JPH0276577A - Monoclonal antibody-producting cell line and monoclonal antibody - Google Patents

Abstract

PURPOSE:To produce a specific monoclonal antibody comprising an immunoglobulin by fusing mouse spleen cells sensitized with a specified antigen with a cell line originated from myeloma and subsequently cloning the fused cells. CONSTITUTION:A cell line originated from myeloma is fused with mouse spleen cells sensitized with an antigen consisting of a combined substance between trinitrobenzene or a derivative thereof and a protein and the fused cells are subsequently cloned to give an immunoglobulin in the supernatant of the cultured solution. The immuniglobuolin-producing cells are monoclonal antibody-producing cells line specifically combined with the trinitrobenzene or the derivative thereof. The monoclonal antibody produced from the cell line and purified permits the measurement of the trinitrobenzene or the derivative thereof utilizing a fluorescence-quenching phenomenon.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel monoclonal antibody-producing cell line and a monoclonal antibody produced by the cell line.

The monoclonal antibodies obtained by the present invention are useful for specifically, highly sensitive, and rapid measurement of minute amounts of trinitrobenzene or trinitrobenzene derivatives by immunoassay. Here, the term "trinitrobenzene derivative" refers to a substance having the following structure.

However, X is -CH3, S03H1COOH.

S　HlN　H2, represents a functional group containing -0H.

Prior Art Antibodies against trinitrobenzene or derivatives of trinitrobenzene have been reported by John Vanland et al. (For example, John Wannlund and Marle, Analytical Biochemistry
ne Deluca, Analytical Bioc
hemistry), 122.385-393 (+98
2)). However, the reported antibodies are polyclonal antibodies obtained by purifying the blood of immunized goats.

We have already created a monoclonal antibody that specifically binds to l-linitrobenzene or a derivative of trinitrobenzene, and using this monoclonal antibody to specifically bind to trinitrobenzene or a derivative of trinitrobenzene with high sensitivity, we performed an in-phase ELISA. was successfully detected. Solid phase method E1, IsA is one of the most widely used immunoassay methods at present, and either antigen or antibody is placed on a solid phase such as polystyrene beads or vinyl chloride 96-well plate 1. It is characterized by being used in a fixed manner. Below, El and IS with antigen immobilized in the same phase
A shows an example of trinitrotoluene, which is a derivative of trinitrobenzene. In the following text, phosphate buffer Zarin (Pho
Spate-Buffered Saline) is abbreviated as PBS.

(A) Antigen coating Bovine serum albumin (BSA) Conjugate (TN) with 0.5 molecules of trinitrobenzene bound per molecule
pH 6-BSA) was diluted with PBS containing 0.04% sodium azide (PBS-Az) and BsACl
i! Antigen solutions of 0 and 1 mg/mL were prepared at different concentrations.

Microplate (96-well plate made of vinyl chloride,
Manufactured by Dynatic Laboratories, DYNATECH
100% antigen solution in LABORATORIES)
μI/well was injected and stored at 20° C. overnight. After removing the antigen solution with an aspirator, PBS containing 0.05″A surfactant (polyoxyethylene glycol sorbitan argyl esters) and 0.04% thorium azide (PBS-AZ-T2O) Te3 It was washed twice and the remaining PBS.AZ-720 was removed with a 7-spirator.

(B) PBS-Az containing blocking I% BSA (BSA-PBS-A
z) was injected at 250 μL/well and left for 1 hour on a home appliance. Thereafter, BSA-PBS-Az was removed using an aspirator. If subsequent experiments were not to be performed on the same day, the sample was stored in this state at 4°C along with a filter paper moistened with water.

(C) Antibody reaction Inject 50 μL/well of TNT solution, and add 50 μL of purified antibody appropriately diluted with BSA-PBS-Az while shaking.
/added more wells. After storing at room temperature for 3 hours, remove the antibody solution with an aspirator and add PBA. AZ-T2O
Wash three times with PBS. AZ-
T2O was removed.

(D) Reaction of second antibody 0, 2 μg/mL goat-derived peroxidase labeled jl anti-mouse IgG antibody (KPL Cat.14180G l
ot. HLIO-5)! % BSA dissolved in PBS solution (second antibody solution), inject 50 μL/well,
It was left at room temperature for 30 minutes. Remove the second antibody solution with an aspirator and use
The plate was washed three times with BS (PBs-T2O), and the remaining PBS-720 was removed using an aspirator.

