JPS63253099A - Monoclonal antibody composed of neutralized human tumor necrosis factor - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody specific to active site of human tumor necrosis factor (hTNF), unable to form a bond with (a) an hTNF obtained by neutralizing cytotoxicity of hTNF against tumor cell and lost its physiological activity, (b) a tumor necrosis factor of animal other than human and (c) lymphotoxin by immunity reaction, belonging to IgG1 subclass and having L- chain type of isotype kappa and isoelectric point of 6.2+ or -0.1. USE:A reagent for quantitative determination of hTNF having activity. PREPARATION:For example, an E.coli transformed with a plasmid containing an hTNF gene is cultured and the produced hTNF is administered to a Balb/c mouse. A spleen cell collected from the immunized mouse is fused with a myeloma cell and the fused cell is screened and cloned to obtain a monoclonal hybridoma. The objective monoclonal antibody is prepared by culturing the hybridoma.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to h) [flW necrosis factor (hereinafter referred to as h-TNF)]
A novel neutralizing monoclonal antibody against (abbreviated as). More specifically, by specifically binding to h-TNF,
The present invention relates to a monoclonal antibody that has neutralizing activity against the cytotoxicity of h-TNF and does not have the ability to bind to human-derived lymphotoxin, and uses thereof.

(Prior art) It is attracting attention as a protein with anti-111m activity [
1! Tumor necrosis factor (TNF) was discovered in 1975 by Carswell et al. as a substance with tumor killing or cancer necrosis activity in the serum of animals sensitized with an immunostimulant (Proc, Natl, Acad, Sc i,
USA, 72: 3666-3670.1975),
After that, TNF has no significant effect on the host (
Various I! necrosis of the tumor and also in vitro
In ro, various transformed cells (M tumorized cells)
Although it kills or stops the growth of cells, it is known to have no effect on normal cells, and is expected to be an anti-tumor or anti-cancer agent. It has been reported that TNF is produced from activated macrophages in vivo (y7 (Ru
t r), HoR, and Gif f
ord), G, E,, Lymphokines (Lymph
okines), Vol. 2, 235-272. (Pick, E. &, Academic Press, New York, 1981); in recent years, substances having TNF activity have been isolated from culture fluids of established macrophage-like cells.

Recently, two groups have used transgenic DNA techniques to reveal the amino acid sequence of the TNF protein (produced by activated human macrophage-like cells) [Pennica D.

et al., Nature, 312 Near 24-729.1984
and Wang, AoM, et al.

Sc 1ence, 228: 149-154.19
[85], both groups isolated human TNF mRNA from activated human macrophage-like cells HL-60, cloned the cDNA, and determined the base sequence, while also purifying TNF from the culture fluid of the cells. By determining the amino acid at the terminal end of the amino group, mature TNF can be expressed as V a l -A r g as shown in Figure 1.
-3e r... and the carboxyl group end is Leu
It is a polypeptide consisting of 157 amino acids ending with , and its precursor is a protein in which a polypeptide consisting of 76 amino acids is added to the amino terminus of the above polypeptide. In addition, they have the above-mentioned human TNF
Polypeptides have also been successfully produced in transformed E. coli.

By the way, TNF has already been used on cancer patients and clinical studies have begun. Under these circumstances, there is a need for a method to quickly and easily detect TNF in a sample, especially TNF in the active state. For example, when administering TNF to a patient, It is necessary to monitor the subsequent blood TNF concentration and manage it to obtain the maximum administration effect. However, TNF measurement has conventionally been carried out using a bioassay using cytotoxicity to L929 cells as an indicator, which requires time and effort, requires skill, and has the problem of not being able to process a large amount of specimen.

Instead of bioassay, it is also possible to measure TNF immunochemically, but for that purpose, active T
It is necessary to use antibodies that specifically react with NF only. Otherwise, denatured and inactivated TNF will be measured, and there is a risk that the measured value will not match the actual TNF activity. However, human TNF (h
- TNF) For monoclonal antibodies, activated h
- Monoclonal antibodies that are specifically reactive with only TNF have not been obtained.In recent years, antibody-producing cells from mice immunized with purified human TNF have been used to generate myeloma cells.
A monoclonal antibody against human TNF has been produced using hybrid cells (hybridoma) with h-TNF (Japanese Patent Application Laid-Open No. 60-208924), but this antibody reacts with h-TNF and also with human-derived lymphotoxin. Not satisfactory in reaction specificity, i.e. h-
A monoclonal antibody that specifically reacts only with TNF (especially active h-TNF) has not yet been obtained.

