JPH10165179A - Monoclonal antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new monoclonal antibody comprising an antibody obtained by transducing a T cell-replacing factor(TRF) into a rat, exhibiting binding property specific to an antigen part of TRF and useful for TRF activity inhibitor, therapeutic agent, etc., for autoimmune diseases. SOLUTION: This monoclonal antibody is obtained by transducing TRF (IL-5) having antigenicity into a rat as a non-human animal, and exhibits binding property specific to antigen parts of plural series of TRF, but does not exhibit binding property to IL-1, IL-2, IL-3 and B cell stimulation factor 1 (BSF-1) and is specifically bound to an antigen substance of TRF derived from a cell line having different origin and is useful as a therapeutic agent, etc., for autoimmune diseases or patients with immune deficiency. The antibody is obtained by administering purified TRF into a rat, collecting a spleen cell after final immunity, fusing the cell with a mouse myeloma cell, cloning the resultant antibody-producing strain and culturing the strain.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a monoclonal antibody which specifically binds to a T cell-replacing factor (hereinafter, referred to as TRF), and more particularly to an inhibitor of TRF activity or TRF. (IL-
No. 5) for treating autoimmune diseases or immunodeficiency patients.

[0002] Excessive secretion of TRF causes autoimmune diseases, and poor secretion of TRF causes immunodeficiency. As a specific use of the TRF activity inhibitor,
Therapeutic agents for autoimmune diseases due to TRF hypersecretion are included. The TRF purified by the monoclonal antibody of the present invention is applied to the treatment of immunodeficiency due to poor secretion of TRF and the treatment of autoimmune diseases induced by the production of anti-TRF antibodies in the patient's own body.

Furthermore, by using the monoclonal antibody of the present invention and purified TRF, it will be possible to elucidate a network of the immune mechanism that has not been solved yet.

[0004]

2. Description of the Related Art Immature B cells are activated by cross-linking antigen receptors with an anti-IgM antibody. TRF binds to activated B cell surface receptors to promote B cell proliferation and differentiation into immunoglobulin secreting cells (Howard,
M., Nakanishi, K. & Paul, WE, Immunol.Rev.78,185-210
(1984) .Kishimoto, T., Ann.Rev., Immunol., 3,133-157 (1
985) .Dutton, RW, Folkoff, R., Hirst, JA et al., Pro.
g. Immunol., 1,355-386 (1971) .Swain, LL, Howard, M.,
Kappler. JW, et al., J. Exp.Med., 158, 822-835 (1983).
See). TRF is a factor secreted by T cells and also promotes the differentiation of mature B cells producing antibodies (Schimp
l, A. and E. Wecker., Nature, 237, NB, 15-17 (1972).
tsu, K., Tominaga, A. & Hamaoka, T., J.Immunol., 1224,24
14-2422 (1980) .Takatsu, K, Tanaka, K., Tominaga, A. et
al., J. Immunol., 125, 2646-2653 (1980) .Nakanishi, K. e.
etal., J. Immunol., 130, 2219-2224 (1983).). TR
Research and development on F is supported by a number of purification or genetic engineering techniques that have been prepared to obtain various interleukins (Muraguchi, A. et al., J.
Immunol., 127, 412-416 (1981) .Takatsu, K., Harada, N., H
ara, T. etal., J. Immunol., 134, 382-389 (1985).
i, T. et al., Nature, 302, 305-310 (1983); Fung, MCet a
l., Nature, 307,233-237 (1984) .Yokota, T., Arai, K.et a
l.Proc.Natl.Acad.Sci.USA, 83,5894-5898 (Aug, 1986) .To
minaga, A., Takatsu, T.et al., Microbiol.Immunol., 30,7
89-798, (Aug., 1986), Noma, Y., Siders, P. et al., Natur.
e, 319, 640-646, (Feb., 1986), etc.). For example, murine T cell fusion cell B151K12 (Takatsu, K. et al.,
Murine TRF secreted from J. Immunol., 125, 2646-2653 (1980) has the following two features. (1) BC
I by L ₁ leukemic B cell line (obtained from University of Texas)
Induction of gM secretion and induction of IgG class anti-DNP antibody production response in vitro by B cells primed with dinitrophenyl (DNP) -ovalbumin (OA) complex. (Takatsu, K., Tanaka, K., Tominaga, A. et al.,
J. Immunol., 125, 2646-2653 (1980) .Takatsu, K., Harada,
N., Hara, T. et al., J. Immunol., 134, 382-389 (1985).
rada, N., Kikuchi, T., Tominaga, A. et al., J. Immunol., 1
34, 3944-3951 (1985). (2) Proliferation activity of BCL ₁ leukemia B cell line. (1) TRF activity, (2) B cell growth factor type II (BCG
Although it may be distinguished from FII) activity in the present specification, since TRF has the activities shown in the above (1) and (2), the activities of the above (1) and (2) are collectively referred to as TRF Let's call it activity. Rat TRF has a molecular weight in the absence of a reducing agent
45,000-60,000 (on gel filtration). Isoelectric point (pI value)
4.7-4.9, B cell stimulating factor 1 (BSF-1), interleukin 2 (IL-2), interleukin 3 (I
L-3), which differed from immune interferon activity (Takatsu, K., Harada, N., Hara, T. et al., J. Immunol).
l., 134,382-389 (1985) .Harada, N., Kikuchi, T., Tominag
a, A. et al., J. Immunol., 134, 3944-3951 (1985))
. It is also reported that the TRF activity has a function of promoting the proliferation and differentiation of eosinophils, and is not restricted to the activities of (1) and (2) described above (Sanderson, C. et al.).
J., et al., Proc. Natl. Acad. Sci. USA, 83, 437 (1985)). On the other hand, regarding TRF other than mice, sheep T
Report on RF (Schimpl, A. and E. Wecker., Nature,
237, NB, 15-17 (1972)) and reports on human TRF (Mura
guchi, A. et al., J. Immunol., 127, 412-416 (1981).
M. et al., J. Exp. Med., 157, 583-590 (1983); Mayer, L. et a.
l., Immunol.Rev., 78,119-135, (1984) .Okada, M.et al.,
J. Immunol., 136, 1288-1294, (1986)). In particular, it has been reported that murine TRF and human TRF are equivalent (Hirano, T. et al., Proc. Natl. Acad. Sci. U.
SA, 82, 5490-5494 (1985)). Recently, it has been proposed to refer to TRF as interleukin 5 (IL-5).
However, in this specification, it is unified as TRF.

