphorases have not been fully utilized in enzyme immunoassays, tissue staining, and the like.

This invention was made in view of the above circumstances, and when an enzyme-labeled bispecific antibody is prepared using B, S, and fungus-derived diaphorase as a labeling enzyme, the enzyme activity is retained at least 80%. The purpose of the present invention is to provide monoclonal antibodies against diaphorases derived from B, S, and bacteria. Manako's invention provides a hybridoma cell line capable of producing the above-mentioned monoclonal antibody and B,
The purpose of the present invention is to provide a method for labeling diaphorase derived from bacteria.

(Means for Solving the Problems) This invention solves the above problems by fusion of lymphocytes prepared from an animal immunized with diaphorase derived from Bacillus stearothermophilus and myeloma cells. The present invention provides a monoclonal antibody reactive with diaphorase derived from Bacillus stearothermophilus, which is produced from a hybridoma.

The present invention also provides a hybridoma cell line that specifically produces this monoclonal antibody, and a labeling method characterized by chemically crosslinking this monoclonal antibody and an antibody against a measuring substance.

This invention will be explained in detail below.

As mentioned above, the monoclonal antibodies of this invention include B, S
, hybridomas prepared by fusion of lymphocytes prepared from animals immunized with fungal diaphorase and myeloma cells can be selected and cultured, but from the point of view of production efficiency, the above hybridomas are Among them B and S.

Cells that specifically produce antibodies against fungal diaphorase are selected, cloned into cell lines, and B and S are obtained by culturing this cell line.

It is preferable to obtain monoclonal antibodies against fungal diaphorase.

The preparation of such a cell line, the method for collecting a monoclonal antibody, and the method for labeling B, S, and fungus-derived diaphorase using the monoclonal antibody can be performed as follows.

(1) Creation of a hybridoma cell line that specifically produces monoclonal antibodies against B, S, and fungus-derived diaphorase: First, in order to create a hybridoma, B, S, and B.

lymphocytes from animals immunized with fungal diaphorase antigen,
Myeloma (bone marrow lll11) to be fused with this lymphocyte
1I cells) are prepared.

Among these, commercially available B and S are used to obtain lymphocytes.

A mammal, preferably a mouse or rat, is immunized with fungal diaphorase (manufactured by Seikagaku Corporation) as an antigen.

Immunization methods such as the amount of antigen to be used, the site of administration, and the use of adjuvants may be in accordance with conventional methods for obtaining antiserum. For example, when using mice, 0.001 to 101 g per mouse, preferably 0.01 to 11 g
The B, S, and fungus-derived diaphorases are first mixed well with an adjuvant (e.g., Freund's complete adjuvant) and administered subcutaneously, intraperitoneally, etc., and after 2 weeks or more, are administered again with an adjuvant (e.g., Freund's complete adjuvant). Complete adjuvant) and mix well and administer subcutaneously, intraperitoneally, etc. Furthermore, after two weeks or more have elapsed, only B, S, and bacterial diaphorase are administered intravenously, subcutaneously, intraperitoneally, etc. to sufficiently immunize the animal. Animals thus immunized are preferably sacrificed 2 to 4 days after the final immunization, and lymphocytes are harvested. For lymphocyte preparation, lungs, lymph nodes, peripheral blood, etc. are used.

The lymphocytes are suspended in a culture medium.

On the other hand, it is preferable to use myeloma derived from the same species as the immunized animal. Furthermore, the myeloma is preferably a drug-resistant mutant strain, and unfused myeloma is preferably one that does not grow in a hybridoma selection medium. Most commonly, 8-azaguanine resistant cell lines are used. This is hypoxanthine-guanine-phosphoribosyltransferase (Hypoxanthin-guanine-phosphoribosyltransferase).
t inguanine phosphoribosy
L transferase) is deficient, and a type of selective medium, hypoxanthine-aminobuterine-thymidine (
HAT) cannot grow on medium. In addition, since it is desirable that the myeloma used does not secrete antibodies itself, for example, commercially available mouse myeloma P3/X63/A
g8.6.5.3 (X63.6.5.3), P3.X6
3・Ag8・Ul (P3U1), rat myeloma 2
10 ・RCY3・Agl・2・3 etc. are preferably used.

