JPH08224083A - Monoclonal antibody - Google Patents

Abstract

PURPOSE: To provide a new monoclonal antibody free from inhibiting action on the activity of lymphokines different from T-cell growth and differentiation factor (TRF), effective for inhibiting TRF activity even at a low antibody concentration and useful for the purification and determination of TRF and as a TRF inhibitor, etc. CONSTITUTION: This new monoclonal antibody does not inhibit the activity of lymphokines different from T-cell growth and differentiation factor (TRF) such as interleukin(IL)-1, IL-2, IL-3 and B-cell stimulation factor 1 (BSF-1) even at an antibody concentration of 10μg/mL, inhibits TRF activity at an antibody concentration of 50mg/mL, specifically bonds to TRF and is useful as an immunoabsorbent for the purification of TRF, a TRF activity inhibiting agent, an agent for the quantitative determination of TRF, etc. The antibody can be produced by immunizing a Wistar rat with purified TRF emulsified with an adjuvant, collecting spleen cell after the final immunization, fusing the cell to a myeloma cell, subjecting the fused cell to selective culture in HAT medium, cloning the obtained hybridoma and culturing the product.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody that specifically binds to T cell-replacing factor (hereinafter referred to as TRF), which is an immunoadsorbent used for purification of TRF, and It is useful as an inhibitor of TRF activity. Specific uses of the immunosorbent include quantitative measurement of TRF in body fluids and purification of TRF. Measurement of TRF in body fluid is essential for diagnosing autoimmune diseases due to TRF hypersecretion and immunodeficiency due to defective TRF secretion. The TRF purified by the monoclonal antibody of the present invention can be used for the treatment of immunodeficiency due to poor TRF secretion and for the treatment of autoimmune diseases induced by the production of anti-TRF antibodies in the patient's own body.

[0002] Specific applications of inhibitors of TRF activity include remedies for autoimmune diseases due to TRF hypersecretion. Furthermore, the use of the monoclonal antibody of the present invention and purified TRF could elucidate an unsolved network of the immune system.

[0003]

BACKGROUND OF THE INVENTION Immature B cells are activated by cross-linking their antigen receptors with an anti-IgM antibody. TRF binds to an activated B cell surface receptor to promote B cell proliferation and differentiation into immunoglobulin secreting cells. (Howar
d, M., Nakanishi, K. & Paul, WE, Immunol. Rev. 78, 185-21
0 (1984) .Kishimoto, T., Ann.Rev., Immunol., 3,133-157
(1985) .Dutton, RW, Folkoff, R., Hirst, JA et al., P
rog. Immunol., 1,355-386 (1971). Swain, LL, Howard,
M., Kappler.JW, et al., J.Exp.Med., 158,822-835 (198
3). See TRF, which is a factor secreted by T cells, also promotes differentiation of antibody-producing mature B cells. (Schimpl, A. and
E. Wecker., Nature, 237, NB, 15-17 (1972). Takatsu, K., T
ominaga, A. & Hamaoka, T., J.Immunol., 124,2414-2422 (1
980). Takatsu, K., Tanaka, K., Tominaga, A. et al., J. Im
munol., 125,2646-2653 (1980). Nakanishi, K. et al., J.
Immunol., 130, 2219-2224 (1983).) Murine T cell fusion cell B151K12 (Takatsu, K. et.
al., J. Immunol., 125, 2646-2653 (1980).), the murine TRF has the following two characteristics. (1)
Induction of IgM secretion by BCL ₁ leukemia B cell line (obtained from University of Texas) and dinitrophenyl (DNP) −
Induction of IgG class anti-DNP antibody production response in vitro by B cells primed with ovalbumin (OA) complex. (Takatsu, K., Tanaka, K., Tominaga, A. Et
al., J.Immunol., 125,2646-2653 (1980). Takatsu, K., Har
ada, N., Hara, T. et al., J. Immunol., 134,382-389 (198
5) .Harada, N., Kikuchi, T., Tominaga, A. et al., J. Immun
ol., 134, 3944-3951 (1985).) (2) Proliferative activity of BCL ₁ leukemia B cell line. (1) is TRF activity, (2) is B cell growth factor type II (BCG
FII) activity in some cases, but since TRF has the activity shown in the above (1) and (2) in the present specification, the activities of (1) and (2) above are collectively referred to as TRF. I will call it activity. The TRF of a mouse has a molecular weight without adding a reducing agent.
45,000-60,000 (on gel filtration). Isoelectric point (pI value)
4.7 to 4.9, B cell stimulating factor 1 (BSF-1), interleukin 2 (IL-2), interleukin 3 (I
L-3), which was different from the immune interferon activity. (Takatsu, K., Harada, N., Hara, T. et al., J. Immu
nol., 134,382-389 (1985). Harada, N., Kikuchi, T., Tomin
aga, A. et al., J. Immunol., 134, 3944-3951 (1985)) Recently, it has been proposed that TRF be called interleukin 5 (IL-5). However, in the present specification, the term TRF is used for the description.