[0005]

In the prior art, since there was no anti-TRF monoclonal antibody, TR
It was difficult to purify, measure, analyze or control inhibition of F. Moreover, a widespread i
in vivo over n vivo or in vitro
Neither could monoclonal antibodies be used in vo. The present invention has been made in view of such problems,
It is an object of the present invention to provide a monoclonal antibody that can be widely used for TRF.

[0006]

The monoclonal antibody of the present invention is an antibody obtained by introducing an antigenic TRF (IL-5) into a mouse as a non-human animal. Demonstrates specific binding to the antigenic moiety, but IL-1, IL-2, IL-3 and BSF-1
Is characterized by exhibiting no binding property. Here, it is more effective that the heterologous TRF includes TRF obtained from animals of different origins. In addition, heterogeneous TRF
More effectively corresponds to the TRF secreted by each of a plurality of cell lines from different sources.

[0007] The monoclonal antibody of the present invention is, for example,
It is produced by culturing the fused cells in vivo and in vitro. Fusion cells can be obtained, for example, by using a spleen cell obtained by immunizing a mammal with TRF and a mouse bone marrow cell, using a cell fusion technique of Keller and Milstein (KOhlerand Milstein, N.K.).
, 256, 495-497 (1975).). The animal from which the TRF used for immunization is derived and the animal to be immunized may be any combination that produces antibodies by an immune response. A clone of a cell that has become capable of producing an antibody against TRF through an immune response is advantageous for stably producing an antibody against TRF. The specificity of the antibody for TRF can result in a monoclonal antibody when the degree of purification of TRF used for immunization is high.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described based on embodiments. Example 1 Preparation of TRF T cell hybridoma B151K12 cell line (Taka
tsu, K. et at., J. Immunol., 125 , 2646-2653 (1980)
PMA (4β-phorbol 12β-myristate 12α)
-acetate (manufactured by Sigma) was serum-free cultured in 3 ng / ml for 48 hours to prepare 225 l of a culture supernatant containing TRF. Next, ammonium sulfate fractionation, anion exchange chromatography (DEAE-cellulose), blue sepharose column chromatography, mono-P column (pharmacia)
Chromatograph, Protein C4 (Vydac)
TRF whose specific activity increased by 1.4 million times by gel chromatography using reverse phase HPLC (manufactured by LKB)
I got Incidentally, the TRF activity was measured using ICL using BCL ₁ cells.
gM plaque forming cell test (hereinafter referred to as PFC test)
Was measured.