The myeloma is cultured in a medium such as Eagle's minimal medium (MEM) RPM I 1610 medium (RPM I 1640) containing serum, preferably fetal bovine serum.

Next, the lymphocytes and myeloma obtained above are each suspended in a medium such as MEM or RPM I 164G and mixed. The mixing ratio at this time can be selected arbitrarily, but preferably lymphocytes: myeloma cells: 1 = 1 to 20:
1, preferably at a ratio of 5:1 to 1o:1.The mixed cells are fused using a fusion promoter.As a fusion method, for example, Immunological Methods Vol. 2, p. Method
ds Vol, II, 1981゜＾cadenic
As the fusion promoter, various polymeric substances, viruses, etc. can be used, but polyethylene glycol (PEG) is preferable.
, Sendai virus may be used. PEG with an average molecular weight of 400 to 20,000 can be used, but preferably one with an average molecular weight of i,ooo to 7,500 can be used.

The concentration used is 40 to 60 vol. % is preferred.

The fused cells are washed to remove the fusion promoter, and then
15vo1. MEM or RP MI containing % serum
Suspend in 164G medium and add 0.5 to 5 to 96-well cultured blood, etc.
Dispense at a ratio of x10'/hole. Furthermore, if a selective medium (eg, HAT medium) is added to each person and the selective medium is replaced as appropriate, unfused myelomas will die after 10 to 14 days, and only hybridomas will grow. Incidentally, lymphocytes cannot grow in vitro for a long time and die after 10 to 14 days.

Next, hybridomas that specifically produce monoclonal antibodies against B, S, and bacterial diaphorases are searched for and selected. As a method for this purpose, ELISA method can be used. That is, for example, commercially available F,LISA
B, S, and bacterial diaphorase are adsorbed onto a solid phase such as a commercial plate, and hybridoma supernatant is added to this, and after washing,
A commercially available labeled antibody (eg, HRP-labeled antibody or 125I-labeled antibody) against immunoglobulin of an immunized animal is added. As a result, B and S were present in the hybridoma supernatant.

If antibodies against fungal diaphorase are present,
This binds to B, S, and fungal diaphorase on the solid phase, and further binds to this labeled antibody against immunoglobulin, resulting in a signal from the label. On the other hand, B in the supernatant,
If there is no antibody against S, bacterial diaphorase, nothing will bind to the solid phase B, S, bacterial diaphorase, and therefore no signal will be obtained.In this way, we can get a clue as to whether or not there is a signal due to the label. As a result, hybridoma selection can be performed.

Next, a hybridoma cell line can be created by selectively culturing the thus obtained hybridomas that specifically produce monoclonal antibodies against B, S, and fungus-derived diaphorase.

In addition, according to this method, III! One type of hybridoma cell line created by fusing m-lymphocytes and mouse myeloma cells was named hybridoma UDIH2F6. This strain was deposited with the Ministry of International Trade and Industry's Microbial Research Institute on July 11, 1990.
It was accepted as No. (FERN P-11601). This UDIH2F6 can be stored frozen almost permanently at -120°C or lower, and is always ready for distribution.

This hybridoma UDIH2F6 can be grown in commonly used media. For example, 5-20% fetal bovine serum
Using RPM11640 or MEM containing RPM11640 or MEM as a medium, the temperature was 37°C and the carbon dioxide concentration was 5vol. It grows well in air containing %. It also has myeloma tumorigenic properties, so it can proliferate in vivo (for example, in syngeneic animals, nude mice, etc.) and produce monoclonal antibodies against B, S, and fungus-derived diaphorases.