On the other hand, by fusion of mouse myeloma cells with mouse lung cells immunized against a specific antigen, they can proliferate and grow in vitro.
A method for producing hybrid cells (hybridomas) that can replicate and produce monoclonal antibodies is described by Köhler and Milstein [Nature, 25
6.495-497, 1975). Hybridomas have the property of secreting homogeneous antibodies (monoclonal antibodies) that are specific only to a particular antigenic determinant, so hybridomas that produce a variety of monoclonal antibodies have been created, and they have been produced to target various viral antigens. It is used as a useful means for detecting and purifying not only bacterial antigens, but also cell surface differentiation antigens, cancer antigens, and trace antigen components in living organisms. Furthermore, monoclonal antibodies against these trace antigen components are also effectively used for diagnosis and testing of various diseases.

A general method for obtaining hybridomas is to first immunize a mammal with an antigen, such as mouse or rat lung cells (hereinafter abbreviated as S) and hyboxanthin-guanine phosphoribosyltransferase-deficient myeloma cells (Myeloma cells). (hereinafter abbreviated as M) in the presence of a fusion accelerator, such as polyethylene glycol 1500:PEG1500
Only the S-M fused cells are grown on a multi-plate in a medium containing T) to kill the M-M fused cells and free M. Next, the antibody of interest is produced from the wells where growth was observed. Cells (hybridoma) are cloned using limiting dilution method.

Next, by culturing the hybridoma in vitro or transplanting it into an animal (for example, intraperitoneally in a mouse), a large amount of monoclonal antibodies against a specific antigen can be obtained.

(Problems to be Solved by the Invention) The present invention specifically binds to h-TNF and neutralizes its cytotoxicity, does not exhibit cross-activity with human-derived lymphotoxin and xenobiotic-derived TNF, and is non-active. Active h-TNF
The purpose of the present invention is to obtain a specific monoclonal antibody against h-TNF that has activity and does not cross-react with other antibodies.

An object of the present invention is to provide a method for selectively measuring active h-TNF using such an antibody by an immunochemical method, such as an ELISA method.

(Specific Means for Solving the Problems) The antibody of the present invention is produced by combining antibody-producing cells extracted from a mammal (preferably a mouse) immunized with h-TNF with 110% of an appropriate animal (preferably a mouse). It is produced by fusing tumor cells, such as myeloma, and culturing hybridomas obtained by cloning the fused cells that produce the antibody of interest.

a) m-production of antibody-producing cells In order to obtain the hybridoma of the present invention, first h-T
Immunize the mammal with NF. Examples of mammals that can be used include mice, rats, rabbits, guinea pigs, etc.
- Immunize by intraperitoneal or subcutaneous inoculation with TNF. Inoculation is repeated several times every 1 to 2 weeks with 10 to 30 pg of antigen/mouse mixed with the same amount of adjuvant per mouse, and the final immunization is 50 to 15 pg of antigen as it is.
0 pg/mouse by intravenous or intraperitoneal injection, 3-4
On day 1, the pancreas is removed and used as antibody-producing cells. h
-TNF may be inducibly produced from human-derived lymphoid tM cells and purified, or may be prepared from a culture of h-TNF-producing microorganisms created by recombinant DNA technology.

b) Preparation of Myeloma Cells There are no particular restrictions on the myeloma cells (myeloma) used, and cell lines from animals such as S. maris, rats, rabbits, and humans can be used, but mouse myeloma cells are usually used. In general, myeloma IIligI hypertrophic strains, such as myeloma, with appropriate selection markers such as hypoxanthine guanine phosphoribosyltransferase deficient (HGPRT-) or thymidine kinase deficient (TK-), such as P
3-X63-Ag8, P3-X63-Ag8-Ul.

Prepare SP2O-Ag14 and X63-Ag8-6.5°3. This am cell is an 8-azaguanine resistant cell line and has the property of not being able to grow in HAT medium.