(E) Substrate reaction and termination 20 mg of 0-phenylenediamine (for selenium detection) was dissolved in 1 On+L citric acid-phosphate buffer (pH 5), and 4 μL of 30% hydrogen peroxide was added immediately before use. Solution) was injected at 100 μl/well and left at room temperature. After 10 minutes, 25 μL/well of 4N sulfuric acid was injected to stop the reaction.

(F) Measurement Absorbance above 92 nm was measured using Tltertek Multfskan MC. Normally, pure water was injected into the first column to serve as a reference value, and a blank value in which only the term (C) was omitted was used at appropriate times.

However, in the solid-phase ELISA described above, since the antigen or antibody is immobilized on a solid phase, it takes at least 2 to 2 hours for the antigen-antibody binding reaction to reach an equilibrium state.
It took 3 hours and a quick measurement completed within a few minutes was not possible.

On the other hand, when an antibody is excited with an excitation wavelength of 280 nm, it generally emits fluorescence with a fluorescence wavelength of 340 nm, but it is known that for some types of antigens, when the antigen and antibody bind, the intensity of the fluorescence emitted by the antibody decreases. This phenomenon is called fluorescence quenching, and is observed, for example, when dinitocyanin and antibodies against it bind (H, N, Eisen, G,
W. Siskind, Biochemistry, 3.9
B. (1964), H.N., Eisen, G.W.

5lsklnd, Blochemistry, 3,9
96 (19G4)). The amount of antigen added can be quantified by measuring the extent to which the fluorescence of the antibody is reduced by adding the antigen. In this case, a monoclonal antibody is used as the antibody, but since both the antigen and the monoclonal antibody are dissolved in a solution and are not fixed, the binding reaction between the two reaches an equilibrium state in a few seconds to several tens of seconds. In addition, separation of antigen-bound monoclonal antibodies and free monoclonal antibodies,
Since so-called B/F separation is also unnecessary, very rapid measurement is possible. If the fluorescence quenching phenomenon is applied to the detection of trinitrotoluene or trinitrotoluene derivatives,
It seems possible to measure specifically, highly sensitively and quickly.

Problems to be Solved by the Invention However, in order to apply the fluorescence quenching phenomenon to the measurement of trinitrobenzene or trinitrobenzene derivatives, it is necessary to first bind specifically to trinitrobenzene or trinitrobenzene derivatives, and as a result of the binding, fluorescence quenching occurs. It was necessary to create a monoclonal antibody that would exhibit this phenomenon.

Means for solving the problem is obtained by fusion and cloning of mouse spleen cells sensitized with an immunogen consisting of trinitrobenzene or a derivative of trinitrobenzene and a protein binding substance and a myeloma-derived cell line, Immunoglobulin produced in the culture supernatant specifically binds to trinitrobenzene or trinitrobenzene derivatives, and the intensity of fluorescence specific to the monoclonal antibody decreases as it binds to trinitrobenzene or trinitrobenzene derivatives. This constitutes a monoclonal antibody-producing cell line characterized by:

Function: By establishing the above cell line, purifying the monoclonal antibody produced therefrom, and using this monoclonal antibody, it becomes possible to measure trinitrobenzene or trinitrobenzene derivatives using the fluorescence quenching phenomenon. Furthermore, monoclonal antibodies with certain characteristics can be provided as long as hybridoma cell lines are cultured.

Example Cells that produce immunoglobulins accumulate within the spleen.

Spleen cells themselves do not have the ability to proliferate, but by fusing with myeloma cell lines, it is possible to create hybridoma cell lines that proliferate and produce antibodies. Select (clon) one hybridoma cell that has the desired properties (for example, the produced antibody exhibits a fluorescence quenching phenomenon upon binding with an antigen) and exhibits high proliferation ability, and culture it to achieve the desired purpose. A monoclonal antibody with the properties is produced.

In the present invention, the antigen is trinitrobenzene or a derivative of trinitrobenzene.

Here, the trinitrobenzene derivative refers to a substance having the following structure, for example.

However, X is -CH3, 5OsH1-COOH1SHl
Indicates a functional group including NH2 and OH.

Since these substances are all low molecules, they act as immunogens only by binding to macromolecules such as proteins. Chicken-derived gamma globulin is a protein suitable for use for the above purpose because it induces an immune response well.

Furthermore, Balb/C strain or A/J strain mice exhibit good immune responses and are therefore suitable for use in the present invention.

Based on these findings, the inventors fused spleen cells from immunized mice with a mouse myeloma-derived cell line, which had high affinity for trinitrobenzene or trinitrobenzene derivatives, and With the combination with the derivative,
We were able to create a cell line that produces a monoclonal antibody whose fluorescence intensity at 340 nm decreases when excited at an excitation wavelength of 280 nm.