[0004]

In the prior art, since there was no anti-TRF monoclonal antibody, TRF was not available.
Was difficult to purify. Also, TR of patients with autoimmune diseases
The blood concentration of F could not be measured. The present invention has been made in view of such problems, and an object thereof is to provide an anti-TRF monoclonal antibody with high specificity.

[0005]

The monoclonal antibody of the present invention does not inhibit the activity of lymphokines of a type different from TRF even at an antibody concentration of 10 μg / ml.
It is characterized in that it inhibits RF activity even at an antibody concentration of 50 ng / ml. Here, lymphokines of a type different from TRF are particularly IL-1 and IL-
2, IL-3 and B cell stimulating factor 1 (BSF-1). Further, the monoclonal antibody of the present invention is characterized by inhibiting the antibody production inducing activity of B cells. Also,
The monoclonal antibody of the present invention is characterized by inhibiting the proliferation and differentiation activity of B cells.

[0006] The monoclonal antibody of the present invention is produced by culturing the fused cells in vivo and in vitro. For example, the fused cells can be obtained by immunizing a spleen cell obtained by immunizing a mammal with TRF and mouse bone marrow cells from a cell fusion technique of Köhler and Milstein, Nature,
256, 495-497 (1975).).

[0007]

EXAMPLES The present invention will be described based on examples. Example 1 Preparation of TRF T cell hybridoma B151K12 cell line (Taka
tsu, K. et at., J. Immunol., 125 , 2646-2653 (1980)
PMA (4 β -phorbol 12 β -myristate 12)
α-acetate, manufactured by Sigma) was serum-free cultured in 3 ng / ml for 48 hours to prepare 225 liters of culture supernatant containing TRF. Next, from this culture supernatant, ammonium sulfate fractionation, anion exchange chromatography (DEAE-cellulose), blue sepharose column chromatography, mono P column (pharma
cia) chromatograph, reverse phase HPLC using protein C4 (Vydac) (LKB) gel chromatograph, purified T with a specific activity increased by 1.4 million times
RF was obtained. Incidentally, TRF activity was measured by IgM plaque forming cells test using BCL ₁ cells (hereinafter referred to as PFC test.).