C) Add 10% C3 (calf serum) to a commonly used cell fusion medium such as Eagle's Minimum Essential Medium (MEM), Dulbecco's Improved MEM, or Roswell Park Memorial Institute (RPMI) 1640.
) or 5% FC3 (real calf
Add serum) + 5% C3 or 10% FC5. Normal maintenance of parent cells can be carried out in any of the above-mentioned media, but lO%FC3 is preferable for the production of hybrid cells.Cell fusion is carried out by combining parent cells, Il tumor cells such as myeloma, and immunized lung cells. (Antibody-producing cells) 1:5 to 1:1
This is done in the presence of a fusion promoter by mixing at a ratio of 0. As a fusion promoter, HVJ (Hemagglutinat
ing Virus or Japan), polyethylene glycol (PEG), etc. In particular, PEG
Approximately 30 to 50% of L500 is good.

d) The cells after HAT selection fusion of bipridoma are diluted appropriately with RPM11640 medium containing 20% FC5, etc., and placed in a microculture plate (usually 96-well type) for 10 s to 1 O6/100. d
/ Plant in a well.

Add HAT selection medium to each well and culture by replacing the medium usually every 1 to 2 days.If an 8-azaguanine resistant strain is used as myeloma cells, unfused myeloma cells and M-M fused cells will be HAT. They die in about 10 days in the culture medium, and since splenocytes are normal cells, they have a limited lifespan, and cannot grow for more than 2 weeks in vitro. All are considered to be S-M hybridomas.

C) Screening of hybridomas Screening mainly uses EL fsA (Enzyme
linked-immunosorbent as
This is carried out by procuring hybridoma clones secreting the antibody of interest using a known method such as the ``say'' method.

r) Cloning Although there is a possibility that two or more types of hybridomas producing different antibodies are growing in each well, cloning is performed using the limit station method, etc., and ultimately the desired monoclonal antibody is produced. Unigenic hybridomas can be obtained.

g) Obtaining monoclonal antibodies The hybridoma obtained above is placed in a culture container (in
viLro) or in vivo. When culturing in vitro, the medium may be the above-mentioned normal medium to which C3 or Fe2 is added, and after culturing in this medium for 3 to 5 days, the antibody of interest can be obtained from the medium supernatant. ±n In vivo culture, myeloma cells and syngeneic animals were treated with bristane (2,
After at least 1 week has elapsed after intraperitoneal administration of mineral oil such as 6°10.14-tetramethylpentadecane, hybridomas are inoculated intraperitoneally and
The ascites that accumulates after a few days is collected and the desired antibody can be obtained from this.

The monoclonal antibody thus obtained is h-T
It can be used for measuring and detecting NF (especially active h-TNF). Cytotoxicity of TNF to L929m cells is a major indicator of TNF, and conventionally, the titer of TNF has been measured by bioassay using L929 cells, but while this method is highly sensitive, it is time-consuming. It takes a while,
There are complications inherent in bioassays. Therefore, the monoclonal antibody of the present invention was used in combination with the h-TNF polyclonal antibody to perform a rapid and convenient enzyme-linked immunosorbent assay (IE).
LISA) was established. The polyclonal antibody used here can be any h-TNF-specific antibody derived from a species different from the animal from which the monoclonal antibody was obtained.

Hereinafter, the present invention will be explained in detail with reference to Examples.

(Example) Example 1 h-TNF monoclonal Fy wood q wood mold 4
) Plasmid pT4TNFST8ro containing the Houou Tohoku X TNF gene
p-transformed E. coli strain W3110/pT4TNF
ST8rop- (Japanese Patent Application No. 60-217740) was used as tetracycline lO1! GC medium containing g/rn1 (2
% glycerin, 3% casamino acids, 0.5% KH2PO4
, 0.2% yeast extract, 0.1% MgSO4・7H20
, 6.5) at 37℃ for 15 hours, 3! Dose (actual volume 2
The cells collected by centrifugation are suspended in 10mM) Lis-HCl buffer (PH8) (hereinafter simply referred to as Tris buffer), which has been cultured using a Jarfer Mentor of ffi).
Cooling high-pressure homogenizer (Man, ton-Gaul)
in Laboratory Hom.

Genizer 15M-87A) at 8000p
After disrupting with s+, centrifugation was performed to obtain a supernatant. The protein precipitate produced by adding ammonium sulfate to 80% saturation was dissolved in Tris buffer, thoroughly dialyzed against the same buffer, and DEAE Toyovar 650C (
(manufactured by Toyo Soda Co., Ltd.) column chromatography. T
The NF-active fraction was eluted by running a sodium chloride solution with a linear concentration gradient from OmM to 300 mM.