Example 1 Hereinafter, the present invention will be described in detail with reference to Examples.

In carrying out the present invention, fusion cells and monoclonal antibodies were evaluated using ELISA in which antigens were immobilized.

First, the experimental procedure of ELISA will be explained. In addition, in the following text, phosphate buffer solution (Phosphate-B)
5aline) is abbreviated as PBS.

(A) Antigen coating Bovine serum albumin (BSA) Funshugate (TN) with 0.5 molecules of trinitrobenzene bound per molecule of bovine serum albumin (BSA)
Antigen solutions with BSA concentrations of 0 and 1 mg/mL were prepared by diluting the antigen solution (pH, 6-BSA) with PBS (PBS-AZ) containing 0.04% sodium azide.

Microplate (96-well plate made of vinyl chloride,
Manufactured by Dynatic Laboratories, DYNATECH
Add 100μ of antigen solution to LABORATORIES).
L/well was injected and stored overnight at 20°C. After removing the antigen solution with an aspirator, wash three times with PBS containing 0.05% surfactant (polyoxyethylene glycol sorbitan alkyl esters) and 0.04% sodium azide (PBS-AZ-T2O). Then, the remaining PBS and AZ-T2O were removed using an aspirator.

(B) PBS-Az containing blocking I% BSA (BSA-PBS-A
z) was injected at 250 μL/well and left at room temperature for 1 hour. Thereafter, BSA-PBS-Az was removed using an aspirator. If subsequent experiments were not to be performed on the same day, the samples were stored in this state at 4°C along with filter paper moistened with water.

(C) Antibody reaction 100 μL/well of antibodies (serum, culture medium, purified antibody, etc.) appropriately diluted with BSA-PBS-Az was injected. After being stored at room temperature for 3 hours, the antibody solution was removed using an aspirator, and the plate was washed three times with PBA and AZ-T2O, and the remaining PBS and AZ-T2O were removed using an aspirator.

(D) Reaction of second antibody 0.2 μg/mL goat-derived peroxidase-labeled anti-mouse IgG antibody (KPL Cat, 1418Hlot,
10-5) dissolved in I% BSA PBS solution (second antibody solution) was injected at 50 µL/well and incubated at room temperature for 30 µL/well.
I left it for a minute. Remove the second antibody solution with an aspirator and o
, containing 05% surfactant (polyoxyethylene glycol sorbitan alkyl esters)
BS-T2O) three times, and the remaining PBS-T2O was removed using an aspirator.

(E) Substrate reaction and termination 0-phenylenediamine (for selenium detection) 20mg
A solution (substrate solution) dissolved in 0 mL of citric acid-phosphate buffer (pH 5) and added with 4 μL of 30% hydrogen peroxide immediately before use was injected at +00 μL/well and left at room temperature. After 10 minutes, 25 μL/well of 4N sulfuric acid was injected to stop the reaction.

(F) Measurement Absorbance of 492 ni or more was measured using Tltertek Multlskan MC. Normally, pure water was injected into the first column to serve as a reference value, and a blank value in which only the term (C) was omitted was used at appropriate times.

Below, an experimental method for producing the monoclonal antibody-producing cell line of the present invention will be described.

1) Preparation of immunogen According to the report of Jon Wannlund et al. (cited above),
The immunogen was synthesized. The subsequent procedure will be described step by step.

(A) Preparation of CGG solution CGG (Calbjochem17GF-936 (1
) 10 (1 mg, (1, IM NaHcOs
(pH 8,4) 2On+] and stirred for 30ml to completely dissolve.

(B) Preparation of TNBS solution 14 mg of sodium trinitrobenzenesulfonate dihydrate (hereinafter referred to as TNBS) was dissolved in 35 m
Dissolved in l.

(C) Synthesis of TNP-CGG Mix the TNBS solution and CGG solution and stir.
Incubation was performed at 7°C. After 2 hours, remove low molecules by gel chromatography and add 32.5 mL of TN.
A P-CGG solution was obtained.

Through the above operations, an immunogen to which 32 TNB molecules were bound per 0001 molecule was obtained.

(D) Preparation of adjuvant emulsion TNP-CGG obtained by synthesis was diluted with PBS to obtain 3 mL of a 1 mg/mL solution. Adjuvant
Take 3 mL of Complete Freund (containing killed human tuberculosis bacteria, Wako, H37RV)-15= while stirring well, and stir with a homogenizer (10,00 Orpm>, TNT-CGG).
The solution solution was added in 3 L portions. It was confirmed that it was sufficiently emulsified and did not spread even when a small amount was dropped on water.