The IgM PFC test was conducted in the following procedure. 50-100 μl of the cultured BCL ₁ cell suspension, 50 μl of 10% suspension of protein A-bound sheep red blood cells and 400 μl of 0.5% agarose solution containing MEM medium were mixed at 45 ° C.,
It was poured on a slide glass and left at room temperature for gelation.
Anti-mouse IgM serum was added to this gel and incubated at 37 ° C. for 2 hours and 30 minutes. After adding guinea pig complement and incubating at 37 ° C for 1 hour, the number of plaques was counted. Anti-D
The NP IgG PFC test was similarly performed using 50 to 100 μl of B cell suspension stimulated with the DNP-OA complex. (B
Since cells secrete IgG, an operation of adding anti-mouse IgG serum instead of the above anti-mouse IgM serum is performed. ) T
RF activity is half-maximal respo when using purified TRF
The amount giving nses was defined as 1 unit (U) and expressed in units per culture solution (U / culture or PFC / culture). Preparation of spleen cells Purified TRF emulsified in complete Freund's adjuvant
Wistar rats were immunized with 3.5 × 10 ⁵ U. Furthermore, Wistar rats were treated with purified TRF 2 × 10 ⁵ U every 20 days for 2
I boosted it. Three days after the final immunization, spleen cells were taken out from Wistar rats and prepared by the following procedure.

The spleen was placed in 5 ml of Eagle's MEM culture solution. Next, 4 ml of the above culture solution was placed in a 10 ml syringe and injected into the spleen. Splenocytes were loosened using tweezers. Then, the cell suspension was put into an injection cylinder and put into and taken out from the injection cylinder three times to make the cell mass fine. The singulated cells were taken out and centrifuged at 1000 to 1200 rpm for washing. The washed cells were suspended in 2 ml of Eagle's MEM culture medium. Cell fusion Rat spleen cells and mouse myeloma cells P3X63-Ag8.653 (ATCC accession number CRL-
1580) and the cell fusion technology of Köhler and Milstein.

The prepared spleen cells (6 × 10 ³ cells) and mouse myeloma cells P3X63-Ag8.653 (3 × 10 ³ cells) were mixed and then centrifuged to pellet the cells. 50% PEG-on the pellet
1 ml of RPMI-1640 culture solution containing 1500 was gradually added with stirring. Next, RPMI-1640 culture solution was gradually added and diluted while stirring. Then, the supernatant is removed by centrifugation and 10%
1 × 10 ⁶ cells in RPMI-1640 culture medium containing FCS
Suspend to 1 ml / ml and add 1 ml to 24-well microplate.
/ Well was dispensed. Next day, 500 μl / well HA
T medium was added. Repeat the same operation every two days thereafter
It was cultured for 14 days. Further, the HAT medium was replaced with HT medium containing hypoxanthine and thymidine, and 500 μl / well was exchanged every 2 days. After 7-10 days, change to HT medium and 10% FC
The RPMI-1640 culture medium containing S was replaced every 500 days with 500 μl / well. Screening Anti-T of hybridoma culture supernatant obtained by the above cell fusion
RF activity was examined by TRF activity inhibition and TRF activity absorption test. TRF activity inhibition test was performed by measuring the IgM PFC test and ³ H-thymidine ⁽³ H-thymidine) uptake. Here, the amount of ³ H thymidine incorporation was measured by culturing 1.5 × 10 ³ BCL ₁ cells in 0.2 ml of a culture medium containing TRF 2U and anti-TRF monoclonal antibody, and ³ H thymidine 0.2 μCi / Wells were added. After washing the cells by centrifugation, ³ H incorporated into the cells was measured by liquid scintillation.

In the TRF activity absorption test, the culture supernatant was added to a microplate to which rabbit anti-rat Ig antibody had been adsorbed, and the mixture was incubated at 37 ° C for 4 hours, washed, and a certain amount of TRF was added, and further incubated at 37 ° C for 4 hours. .
The residual TRF activity of the supernatant was measured. As a result, 61 out of the 479 wells containing the hybridoma were anti-TR.
It showed F activity. Furthermore, from 61 wells, 1 well showing reproducible inhibition and absorption activity was selected. Cloning Hybridomas contained in one selected well were cloned by limiting dilution method to obtain hybridomas TB13 and NC.
17 was obtained. Purification of monoclonal antibody An anti-TRF monoclonal antibody was obtained from hybridomas TB13 and NC17 by the following procedure.