Subsequently, the TNF active fraction was added to Tris buffer (pH 7).
After dilution, the mixture was applied to a DEAE CEF yO-XCL-6B (manufactured by Pharmacia) column, and a sodium chloride solution having a linear concentration gradient from OmM to 200mM was passed therethrough to obtain TNF activity. In this purification step, SDS PAGE
An almost single band of a protein with TNF activity was obtained by analysis using 100% protein.
-4B (manufactured by Pharmacia) column for purification. In addition, the TNF preparation obtained by the above re-chromatography was added to Sephacryl 3, which is a gel filtration carrier.
200 was used to further increase the purity.

The final TNF preparation was purified by reverse high performance liquid chromatography (Shimadzu Corporation! 52) using a Microponder Bafuku CI8 column (Waters Corporation), and then automated amino acid analysis 23 (Hitachi Corporation). Made in Japan 835-
Amino acid analysis was carried out according to the conventional method using the following method. The results showed good agreement with the amino acid composition expected from the amino acid sequence shown in FIG.

In addition, the amino acid sequence of the amino acid terminal of the purified protein was determined using a gas phase protein sequencer Mo according to the Edman method.
del 470A (Applied Biosys
As a result of analysis using TEM), it was found that the amino acid sequence completely matched the amino acid sequence shown in FIG. Hereinafter, the h-TNF obtained here will be referred to as r,h-TNF.

(b) R,h-TNF (10ag) purified by Freund
Suspended with complete adjuvant (FCA) and intraperitoneally administered to 4-5 week old BaIb/c mice.
n/mouse was administered. Booster immunizations were given twice at 2-4 week intervals with the same amount of r and h-TNF as the initial immunization.
The suspension was suspended in incomplete adjuvant (FICA) and administered intraperitoneally to Balb/e mice at 5 #g/mouse. Three weeks after the third immunization injection, r,h-TNF was dissolved in phosphate buffered saline (PBS) and administered at 10n/mouse for final immunization.

(c) MMxfL 1lII! from mice 3 days after final immunization! After removing the viscera and cutting it into pieces, the lung cells obtained by attenuation with a stainless mesh were transformed into mouse myeloma cells (P3-X63
-Ag8U1: Abbreviation P3U1) and mixed at a ratio of 4:1, 5
Cell fusion was performed using 0% polyethylene glycol 1540 (manufactured by BDH). Suspend myeloma cells in 10% FC3-RPM medium to approximately 106 cells/- and place them in a 96-well microculture plate. /well was dispensed.

One day later, 100 μl of HAT medium was added to each well, and half the amount was replaced with IAT medium every 3 days. From around the 5th day, growth of hybridomas was observed in some wells. After two weeks, hybridomas had grown in almost all the wells.

(d) Progression of m-type cells ζkyuuni 379'r, h-TNF
in 0.1M sodium bicarbonate buffer (PH9,6
) was adjusted to 10 ug/ml and dispensed into a 966 microplate (manufactured by Costar) in the amount of food/well. After leaving the plate overnight at 4°C, the supernatant was removed and the remaining protein binding sites in the wells were blocked with PBS containing 1% ovalbumin.
PBS containing Tween 20 (abbreviated as P B
After washing with 5-T), the hybrid cell culture supernatant was diluted with 10011/
Dispense into wells, incubate at 37°C for 2 hours, and then remove the plate.
Washed three times with BS-T and added alkaline phosphatase-labeled goat 1gG anti-mouse summer gG (H) (PL Bioc
Manufactured by Herticals, Lot #121 2
) 0) I O00M rare Y'! '! Aj, 'E 1
00p! / well. After incubating at room temperature for 2 hours,
Washed with PBS-T and treated with 4-methylumperiphery phosphatase (4-methylumbellifer).
ryl-phosphophaLase) 1.7■150p
Then, after incubating at room temperature for 10 minutes, IMK2HP04-KOH (9HL0.3) was added to L5Qd/well to stop the reaction, and the fluorescence intensity of the reaction product was measured using corona multispectrofluorometry. The amount of anti-h-TNF antibody secreted was measured using a meter (excitation wavelength: λ365nm, extinction wavelength: λ450nm)a Wells in which strong antibody activity was observed were cloned by limiting dilution method, and one clone was isolated. The monoclonal antibody obtained by culturing this hybridoma was named MAB-3810.

MAB-3810 is recombinant human TNF (r.

It is a monoclonal antibody obtained from a bilidoma of mouse myeloma and antibody-producing cells of a mouse immunized with h-TNF.
Production cells (U937 cells: ATCCCRL 1593
), the neutralizing activity against h-TNF purified from the culture supernatant was compared.