2) Preparation of hybridoma cells (A) Immunization of mice 200 μL of the adjuvant emulsion was injected into the abdominal cavity of 20 mice (Balb/c) about 8 weeks old.

(B) Check for antibody production From mice 28 days after the immunization injection, 50 to 100 μL of blood was collected from the eye vein into a centrifuge tube.

When the serum was centrifuged and screened using ELISA, production of anti-trinitrobenzene antibodies was confirmed in all mice.

(C) Mouse boost (B) 2 with particularly high titer in screening
A boost (injection of a weak immunogen) was given to two mice to enlarge their spleens. T as an immunogen
0.5 mg/m obtained by diluting NP-CGG with PBs
The L solution was used as is without the addition of adjuvant. 6 weeks after immunization, this immunogen 10
0 μL was injected into the peritoneal cavity of the mouse.

(D) Spleen cells of mice 3 days after cell fusion boost were removed,
A mouse myeloma-derived cell line (P3XG
3-Ag8. G53). Spleen cells from the same mouse were used as feeders (cells that supply growth factors) and cultured in AT medium containing 10% fetal bovine serum on five 96-well plates. One week later, the medium was replaced with IIT medium containing 10% fetal bovine serum.

3) Preparation of monoclonal antibodies (A) Cloning Screening was performed by ELISA method, and the top 5 wells with the highest titers were selected. 1 per well
The mixture was diluted (limiting dilution) to a concentration containing 50 cells, and dispensed into 10 96-well microplates.

Initial proliferation was promoted using 4-week-old mouse (Balb/c) thymocytes as feeders. Proceed with the culture while increasing the plate size, and perform ELISA on the supernatant at appropriate times.
Screening was repeated, and cell lines showing high titer for TNT and good growth were selected and cultured in 200 ml until a concentration of 5×10 6 cells/ml was reached.

(B) Protein A-Sepha manufactured by Pharmacia
Monoclonal antibodies were purified from cell culture supernatants by affinity chromatography using rose CL-4B. This monoclonal antibody was compared with standard protein by SDS polyacrylamide gel electrophoresis.
The purified antibody has a heavy chain with a molecular weight of about 50,000 and a molecular weight of about 20.00.
It was confirmed that it was an IgG consisting of 0 L chains.

(C) The purified monoclonal antibody was diluted with PBS to prepare a 10-7M solution. This solution 380 μm and T
20 μm of a solution of NT dissolved in PBS was added, the mixed solution was excited at 280 nm, and the fluorescence intensity at 340 nm was measured. Concentration of added TNT solution: 10-6M, 10-7
M, 10-8M, and 10''M were selected.Finally, a cell line was selected that produced a monoclonal antibody with a particularly remarkable decrease in fluorescence intensity when TNT was added.

(D) The final sorted cell line was separated by centrifugation of the supernatant and treated with Fe2:DMSO at a concentration of 5X 10”/mL.
=9: It was suspended in ImL of a solution of I, frozen at -80°C, and then transferred to liquid nitrogen for long-term storage.

Effects of the Invention By purifying the monoclonal antibody produced from the monoclonal antibody producing cell line of the present invention and using this monoclonal antibody, it becomes possible to measure trinitrobenzene or trinitrobenzene derivatives using the fluorescence quenching phenomenon.

Claims (4)

[Claims] (1) Obtained by fusion and cloning of mouse spleen cells sensitized with an immunogen consisting of trinitrobenzene or a trinitrobenzene derivative and a binding substance to a protein, and a myeloma-derived cell line; The immunoglobulin produced by the monoclonal antibody specifically binds to trinitrobenzene or a derivative of trinitrobenzene, and the intensity of fluorescence specific to the monoclonal antibody decreases as it binds to trinitrobenzene or a derivative of trinitrobenzene. Monoclonal antibody-producing cell line. (2) The monoclonal antibody-producing cell line according to claim 1, wherein the mouse sensitized with the immunogen is of the Balb/c strain or the A/J strain. (3) The monoclonal antibody-producing cell line according to claim 1, wherein the immunogen consists of a binding substance of trinitrobenzene or a derivative of trinitrobenzene and chicken-derived gamma globulin. (4) The monoclonal antibody produced by the monoclonal antibody-producing cell line according to claim 1, which specifically binds to trinitrobenzene or a derivative of trinitrobenzene, and upon binding to trinitrobenzene or a derivative of trinitrobenzene, has a unique fluorescence of the antibody. A monoclonal antibody characterized by a decrease in the intensity of.