[0009] The IgM PFC test was performed in the following procedure. 50 to 100 μl of the cultured BCL ₁ cell suspension, 50 μl of a protein A-conjugated sheep red blood cell 10% suspension and 400 μl of agarose 0.5% solution containing MEM medium were mixed at 45 ° C.
It was poured on a slide glass and left at room temperature to gel.
Anti-mouse IgM serum was added to the 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 NPIgGPFC test was performed in the same manner using 50 to 100 μl of the B cell suspension stimulated with the DNP-OA complex. (Because B cells secrete IgG, an anti-mouse IgG serum is added instead of the anti-mouse IgM serum described above.) TR
F activity was determined by half-maximal response when purified TRF was used.
Unit per culture solution, with the amount giving es as 1 unit (U)
(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. Wistar rats were further purified with 2 × 10 ⁵ U of purified TRF every 2 days for 20 days.
Boosted. Three days after the final immunization, spleen cells were removed from Wistar rats, and spleen cells were prepared by the following procedure.

[0010] The spleen was placed in 5 ml of Eagle's MEM culture.
Next, 4 ml of the above culture solution was taken in a 10 ml syringe and injected into the spleen. Splenocytes were loosened using forceps. Thereafter, the cell suspension was placed in the syringe and taken out of the syringe three times to reduce the cell mass. Take out singulated cells
It was centrifuged at 1000-1200 rpm and washed. Wash the washed cells with Ea
The cells were suspended in 2 ml of gle's MEM culture solution. Cell fusion Rat spleen cells and mouse myeloma cells P3 X63-Ag8.653 (ATCC accession number CRL-
1580) and Koehler and Milstein's cell fusion technology.

After mixing 6 × 10 ³ prepared spleen cells and ³ × 10 ³ mouse myeloma cells P3 X63-Ag8.6533 × 10 ³ cells, the cells were pelleted by centrifugation. Add 50% PEG- to the pellet.
1 ml of RPMI-1640 culture solution containing 1500 was gradually added with stirring. Next, the RPMI-1640 culture solution was gradually added and diluted with stirring. Then remove the supernatant by centrifugation and remove 10%
1 × 10 ⁶ cells / RPMI-1640 culture solution containing FCS
1 ml / ml in a 24-well microplate.
Each well was dispensed. The next day, 500 μl / well of HAT medium was added. Thereafter, the same operation was repeated every two days, and the cells were cultured for 14 days. In addition, replacing HAT medium with hypoxanthine,
The HT medium containing thymidine was replaced every two days by 500 μl / well. After 7 to 10 days, the culture medium was further replaced with an HT medium and the culture medium of RPMI-1640 containing 10% FCS was exchanged every two days with 500 μl / well. Screening Anti-T of the hybridoma culture supernatant obtained by the above cell fusion
RF activity was determined by TRF activity inhibition and TRF activity absorption tests. TRF activity inhibition test was performed by measuring the IgMPFC 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 BCL1 cells in 0.2 ml of a culture solution containing TRF2U and anti-TRF monoclonal antibody, and adding 0.2 μCi / well of ³ H-thymidine 6 hours before the end of the culture. did. 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 on which a rabbit anti-rat Ig antibody had been adsorbed and incubated at 37 ° C. for 4 hours. After washing, a certain amount of TRF was added and the mixture was further incubated at 37 ° C. for 4 hours. .
The residual TRF activity of the supernatant was measured. As a result, 61 out of 479 wells containing hybridomas had anti-TR
F activity was exhibited. Further, one well showing reproducible inhibition and absorption activities was selected from the 61 wells. Cloning The hybridoma contained in one selected well was cloned by the limiting dilution method, and hybridomas TB13 and NC were used.
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 the hybridoma TB13 was applied to an affinity column using a goat anti-rat IgG antibody-bound agarose. The adsorbed fraction was eluted with a borate buffer containing 3 MKSCN at pH = 8.0 to purify the anti-TRF monoclonal antibody TB13. In addition, mass purification was performed by the following operation. After injecting 131 × 10 ⁷ hybridomas TB into the abdominal cavity of BALB / Cnu / nu mice, about 15
After about 20 days, ascites was collected, and an IgG fraction was obtained by gel filtration using reverse phase HPLC to purify the anti-TRF monoclonal antibody TB13.