The culture supernatant of hybridoma TB13 was applied to an affinity column using agarose conjugated with goat anti-rat IgG antibody. The adsorbed fraction was eluted with borate buffer pH = 8.0 containing 3M KSCN to purify anti-TRF monoclonal antibody TB13. The large-scale purification was carried out by the following procedure. About 15 to 20 days after the injection of 1 × 10 ⁷ hybridoma TB13 into the abdominal cavity of BALB / C nu / nu mice, ascites was collected and the IgG fraction was obtained by gel filtration using reverse phase HPLC to obtain the anti-TRF monoclonal antibody. Antibody TB13 was purified.

The anti-TRF monoclonal antibody NC17 was also purified by the same procedure using the hybridoma NC17. Example 2 (Physicochemical and immunological properties of monoclonal antibodies TB13 and NC17) Identification of immunoglobulin class The immunoglobulin class of anti-TRF monoclonal antibodies TB13 and NC17 was determined by the Ouchterlony method. Anti TR
F monoclonal antibodies TB13 and NC17 are both Ig
It was G ₁ . The molecular weights of the molecular anti-TRF monoclonal antibodies TB13 and NC17 are shown in Table 1 below.

[0014]

【table 1】

[0015] Inhibition To examine the specificity of the specific anti-TRF monoclonal antibody, anti-TRF monoclonal antibody TB13, NC17 is TRF, a BSF-1, IL-1, IL-2, the activity of the lymphokine IL-3 I checked whether to do it. Table 2 shows the results. As can be seen from Table 2, TB13, NC
Each of the 17 anti-TRF monoclonal antibodies is BSF-1,
IL-1, IL-2, IL-3 activity was not inhibited and TRF
Only activity was inhibited.

[0016]

[Table 2]

Note: a) BC for TRF (2 U / ml)
The response of L ₁ cells was determined by the number of IgM PFC cells after 2 days of culture. b) was measured with a radiation uptake of ³ H-thymidine ⁽³ H-thymidine) after 3 days culture. c) ³ H thymidine incorporation was measured by radiation dose after 1 day of culture. PC61 (obtained from Swiss Cancer Institute) (Lowenthal, JW,
Zubler, R, H., Nabholz, M. & MacDonald, RH, Nature (Lon
Don), 315, 669-672 (1985).) is a rat monoclonal antibody against the anti-interleukin 2 receptor (α-IL-2 receptor). 18F10 (obtained from NIH) (Ohara, J. & Paul, WE, Nature (London), 315,333-
336. (1985).) Is an IgG ₁ class rat monoclonal antibody against BSF-1. WEHI-3
(Obtained from WEHI, Australia) is a T cell line that produces IL-3. PC61 50 ng / ml
Is a T cell line GY-1 (Takatsu, K., Harada, N., Hara,
T., et al., J. Immunol., 134, 382-389 (1985).). 18 at 30,000 times dilution
F10 inhibits proliferation of resting B cells by BSF-1 in the presence of anti-IgM antibody. Specificity was examined by IgM secretion of BCL ₁ cells by TRF, BSF-1 + anti-IgM.
Resting B cell stimulation by antibodies (Ohara, J. & Paul, W.
E., Nature (London), 315, 333-336 (1985).), IL-
1 + PHA stimulates thymocytes (Mizel, SBImmuno
Rev., 63, 51-72 (1979).), GY-by IL-2
Proliferation of 1 cell, T cell line FDCP- by IL-3
1 Stimulation (Kikuchi, Y., Kato, R., Sano, Y., Takahashi,
H., et al., J. Immunol., 136, 3553-3560 (1986).)
Was tested whether various monoclonal antibodies inhibited. TR obtained from titer T cell hybridoma B151K12
F has the effect of differentiating BCL ₁ cells into IgM-secreting cells and increasing the proliferation, so IgM using BCL ₁ cells was used.
PFC and growth tests were performed.