(2) Collection of large amounts of monoclonal antibodies against B, S, and fungus-derived diaphorases: By culturing the hybridoma cell line created as described above, it is possible to collect large amounts of monoclonal antibodies against B, S, and fungus-derived diaphorases. . There are two main methods for collecting monoclonal antibodies. One method is to culture in a culture container such as a flask using a medium, and collect the antibody from the supernatant. For example, 5 to 10 vol. Planting 0.5-5X10' hybridoma cell lines in MBM or RPM11640 medium containing 10% serum produces 10-20 cells in 2-4 days.
This is a method in which antibodies are collected from the supernatant after culturing. Another method is to inoculate the hybridoma cell line thus cultured in a culture vessel into a syngeneic animal. That is, hybridoma cell line 10
In this method, 5 to 10 cells are administered subcutaneously or intraperitoneally to a syngeneic animal, and after 7 to 20 days, when the hybridoma cell line proliferates and the tumor becomes large, serum and ascites are collected. When administering intraperitoneally, a larger amount of ascites can be obtained by administering mineral oil such as 2.6,10.14-tetramethylpentadecane in advance (3 to 7 days).

The antibody thus obtained can be purified and used if necessary. For example, purification can be carried out using means commonly applicable to proteins, such as ammonium sulfate fractionation, ion exchange chromatography, and affinity taromadography with protein A immobilized.

In this way, monoclonal antibodies against B, S, and bacterial diaphorases can be easily obtained in large amounts.

(3) Preparation of enzyme-labeled bispecific antibodies using monoclonal antibodies: First, antibodies against B, S, and bacterial diaphorase and antibodies against the analyte are mixed with a chemical reagent used for normal protein crosslinking (for example, N-succinimidyl).・Maleimide carboxylate, ortho-phenylene dimaleimide, etc.) is used to create a bispecific antibody, and this bispecific antibody is further treated with 1 to 100 times the amount of B, S, or bacterial diaphorase. Preferably 2 to 10 culture volumes, 1
) H4-10, preferably mixed in a buffer solution in the pH range of 6 to 8, using methods commonly used for protein separation (e.g.
(gel filtration, ion exchange chromatography, etc.) can produce B, S, and bacterial-derived diaphorase-labeled antibodies.

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

Example 1 (Creation of hybridoma) Hybridomas producing monoclonal antibodies against B, S and fungal diaphorase were created.

B, S, and fungus-derived diaphorase (obtained from Seikagaku Corporation) were mixed and emulsified in a ratio of 1:1 with complete Freund's adjuvant (obtained from Semi-Kagaku Kagaku) to 8-week-old mouse Ba1b/c (obtained from Oriental Bioservices). , administered intraperitoneally, and 3 weeks later, 50 μg of B, S, and bacterial diaphorase were intravenously injected for booster immunization. Three days later, the lungs were removed and MBM
It was loosened and suspended in a medium (obtained from Semi-Kagaku) and washed. On the other hand, mouse myeloma X63.6.5.3 (obtained from Kyoto University) was cultured for 2 days, and cells in the logarithmic growth phase were collected by centrifugation. Myeloma X6 with 108 spider cells
After mixing with 3,6,5,3,107 and pelleting by centrifugation, 50% PEG4000-R was added in a water bath at 37°C.
Gradually add 1 ml of PM11640 (obtained from Gibco) over 1 minute, and after stirring gently for 1 minute, add 9 m
1 of RPM11640 medium was gradually added to P E 0
Diluted 4000. Remove the PEG solution by centrifugation, and add HAT medium 20 containing 10% tallow serum to a pellet.
ml was added and 0.1 ml was dispensed into two 96-well culture dishes (obtained from Corning). 4°8.11
100 times, discard half of the culture medium and add new H.
AT medium was added.

As a result, after 14 days, hybridoma growth was observed in 65 wells of 192 large cells.