The anti-TRF monoclonal antibody NC17 was purified in the same manner using 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. Molecules The molecular weights of the anti-TRF monoclonal antibodies TB13 and NC17 are shown in Table 1 below.

[0015]

[Table 1]

[0016] 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 was BSF-1,
It does not inhibit IL-1, IL-2, IL-3 activities,
Only activity was inhibited.

[0017]

[Table 2]

Notes: a) BCL for TRF (2U / ml)
The response of _one cell was determined by the number of IgMPFC cells after two days of culture. b) was measured with a radiation uptake of ³ H-thymidine ⁽³ H-thymidine) after 3 days culture. c) The incorporation of ³ H-thymidine was measured by radiation dose after one day of culture.
PC61 (obtained from Swiss Cancer Institute) (Lowenthal, JW, Z
ubler, R, H., Nabholz, M. & MacDonald, RH, Nature (Lond
on), 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, 333-33
6. (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 was obtained from T cell line GY-1 (Takatsu, K., Harada, N., Hara, T., et.
al., J. Immunol., 134, 382-389 (1985).
Inhibits the proliferative response. At a 30,000-fold dilution, 18F10 inhibits proliferation of resting B cells by BSF-1 in the presence of anti-IgM antibodies. Examination of specificity is performed by BC using TRF.
L ₁ cells IgM secretion, resting B cell stimulatory effects of BSF-1 + anti-IgM antibody (Ohara, J. & Paul, WE, Nature
(London), 315, 333-336 (1985).), IL-1 + PH
A stimulates thymocytes (Mizel, SBImmunol. Rev., 6
3, 51-72 (1979)), proliferation of GY-1 cells by IL-2, stimulation of T cell line FDCP-1 by IL-3 (Kikuchi, Y., Kato, R., Sano, Y. , Takahashi, H., et a
l., J. Immunol., 136, 3553-3560 (1986).) was tested to determine whether various monoclonal antibodies inhibit it. TR obtained from titer T cell hybridoma B151K12
F is to differentiate BCL ₁ cells into IgM secreting cells, because it has the effect of enhancing the growth, igmp with BCL ₁ cells
An FC test and a proliferation test were performed.

FIGS. 1A and 1B are graphs showing the ability to inhibit TRF activity when anti-TRF monoclonal antibodies TB13 and NC17 are added to a BCL ₁ cell culture solution. Exam is in viv
o BCL ₁ cells grown in the line 1.5 × 10 ⁵ cells / 0.2ml
Was incubated with TRF2U in 200 μl of culture containing various concentrations of anti-TRF monoclonal antibody TB13 or NC17 or anti-BSF-1 monoclonal antibody 18F10.

FIG. 1A shows reverse IgMPFC after 2 days of culture.
The number of IgM producing cells was determined by the test. In the figure, the symbol ▼ is IgMPFC measurements in the medium cultured only BCL ₁ cells, ○ is measured at TRF addition, △ is TRF + TB13
, T: TRF + NC17, □: T
The value measured with RF + 18F10 (in vivo cultured ascites extraction), and the symbol 表 す represents the value measured with TRF + TB13 (in vivo cultured ascites extraction). In FIG. 1B, 3H thymidine incorporation was measured after 2 days of culture. In the figure, the symbol ▼ is B
Radiation measurement value when cultured CL ₁ cells alone, is ○ T
Measured value at RF addition, Δ represents measured value at TRF + TB13, ● represents measured value at TRF + NC17, □ represents TRF + 1
8F10 (in vivo culture ascites extraction) represents the measured value, respectively.

[0021] The anti-BSF-1 monoclonal antibody 18F10 is IgM of rat IgG ₁ class, which was carried out for the purpose of comparison
It had no secretion inhibition (FIG. 1A) and no inhibition of BCL1 cell growth (FIG. 1B). In addition, the anti-TRF monoclonal antibodies TB13 and NC17 both had a strong IgM of 40 ng.
Inhibition of secretion (FIG. 1A) and growth inhibition of BCL ₁ cells (FIG. 1B) were shown.