1A and 1B are graphs showing the ability to inhibit TRF activity when anti-TRF monoclonal antibodies TB13 and NC17 were added to a BCL ₁ cell culture medium. The test is in vivo
o BCL ₁ cells grown in line 1.5 × 10 ⁵ cells / 0.2
It was performed after incubating ml with TRF2U in 200 μl of culture medium containing various concentrations of anti-TRF monoclonal antibody TB13 or NC17 or anti-BSF-1 monoclonal antibody 18F10.

In FIG. 1A, reverse IgM PF was observed after 2 days of culture.
The number of IgM producing cells was determined by the C test. Symbol in the figure ▼
Is the measured value of IgMPFC in the medium in which only BCL ₁ cells were cultured, ◯ is the measured value with the addition of TRF, and Δ is the TRF + TB1.
3 indicates the measured value, ● indicates the measured value at TRF + NC17, □ indicates the measured value at TRF + 18F10 (in vivo cultured ascites extraction), and ■ indicates the measured value at TRF + TB13 (in vivo cultured ascites extraction). In FIG. 1B, 3H thymidine incorporation was measured after 2 days of culture. In the figure, symbol ▼ is B
Radiation measurement value when culturing only CL ₁ cells, ○ means T
Measured value with RF addition, △ is measured value with TRF + TB13, ● is measured value with TRF + NC17, □ is TRF + 1
8F10 (in vivo cultured ascites extraction) represents the measured values.

For comparison, rat IgG ₁ class anti-BSF-1 monoclonal antibody 18F10 was IgM.
It had no secretion inhibition (FIG. 1A) and BCL ₁ cell growth inhibition (FIG. 1B). The anti-TRF monoclonal antibodies TB13 and NC17 both had strong Ig at 40 ng.
Inhibition of M secretion (FIG. 1A) and growth inhibition of BCL ₁ cells
(Fig. 1B).

Next, the anti-TRF monoclonal antibody TB1
3, it was examined whether NC17 inhibits the TRF-induced anti-DNP IgG production response. The B cells stimulated with the DNP-OA complex showed the result T of the anti-DNP IgG PFC test.
98 PFC / culture without RF addition, purified TRF3
It showed 638 PFC / culture when U / ml was added. Anti TR
F monoclonal antibody TB13 50 ng / ml was added on the first day of culture, and the anti-DNP IgG PFC test result was 126P.
It was FC / culture and clearly inhibited the anti-DNP IgG production response. Similar results were obtained with NC17. This indicates that the anti-TRF monoclonal antibodies TB13 and NC17 inhibit TRF activity even in the experiment using B cells sensitized with DNP. Example 3 (Application example of anti-TRF monoclonal antibody) Application to immunoadsorbent 2.9 mg of a mixture of anti-TRF monoclonal antibodies TB13 and NC17 was applied to bromine cyanide-activated Sepharose CL-4.
What was bound to 3 g of B was used as an immunoadsorbent.

Reversed-phase HPLC (Parmacia) 10 ml of hybridoma cell B151K12 culture supernatant (1.2 × 10 ⁴ U sample) obtained by gel filtration was applied to the above affinity column (3 ml packed volume) to collect the effluent. After that, 50 ml of IM NaCl solution, 100 m of PBS
1, the affinity column was washed with 20 ml of water. Then, 20 ml of 0.8 M acetic acid solution was flown to elute the TRF bound to the affinity column. The eluate was freeze-dried and then redissolved in 0.8 ml of water. The TRF activity was measured by the IgM PFC test using BCL ₁ cells.
The measurement results are shown in Table 3.