Example 2 (Creation of hybridoma cell line) A hybridoma cell line that specifically produces monoclonal antibodies against B, S, and fungus-derived diaphorases was created.

First, among the hybridomas obtained in Example 1, B, S,
A cell line that produces antibodies against bacterial diaphorase was
The search was performed using the LISA method.

First, 50n was added to an ELISA plate (Corning).
B dissolved in N sodium carbonate shock solution, EIH9,6,
S', diaphorase derived from fungus was dispensed in 50 μj for each formula, and left at a temperature of 25° C. for 2 hours. After removing this solution, a large amount of 201M sodium phosphate buffer (pH 7, 2) containing 1.0% BSA, 0, 15M sodium chloride
) and left at a temperature of 25° C. for 1 hour. Furthermore, this liquid was removed and 0.2% Tween 20.0.2% B
Each well was thoroughly washed with a washing solution consisting of SA, 201N sodium phosphate buffer (pH 7.2) containing 0.15 M sodium chloride, and B, S, and fungus-derived diaphorase-immobilized plates were prepared.

Next, 50 μm of the supernatant of the hybridoma obtained in Example 1 was added to this plate, and after being left at a temperature of 25°C for 2 hours, it was thoroughly washed with a washing solution, and HRPi! 50 μm of 1 μz/1 ml of goat anti-mouse IgG (purchased from Funakoshi) was added to each formula and left at a temperature of 25° C. for 2 hours. moreover,
After washing, add 50μ of peroxidase measurement reagent (manufactured by Bio-Rat) and react at 25°C for 20 minutes.
Veroxidase activity was stopped by the addition of 50 μm of 10% 5DS, and the absorbance at 415 ni (A415
) was measured. As a control, only HAT medium was used instead of hybridoma supernatant.

As a result, a supernatant was obtained that showed a higher signal than the signal when no supernatant was added, which was used as a control. The strain in the well of this supernatant was selected and cloned using the limiting dilution method, resulting in the monoclonal hybridoma cell line UDIH2F6.
I got it.

Example 3 (Collection of monoclonal antibodies against B, S, and fungus-derived diaphorases) The hybridoma cell line UDIH2F6 obtained in Example 2 was cultured, and monoclonal antibodies against B, S, and fungus-derived diaphorases were collected.

First, UDIH2F6 was added to RP M containing 10% tallow serum.
I 1640 medium, and when the cell concentration reached 2×10′ cells/ml, 300 ml of the culture was centrifuged, and the crude antibody fraction was separated from the supernatant by 50% saturated ammonium sulfate fractionation and dialyzed. Afterwards, it was adsorbed onto protein A-Sepharose (DH9, 0), and citrate buffer (pH 3,
0> to obtain B, S, and an antibody against bacterial diaphorase (hereinafter referred to as UDIA2F6) 6,4■.

Next, this UDIA2F6 was added to a reagent system for measuring diaphorase activity, and its degree of inhibition against B, S, and bacterial diaphorase was measured. i.e. 500 nH) reethanolamine-HCl (pH, 7,0) 60 μj, 5
100 μm of UDIA2F6 was added to an activity measuring reagent system consisting of IIMNADH (60 μj), 50 mH ethylenediaminetetraacetic acid (60 μm), 5% cetyltrimethylammonium bromide (60 μm), and H2O (200 μm), and this was incubated at a temperature of 30° C. for 3 minutes.
Add 60μm of H nitrofluoritetetrazolium,
React for 10 minutes at a temperature of 550°C, stop the reaction by adding 50μm of 1N HCl,
The absorbance (Asso) at na was measured.

Ass obtained in this way. (Ass.

UDIA2F6) and A s s when the same amount of buffer was added instead of tJDIA2F6 as a control.

The degree of inhibition was determined by dividing by (A sso cont).

As a result, UDI for B, S, fungal diaphorase
The degree of inhibition of A2F6 was 0.8.