Next, the anti-TRF monoclonal antibody TB1
3, It was examined whether NC17 inhibits the anti-DNP IgG production response induced by TRF. B cells stimulated with the DNP-OA conjugate were tested for TRF by anti-DNP IgGGPFC test.
Shows 98PFC / culture without addition, purified TRF3U / ml
The addition showed 638 PFC / culture. When 50 ng / ml of the anti-TRF monoclonal antibody TB13 was added on the first day of culture, the anti-DNP IgGGPFC test result was 126 PFC / culture, which clearly inhibited the anti-DNP IgG production response. N
Similar results were obtained with C17. This indicates that the anti-TRF monoclonal antibodies TB13 and NC17 inhibit TRF activity in experiments 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 activated with bromine cyanide-activated Sepharose CL-4B
Those bound to 3 g were used as immunosorbents.

Reverse phase HPLC (Parmacia) An effluent obtained by flowing 10 ml (1.2 × 10 ⁴ U sample) of the culture supernatant of the hybridoma cell B151K12 obtained by gel filtration through the affinity column (3 ml packed volume) was collected. Thereafter, the affinity column was washed with 50 ml of 1M Nacl solution, 100 ml of PBS and 20 ml of water. Thereafter, 20 ml of a 0.8 M acetic acid solution was flowed to elute the TRF bound to the affinity column. The eluate was freeze-dried and redissolved in 0.8 ml of water. Incidentally, TRF activity was determined by IgMPFC test using BCL ₁ cells. Table 3 shows the measurement results.

[0024]

[Table 3]

As can be seen from Table 3, as a result of applying the roughly purified hybridoma B151K12-producing TRF to the affinity column, 138 U of TRF was eluted.
10.8 × 10 ³ U of TRF eluted with the M acetic acid solution. Normal rat Ig was used as a control column in Sepharose CL.
A similar test was conducted using the sample bound to -4B, and as a result, all TRFs were discharged. Application of TRF to physicochemical properties elucidation Purified B151K12-produced TRF1 by chloramine T method
× 10 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 was
RF monoclonal antibody was bound to TB13-bound CL-4B beads. CL-4B ¹ on the beads ²⁵ I-labeled TRF
The complex of the anti-TRF monoclonal antibody TB13 and
-The presence of mercaptoethanol (hereinafter referred to as 2ME)
¹²⁵ I-labeled TRF was dissociated with a buffer containing 2% SDS in (+) and in the absence (-). S in the eluted fraction
DS-PAGE was performed. ¹²⁵ I-labeled TRF was detected by autoradiography. FIG. 3A shows the result.

In the figure, number 1 is in the absence of 2ME (−2M
The autoradiograph of the fraction from which ¹²⁵ I-labeled TRF was dissociated from the CL-4B beads in E) is shown. Number 2 is -2M
E is anti-TRF monoclonal antibody TB13-bound CL-4
6 shows an autoradiograph when normal rat Ig-bound CL-4B beads were used instead of B beads. Number 3 is 2
The autoradiograph of the fraction from which ¹²⁵ I-labeled TRF was dissociated from anti-TRF monoclonal antibody TB13-bound CL-4B beads in the presence of ME (+ 2ME) is shown. Number 4
Shows an autoradiograph using + 2ME and normal rat Ig-bound CL-4B beads.

The gel after SDS-PAGE of the fraction eluted from the CL-4B beads conjugated with anti-TRF monoclonal antibody TB13 under -2ME was sliced every 2 mm along the direction of electrophoresis, and the substances contained in each sliced gel Was eluted electrophoretically. The TRF activity of the eluate was determined using IgMP.
Measured by FC test. FIG. 3B shows the result. FIG. 3A
From the results, the number 1 and the number 2 shown as a control clearly show that the main fraction had a molecular weight of 44,000 to 48,000 under non-reducing conditions. No. 3,4, molecular weight 44,000-48,00 under reduction
The main fraction at 0 (under -2ME) has a molecular weight of 23,000 to 26,000
(+2 ME), it was confirmed that TRF was a dimer of a substance having a molecular weight of 23,000 to 26,000. Moreover, in FIG. 3B, the molecular weight is 44,000 to 48,000, particularly
Strong TRF activity was observed in 6,000 fractions, and the anti-TRF monoclonal antibody TB13 had a molecular weight of 4 with TRF activity.
It was demonstrated to specifically bind to 4,000 to 48,000 proteins.