[0023]

[Table 3]

As is clear from Table 3, when the roughly purified hybridoma B151K12-producing TRF was applied to the affinity column, 138 U of TRF was flowed out.
10.8 × 10 ³ U of TRF was eluted with the M acetic acid solution. As a control column, normal rat Ig was treated with Sepharose CL
As a result of carrying out a similar test by preparing one bound to -4B, all the TRFs flowed out. Application to elucidation of physicochemical properties of TRF Purified B151K12 produced TRF1 by chloramine T method
X10 ³ U was labeled with ¹²⁵ I. Unbound ¹²⁵ I was removed from ¹²⁵ I labeled TRF using Sephadex G-25 (Pharmacia). Next, ¹²⁵ I-labeled TRF
Anti-TRF monoclonal antibody TB13 binding CL-4B
Attached to beads. The complex of ¹²⁵ I-labeled TRF on the CL-4B beads and the anti-TRF monoclonal antibody TB13 is a buffer containing 2% SDS in the presence (+) and the absence (−) of 2-mercaptoethanol (hereinafter referred to as 2ME). ¹²⁵ I-labeled TRF was dissociated by. SDS-PAGE was performed on the eluted fractions. ¹²⁵ I labeled TR
F was detected by autoradiography. The result is shown in FIG. 3A.

In the figure, the number 1 is in the absence of 2ME (-2M
The autoradiograph of the fraction from which ¹²⁵ I-labeled TRF was dissociated from CL-4B beads in E) is shown. Number 2 is -2
ME-anti-TRF monoclonal antibody TB13 binding CL-
6 shows an autoradiograph when normal rat Ig-bound CL-4B beads were used instead of 4B beads. No. 3 was ¹²⁵ I-labeled TR from CL-4B beads bound to anti-TRF monoclonal antibody TB13 in the presence of 2ME (+ 2ME).
The autoradiograph of the fraction which dissociated F is shown. No. 4 shows an autoradiograph using normal rat Ig-bound CL-4B beads at + 2ME.

The fraction eluted from the anti-TRF monoclonal antibody TB13-bound CL-4B beads under the above-mentioned -2ME was sliced every 2 mm in the gel after SDS-PAGE after the SDS-PAGE, and the substances contained in each sliced gel Was electrophoretically eluted. The TRF activity of the eluate was changed to IgM
It was measured by the PFC test. FIG. 3B shows the result. It is apparent from the number 1 and the number 2 shown as a control in FIG.
Met. Molecular weights under reduction from Nos. 3 and 44,000 to 48,000
The main fraction of 00 (-2ME lower) has a molecular weight of 23,000 to 26,000 (+
TRF has a molecular weight of 2
It was recognized as a dimer of 3,000 to 26,000 substances. Moreover, in FIG. 3B, strong TRF activity was observed in the fraction of molecular weight 44,000 to 48,000, particularly about 46,000, and the anti-TRF monoclonal antibody TB13 had a molecular weight of 44,000 to 48,0.
It was demonstrated to bind specifically to the 00 protein.

The test results in FIG. 1 and Table 2 also show the relationship between TRF and several lymphokines. Anti-TRF
Monoclonal antibodies TB13 and NC17 are 50 ng / ml
IL-, although it significantly inhibits TRF activity in
It does not inhibit the activities of 1, IL-2, IL-3, and BSF-1 lymphokines even at a concentration of 10 μg / ml. From the above results, TRF is BSF-1, IL-1, IL-2, I.
It was found to be different from L-3 and to act early. This indicates that the TRF preparation has no interferon activity (Takatsu, K., et al.,
J. Immunol., 134, 382-389 (1985).) Shows that TRF is a unique lymphokine. Example 4 The TRF used in the above examples is the hybridoma B1.
Since it was obtained from the 51K12 cell line, it was examined whether the anti-TRF monoclonal antibody of the present invention inhibits TRF activity originating from different cell lines.