Example 4 (Preparation of diaphorase-labeled antibody) First, 4.0 μg of UDIA2F6 was dialyzed against 100 mM citrate buffer (pH, 3,5), 25 μg of pepsin was added, and the mixture was incubated at a temperature of 37° C. for 6 hours. Next, this was subjected to Utrogel AcA44 column chromatography to separate UDIA2F6F (ab')2, which was further reduced to obtain UDIA2F6Pab'.

Further, the same operation as above was performed on anti-carcinoembryonic antigen (CEA) (obtained from Medix Biogemica) to obtain CEAFab'.

Next, UDIA2F6Fab' 2. Add 100 μm of orthophenylene dimaleimide to Oq and incubate at 30'C for 10 minutes to introduce a maleimide group into the thiol group of Fab', and add anti-CEA antibody 2.0■ to this UDIA2F67 reimidized Fab'. , bispecific antibodies against B, S, fungal diaphorase and carcinoembryonic antigen were obtained using a Kurtrogel AcA44 column.

Then, diaphorase is added to this bispecific antibody,
A diaphorase-labeled antibody was created.

Example 5 (Enzyme immunoassay using B, S, and fungus-derived diaphorase-labeled antibodies) Sandwich enzyme immunoassay was performed using the B, S, and fungus-derived diaphorase-labeled anti-CEA antibodies prepared in Example 4.

First, anti-CEA antibody immobilized E was prepared in the same manner as in Example 2.
A LISA plate was prepared, and 50 μm of CEA at concentrations of 0.5.10 and 20 μf/■ and 50 μm of the above-mentioned B, S, and bacterial-derived diaphorase-labeled anti-CEA antibodies were added.

As a comparative example, instead of the above antibody, anti-CEA antibody Fab' and diaphorase were directly cross-linked with N-succinimidyl-maleimidocarboxylate (concentration 1).
50 μJ of tt t/mI) was added.

Next, this ELISA plate was incubated at a temperature of 37° C. for 3 hours to perform an antigen-antibody reaction. That is, after removing the liquid phase of the grate and thoroughly washing the plate with a washing solution similar to that described in Example 2, 50i1 reethanolamine-HCI, 5iHNADH, 0.5% cetyltrimethylammonium bromide, and 511M ethylenediamine were added. A diaphorase measurement solution consisting of tetraacetic acid was added and reacted for 30 minutes, then 150 μm of INHe was added to stop the reaction, and each A s so was measured.

The results are as shown in Table 1 and FIG.

As is clear from Table 1 and FIG. 1, the enzyme immunoassay using the enzyme-labeled bispecific antibody of the present invention is different from the conventional enzyme immunoassay shown as a comparative example. ! It was possible to detect and quantify CBA with much higher accuracy than methods using specific antibodies.

Of course, the present invention is not limited to the above examples, and it goes without saying that various embodiments are possible with respect to the detailed procedures and operations, the scope of application, etc.

(Effects of the Invention) As explained in detail above, the monoclonal antibody of the present invention can be used to label B, S, and bacterial diaphorases as widely useful labeling enzymes for enzyme immunoassays, tissue staining, etc., without substantially impairing their activities. It becomes possible to use it. Moreover, by establishing a hybridoma cell line that specifically produces this monoclonal antibody, it becomes possible to easily obtain a large amount of monoclonal antibodies against B, S, and fungus-derived diaphorases.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the correlation between the amount of CBA and the absorbance when the amount of CBA in a sample is measured by enzyme immunoassay. Diagram

Claims (3)

[Claims] (1) Bacillus stearothermophilus produced from a hybridoma obtained by fusing myeloma cells with lymphocytes prepared from an animal immunized with diaphorase derived from Bacillus stearothermophilus A monoclonal antibody with specific reactivity to diaphorase. (2) The monoclonal antibody-producing hybridoma cell line according to claim (1). (3) A labeling method in which the monoclonal antibody according to claim (1) and an antibody against a measurement substance are chemically crosslinked.