JPH01171495A - Monoclonal antibody and quantitative determination method using said antibody - Google Patents

Abstract

PURPOSE:To perform quantitative determination of a follicle-stimulating hormone releasing protein (FRP) useful for the remedy and diagnosis of climacteric disturbance, by using an antibody obtained by preparing a mouse monoclonal antibody from a specific factor. CONSTITUTION:Human leukocyte is cultured in the presence of a prescribed differentiation-inducing substance to obtain a human differentiation-inducing factor (BUF-3) (A) having the activity to differentiate and mature a mouse leukemia cell to normal cell. A mouse is immunized with the component A and the spleen cell of the mouse is fused with a myeloma cell to obtain a monoclonal antibody (B) having the following properties. Molecular weight, 900,000; class of immunoglobulin, IgM; activity, specifically bonding with BUF-3(FRP); etc. The component B is adsorbed to an insoluble carrier, an FRP having unknown concentration is added to and reacted with the component B and the follicle-stimulating hormone releasing protein is determined by an isotope-labeled immunoassay.

Description

Detailed Description of the Invention <Industrial Application Field> The present invention relates to a mouse monoclonal antibody against human differentiation-inducing factor (hereinafter referred to as BUF-3) and a method for producing follicle-stimulating hormone-releasing protein (hereinafter referred to as FR) using the monoclonal antibody.
(denoted as P).

<Prior art> The present inventors have been conducting research on the human differentiation-inducing factor BUF-3 (Japanese Patent Application No. 15003/1986).
3 and “Human differentiation inducing factor BUF-3J,” published by Ajinomoto Co., Ltd., filed on October 27, 1986. On the other hand, Nature magazine (Nature, 321 y724-72)
5. (1986), Nature, 321.776-
782 (1986)), there is a report on follicle-stimulating hormone releasing protein (hereinafter referred to as FRP), which coincidentally is the same substance as BUF-3.

In other words, the preparation of a monoclonal antibody against 9BUP-3 means the preparation of a monoclonal antibody against FRP, so it is thought that the scope of use will be further expanded as described below.

Follicle stimulating hormone (hereinafter referred to as FSH) released by the action of FRP is an essential hormone along with luteinizing hormone (hereinafter referred to as LH) for guiding oocytes into mature eggs.

Therefore, a contraceptive effect can be expected by keeping the FSH level low. The contraceptives currently in use are steroid-based and affect the central nervous system, so they are bound to have side effects. However, if monoclonal antibodies against FRP can be used, the regulation of FSH levels will work more directly on oocytes. There are fewer worries about side effects.

It has also been found that when excess FSH occurs during menopause, symptoms of menopausal disorder occur.

As described above, if monoclonal antibodies against FRP can be obtained, they can be used for contraception and treatment of menopausal disorders through regulation of FSH levels.

In addition, the method for quantifying FRP using this monoclonal antibody can be more conveniently used in diagnosing infertility and menopausal disorders than conventional methods.

However, since FRP is a human hormone.

It was not possible to obtain it in large quantities and therefore it was difficult to prepare the antibody.

<Problems to be Solved by the Present Invention> The problem to be solved by the present invention is to develop monoclonal antibodies against BUF-3K and BUF-3′t using this monoclonal antibody.
It also provides a method for quantifying FRP.

Means for Solving the Problems〉 As a result of intensive research to solve the above problems, the present inventors have solved the problems of the present invention by noting that FRP (!-BUF-3) are the same substance. Settled.

That is, the present invention was completed by discovering a method for quantifying 9FRP using a monoclonal antibody against BUF-3 in an isotope-labeled immunoassay and a monoclonal antibody against BUF-3K produced by human leukemia cells. .

The present invention will be explained in detail below.

FRP was first extracted from pig follicles and its structure was determined. (Nature, 321.724-725
.

and 776-782 (1986)).

Human FRP also has exactly the same structure as pig FRP.

These are β, -β, and homodimer structures.

Coincidentally, FRP is also the same as the human differentiation factor BUF-3, which is produced by culturing or inducing human leukemia cells in the coexistence of a specific differentiation-inducing substance, whose structure we have previously determined. be.

BUF-3 has the following physical and chemical properties.

That is, (1) Molecular weight: 16 ± 1 kd (in the presence of mercaptoethanol with 1.0 coefficient, 5DS-electrophoresis method) 25 ± 1 kd (in the absence of mercaptoethanol, 5
DS-electrophoresis method) (b) Isoelectric point: pH 6,3 ± 0.2 (chromatofocusing method) pH 7,3 (isoelectric focusing method) (e) pH stability: pH 2,0 to 10 Stable in the range of .0 (d) Thermal stability: Stable when heated at 65℃ for 60 minutes (・)
Organic solvent stability: Stable to lower alcohols and acetonitrile (f) Protease resistance: ! Completely inactivated by lonase treatment.

(g) Specific activity: 2 X I O' U/■ Protein Lys
Amp Ile Gly Trp Asn Asp
Trp Ilm lieAla Pro Ser Gl
y Tyr Hlm Ala Asn Tyr Cys
BUF activity was measured using the known enzyme-labeled immunosorbent assay (hereinafter referred to as EL).
A monoclonal antibody was bound to the antigen adsorbed to the tn7' rate according to the ISA method (abbreviated as ISA method), and this was enzyme-labeled with 2
The antibody activity is measured by reacting with the next antibody and using an enzyme color reaction. Alternatively, a known radioimmunoassay method that labels the antigen with a radioisotope may be used.