Recently, the inventors of the present invention have a cDNA having TRF activity.
A was successfully isolated. Isolated PSP6K-mTRF2
3 was cut with SalI to obtain a linear plasmid DNA.
Next, mRNA was synthesized with SP6 RNA polymerase. Synthesized RNA from Xenopus oocytes
( Xenopus oocytes), and incubated at 20 ° C for 36 hours. Recombinant TRF (rTRF), a product of oocytes translated PSP6K-mTRF23, stimulates proliferation of BCL ₁ cells and differentiation into IgM secreting cells. In order to examine the inhibitory effect of anti-TRF monoclonal antibody NC17 on the TRF activity of rTRF, 1.5 × 10 ⁵ /0.2 ml of BCL ₁ cells in the presence and absence of anti-TRF monoclonal antibody NC17 were used to rT.
Incubated with RF. After 2 days of culture, an IgM PFC test using protein A was performed to determine the number of IgM secreting cells. The results are shown in Figure 2.

FIG. 2 shows the anti-TRF monoclonal antibody NC1.
7 is a graph showing the ability of rTRF to inhibit TRF activity.
In the figure, symbol ▼ indicates IgM P obtained by culturing only BCL ₁ cells.
FC measured value, ○: measured value with rTRF (1 U / ml) added, ●: measured value with rTRF (3 U / ml) added, □: measured value with rTRF (1 U / ml) + NC17 added, △
Shows the measured values with rTRF (3 U / ml) + NC17 added.

From FIG. 2, it is apparent that the anti-TRF monoclonal antibody NC17 inhibits the TRF activity of rTRF depending on the amount added. Similarly, an anti-DNP IgG PFC test was performed, and the anti-TRF monoclonal antibody NC17 showed rTR.
It was examined whether it inhibits F-induced IgG secretion. DN
B cells stimulated with the P-OA complex showed 169 PFC / culture without addition of rTRF and 1003 PFC / culture with addition of 3 U / ml of rTRF. The anti-TRF monoclonal antibody NC17 (1 μg / ml) was added on the first day of the culture to show 328 PFC / culture, which was clearly anti-D.
Inhibited NPIgG secretion.

The above results indicate that the anti-TRF monoclonal antibodies TB13 and NC17 specifically act on different TRFs and inhibit the TRF activity with good reproducibility. As described above, the monoclonal antibody of the present invention
Reacts with all substances that have RF activity. Therefore, TR
It is useful as a probe for purifying F and elucidating the molecular properties of TRF.

The monoclonal antibody of the present invention is TR
Being a potential inhibitor of F activity would allow the role of TRF to be clarified as it would allow the study of a wide range of in vitro and in vivo immune responses.

[0033]

INDUSTRIAL APPLICABILITY The monoclonal antibody of the present invention is TRF.
A different type of lymphokine activity is not inhibited even at a high concentration, and TRF activity is inhibited even at an extremely low concentration, so that a highly specific monoclonal antibody can be provided.

[Brief description of drawings]

1A and 1B show anti-TRF monoclonal antibody T.
It is a graph which shows the ability of B13 and NC17 to inhibit TRF activity.

FIG. 2 is an anti-TRF monoclonal antibody NC17.
Is a T of recombinant TRF manufactured by gene recombination technology
It is a graph which shows RF activity inhibitory ability.

FIG. 3A is a photograph showing an autoradiograph of ¹²⁵ I-labeled TRF. FIG. 3B shows TRF at each position of the gel after SDS-PAGE corresponding to FIG. 3A No. 1.
It is a graph which shows activity.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/577 G01N 33/577 B (C12P 21/08 C12R 1:91)

Claims (4)

[Claims] 1. A monoclonal antibody characterized in that it does not inhibit lymphokine activity of a type different from TRF even at an antibody concentration of 10 μg / ml, but inhibits TRF activity even at an antibody concentration of 50 ng / ml. . 2. A lymphokine of a type different from TRF is IL-1, IL-2, IL-3 and B cell stimulating factor 1.
The monoclonal antibody according to claim 1, which is (BSF-1). 3. The monoclonal antibody according to claim 1, wherein the antibody inhibits B cell antibody production-inducing activity. 4. The monoclonal antibody according to claim 1, which inhibits the proliferation and differentiation activity of B cells.