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 were tested at 50 ng / ml.
Despite significantly inhibiting RF activity, IL-1,
It does not inhibit the activity of each lymphokine of IL-2, IL-3 and BSF-1 even at a concentration of 10 μg / ml. From the above results,
TRF was found to be different from BSF-1, IL-1, IL-2 and IL-3 and to act early. This is a previous report (Takatsu, K., et al., J. Immunol) showing that the TRF preparation has no interferon activity.
l., 134, 382-389 (1985).) indicates that TRF is a unique lymphokine. Example 4 The TRF used in the previous examples was 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. It was examined whether the activity of the recombinant TRF produced by the genetic modification technique was inhibited.

Recently, the present inventors have proposed a cDN having TRF activity.
A was successfully isolated. Isolated PSP6K-mTRF2
3SalITo obtain a linear plasmid DNA.
Here, the PSP6K-mTRF23 is the same as the cell line described above.
Cell line with a different origin from B151K12 2.19
Is a recombinant product of TRF derived from P. eta (Sideras, P. eta
l., Eur.J.Immunol., 15,586-593 (1985) .Noma, Y., Sidera
s, P. et al., Nature, 319, 640-646, (see Feb., 1986).
Next, mRNA is synthesized using SP6 RNA polymerase.
Was. Xenopus oocytes (
Xenopusoocytes) and incubate at 20 ℃ for 36 hours.
I did it. Recombinant TRF (rTRF), or oocyte
Produced by translating PSP6K-mTRF23 into cells
Is the BCL₁Cell proliferation and differentiation into IgM secreting cells
Stimulates. Anti-TRF module for TRF activity of rTRF
To examine the inhibitory effect of the noclonal antibody NC17
In the presence of anti-TRF monoclonal antibody NC17,
1.5 x 10 BCL1 cells in the absence of ^Five/ 0.2ml rTR
Cultured with F. After 2 days of culture, using protein A
IgMPFC test to determine the number of IgM secreting cells
Was. The results are shown in FIG.

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 an IgMP obtained by culturing only BCL1 cells.
FC measured value, ○: measured value with addition of rTRF (1 U / ml),
● indicates the measured value with rTRF (3U / ml) added, □ indicates rTRF
(1U / ml) + Measured value with addition of NC17, ■ indicates rTRF (
3U / ml) and the measured value when NC17 was added are shown.

FIG. 2 clearly shows that the anti-TRF monoclonal antibody NC17 inhibits the TRF activity of rTRF depending on the amount added. Similarly, an anti-DNPIgGPFC test was performed to examine whether the anti-TRF monoclonal antibody NC17 inhibits rTRF-induced IgG secretion. DNP-
B cells stimulated with the OA complex had 16 cells without rTRF.
9 PFC / culture, 10 rFRF at 3 U / ml addition
03 PFC / culture was indicated. The anti-TRF monoclonal antibody NC171 μg / ml was added on the first day of culture to give 328 PF.
C / culture and clearly inhibited anti-DNP IgG secretion.

The above results indicate that the anti-TRF monoclonal antibody NC17 has a TR
It shows that it specifically binds to F and inhibits its activity with good reproducibility. As mentioned above, the monoclonal antibodies of the present invention can react with TRF from cells of all origins having TRF activity. In addition, the fact that the monoclonal antibody of the present invention is an inhibitor of TRF activity makes it possible to examine a wide range of in vitro and in vivo immune responses, so that the role of TRF will become apparent.

[0033]

The monoclonal antibody of the present invention has a TRF
It can be used as a wide range of antibodies.

[Brief description of the drawings]

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

FIG. 2 shows anti-TRF monoclonal antibody NC17.
Is T of recombinant TRF manufactured by genetic engineering 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.
It is a graph which shows activity ability.

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/577 G01N 33/577 B (C12P 21/08 C12R 1:91)

Claims (3)

[Claims] 1. An antibody obtained by introducing TRF (IL-5) having antigenicity into a mouse as a non-human animal,
Shows specific binding to the antigen portion of different TRFs, but does not include IL-1, IL-2, IL-3 and BS
A monoclonal antibody having no binding to F-1. 2. The monoclonal antibody according to claim 1, wherein the heterologous TRF comprises TRF obtained from an animal of a different origin. 3. The monoclonal antibody according to claim 1, wherein the heterologous TRF corresponds to a TRF secreted by each of a plurality of cell lines obtained from different sources.