10% each of h-TNF and r, h-TNF in FC3-ME
M to 256U/mj: MAB-38I made by A
Mixed with Q etc. After standing at 4℃ overnight, L92911
When the neutralizing activity was examined by bioassay using one cell, both showed neutralizing activity against cytotoxicity. In order to examine the reactivity in more detail, h-TNF and r, h-
2. TNF with 10% FC3-MEM. 600U
/lnl was mixed with equal amounts of MAB-3310 at various concentrations of 0°5 to 500μ/d, left overnight at 4°C, and bioassay using L929 was performed to determine whether both T.
The neutralizing activity of MAR-3B10 against NF was investigated. The results are shown in Figure 2. Although the neutralizing activity against h-TNF was slightly stronger, it was within the relative error range at the sample protein concentration standardization stage in the bioassay, and MA
The activity neutralization reactivity of R-3B10 to h-TNF and r,h-TNF was approximately the same.

Human TNF (h-TNF), tJ1 recombinant human TN
F (r,h-TNF), mouse TNF (m-TN
F), recombinant mouse TNF (r, m-TNF), and rabbit TNF (r TNF) were each mixed in PBS at 2
MAR-3B 1 prepared to 56 units/rnl
0 or h-TNF (hereinafter this antibody is referred to as PAB-R5). After standing overnight at 4°C, the cytotoxicity of various TNFs against L929 was determined by the Pyofusei method using L929.
The neutralizing activity of AB-3810 was investigated. The result is the first
Shown in the table.

Table 1 P of various TNF on L929929 cytotoxicity
Neutralizing activity of AB-R5 and MAb-3810 MAB-3B l O and P A B -R5 show neutralizing activity only against human TNF (h-TNF, r, h-TNF), and xenobiotic @IJ ( TN derived from mouse, rabbit)
It showed no neutralizing activity against F.

When measuring the titer of TNF by immunoassay using an anti-TNF antibody, it is especially important to use an antibody that can distinguish between active and inactive TNF. Therefore, in order to investigate the reproducibility and usefulness of titer measurement of activated TNF by ELISA method using the monoclonal antibody of the present invention, we tested rA-denatured TNF (inactive TNF) using ELISA method.
We examined whether cross-reactivity was exhibited using Method A and compared it with the bioassay. 50yg/- of r,h-TNF was prepared in PBS and 30.60.90,120 and 18 at 70°C.
Incubate for 0 minutes. After ink evacuation at each time, each sample was immediately water-cooled and heated to 10.00 Orp.
centrifugation for 5 minutes.

Next, the supernatant was subjected to an ELISA method and a cytotoxicity test using L929 cells. The results are shown in FIG. 3, and clearly showed a positive correlation between the ELISA results and the bioassay results. MAB-3810
The fact that MAB did not bind to human TNF denatured by heating at 70°C, that is, inactivated human TNF,
Considering that -3B10 has the activity of neutralizing the cytotoxicity of TNF to L929 cells, M
This strongly suggests that AB-3810 specifically recognizes only the part involved in the biological activity of TNF.

Human-derived lymphotoxin (h-LT) and recombinant hydrinphotoxin (r, h-LT) were each incubated in PBS for 6 hours.
．． Prepared to 400 U/ml and IOB/mff1, 100 tri was added with rabbit-derived anti-lymphotoxin antibody (
PAB-Rabbit anti-hLT) or M
Mixed equally with AB-38IO. After standing at 4℃ overnight, L
The neutralizing activity of MAR-3810 against the L929 cytotoxicity of each lymphotoxin was examined by bioassay using 929.

The results are shown in Table 2.

Table 2 MA on L929 cytotoxic effect of h-LT
Neutralizing activity of R-3310 MAB-3810 is effective against human-derived lymphotoxin and ME.
It showed no neutralizing activity against recombinant lymphotoxin.

Example 3-1 Measurement of physical properties of 1π or less was carried out using the monoclonal antibody (MAR-3810) purified in Example 1.

1) Antibody class and isotype MAB-3810 class and isotype at 1°5%F
When examined by immunoelectrophoresis using a 4-layer agarose gel, Zab class was IgG, and LmO type was g.
It was an antibody with tl+.

2) Isoelectric point The isoelectric point was measured by isoelectric focusing using a single-layer polyacrylamide gel with a state range of 3.5 to 9.5. As a result, the isoelectric point of MAB-3810 was 6°2±0.1.

Example 4 Basic measurement method h-TN measurement using ELrSA In order to quickly and conveniently measure h-TNF, rabbit h
-We investigated improvements to the ELrSA method using a TNF polyclonal antibody (PAB-R5) and biotin-labeled MAB-3810.

Coating buffer (Na2CO3: 1.59g
/ l, Na HCOs = 2-93 g /
', pH 9.

After adjusting PAB-R5 to 20 females/- using 6), 100 pK/well was dispensed into a 96-well microculture plate. After standing at 4°C overnight, the supernatant was discarded, and 150p1/well of PBS containing 1% ovalbumin was dispensed, followed by standing at 37°C for 1 hour.

TNF prepared at various concentrations was dispensed at 100 aj/well onto a plate coated with PAB-R5, and reacted at 37°C for 2 hours. 0.1% Tween 2
After washing three times with PBS containing 0 (PBS-T), biotin-labeled MAB-38IO (2,000-fold dilution) was added to 10011! / well and reacted at 37°C for 2 hours. Then, after washing three times with PBS-T, streptavidin-biotinylated peroxidase (Amersham
100 volumes/well of 100 volumes of diluted solution (manufactured by Nippon Steel & Co., Ltd.: i, ooo times diluted solution) were dispensed and allowed to react at room temperature for 1 hour. Add 0-phenylenediamine (0.43u/ml, pH 4°8) and ■ (2
0□ (0,002%) prepared in 0.1 M citrate buffer was added at 200 d/well and allowed to react in the dark at room temperature for 30 minutes.
/well was added and stopped. Guinatec absorbance
Measurement was performed using a Micro Elyzer autoreader (490nw).

As shown in FIG. 4, h-TNF could be detected in a concentration-dependent manner in the range of 2 to 60 ng/-. From this, it was found that the measurement sensitivity was approximately the same as in the case of PIOASEI using L929, and it was therefore confirmed that TNF in an unknown sample can be detected quickly and reliably by this method.

(Effects of the Invention) The monoclonal antibody of the present invention has neutralizing activity against the cytotoxicity of human TNF, does not bind to inactive TNF or xenobiotic TNF, and exhibits cross-reactivity with human-derived lymphotoxin. do not have.

That is, since it reacts only with biologically active human TNF, it becomes possible to easily measure human TNF activity instead of conventional complicated bioassays.
By ELISA, using with polyclonal antibodies,
Specific human TNF levels can be detected. In addition, using the monoclonal antibody specific for human TNF in the present invention, human) TNF $i? m, it is also possible to perform immunology conventions ■.

[Brief explanation of the drawing]

Figure 1 is a sequence diagram showing the amino acid sequence of human TNF, Figure 2 is a graph of the neutralizing activity of MAR-38I O against the cytotoxicity of human TNF and recombinant human TNF, and Figure 3 is a graph showing the neutralizing activity of MAR-38IO against the cytotoxicity of human TNF and recombinant human TNF. Figure 4 shows a bioassay and ELISA assay curve graph of denatured human TNF.
g of human TNF by EL[SA in combination with -3810
A Q-curve graph is shown. Patent Applicant Suntory Ltd. Agent Patent Attorney Kyozo Atsushi - 5 Famous Ingredients + 10
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Claims (5)

[Claims] (1) The following properties: a. Neutralizes the cytotoxicity of human tumor necrosis factor to tumor cells; b. Inactive human tumor necrosis factor and non-human animal tumor necrosis factor that have lost their cytotoxicity to tumor cells. What is
Does not bind by immune reaction; c, Does not bind to lymphotoxin by immune reaction; d, Subclass is IgG_1 and L chain type is isotype κ; e, Isoelectric point is 6.2 ± 0.1; A specific monoclonal antibody against the active site of human tumor necrosis factor. (2) Formula I obtained by recombinant DNA: [Gene sequence is available] From a hybridoma of mouse lung cells and mouse myeloma cells sensitized with human tumor necrosis factor expressed by the amino acid sequence (I) The monoclonal antibody according to claim 1, which is obtained. (3) The monoclonal antibody according to claim 1, which is represented by the name MAB-3B10. (4) A method for measuring the activity of human tumor necrosis factor using the monoclonal antibodies according to claims 1 to 3. (5) The method according to claim 4, which is an ELISA method.