Furthermore, if this monoclonal antibody is labeled with an enzyme such as oxidase, alkaline phosphatase, β-galactosidase, etc., the measurement can be simplified since a secondary antibody is not required when used in the ELISA method.

To develop monoclonal antibodies, mice are first immunized with BUF-3 as an antigen according to a conventional method.

The fused cells (hybridoma) obtained by fusing the lung cells of the immunized mouse with myeloma are selected and cloned, and then in vitro or in vivo.
nvlvo), and the culture with 4?4? Collect different monoclonal antibodies.

The monoclonal antibody thus obtained is purified as necessary. The purification method may be a combination of known methods such as salting out, dialysis, ion exchange chromatography, and affinity chromatography. The monoclonal antibody thus obtained has the following properties.

■Type of immunoglobulin: IgM ■Molecular weight: 900,000 ■The obtained monoclonal antibody is specific for K BUF-3
(FRP) binds to protein.

Next, the radioimmunoassay method (hereinafter referred to as R
(abbreviated as IA) BUF-3 or FRP by K (hereinafter BUF-3 or F'RP will be referred to as BUF-3/FR
The method for quantifying P (abbreviated as P) is described below.

First, the above monoclonal antibody is adsorbed onto a solid phase. Here, a certain amount of labeled BUF-3 and an unknown concentration of unlabeled B
When UF-3/FRP is added, competition occurs between the two,
Labeled BUF-3/FRP depending on the concentration of unlabeled BUF-3/FRP
-3 binding to the antibody is reduced, free BUF-3
By measuring the radioactivity after removing the unlabeled BU,
The amount of F-37FRP can be quantified.

Of course, the monoclonal antibody of the present invention can also be used for purification of BUF-3/F'RP by affinity chromatography.

That is, a BUF-3/FRP mixture is added to a gel in which the obtained monoclonal antibody is bound to an insoluble carrier, unbound contaminants are washed away, and then BUF-3/FRP is eluted.

<Effects> By focusing on the fact that F'RP and BUF-3 are the same substance, it was possible to prepare a monoclonal antibody against FRP from BUF-3, which could not be obtained conventionally.

The monoclonal antibody according to the present invention and the method for quantifying FRP content in a sample using this antibody can be used in (1) treatment and diagnosis of menopausal disorders, (2) diagnosis of contraception and infertility.

The present invention will be explained more specifically based on Examples.

<Example 1> 5 RPMI-1640 sterile medium with beef tallow serum 5
．． A 201-volume spinner flask was filled with THP-1 cells, which are human monocytic leukemia cells, in this medium.
The mixture was suspended at a density of 10 pieces/go. This was heated to 37℃ for 4
After culturing for days, the resulting culture solution was centrifuged, and TIP-1
Cells were harvested aseptically. The cells were placed in another spinner flask using the above RPMI-1640 without serum.
Medium 5. Transfer to OL, add 1014/m of TPA to this, and gently stir the liquid (100 r, p, m).
Culture (induction) was performed at 37°C for 2 hours.

The culture fluid thus obtained was centrifuged to separate and remove the cells to obtain a culture fluid with an activity of 20 units/g.

Ammonium sulfate was added to 100 μm of the culture solution obtained in this way (70 μm
the resulting precipitate was centrifuged (10, OOOrp).
m, for 10 minutes) and a small amount of 0.05M)
After dissolving in Lis-hydrochloride buffer (pH 7, 8), it was thoroughly dialyzed against the buffer. DEAE-Toyo-T-#650M (7x
70 cm). Add this column to the same buffer solution 5.
．． After washing with O-01, elution was carried out with the same buffer containing O-0.2M sodium chloride. The differentiation-inducing activity was eluted with 0.1M sodium chloride. This active fraction was collected and saturated with solid ammonium sulfate for 70 scratches, and further ammonium sulfate was precipitated. The precipitate was collected by centrifugation and dissolved in 20-ounces of water. Add 201117 80% saturated ammonium sulfate solution to this solution, and add 201117 80% saturated ammonium sulfate solution
．． The column was loaded onto a Butyl Toyovar 650M column (25 x 30 cIL) pre-equilibrated with 05M Tris-HC1 buffer (pH 7.7). After stepwise lowering the ammonium sulfate concentration, elution was performed with 30 ml of ethanol, and the differentiation-inducing active substance was eluted. FIG. 1 shows the elution curve of hydrophobic chromatography using this butyl-toyol. The active fractions were collected and concentrated under reduced pressure to remove ethanol, and the concentrate was dialyzed against 0.05M) Lys-HC1 buffer (PH7,7)K. The dialysis solution was subjected to filtration using a Su-Mother-41's (Glufiltration applicable column manufactured by Pharmacia) equilibrated with the same buffer solution. The elution pattern after filtration is shown in FIG. As shown in Figure 2, the elution time of the differentiation-inducing active substance for KF5-5 was 56
．． 0 minutes and BUP based on standard protein elution time.
The molecular weight of -3 was calculated to be 10±0.5 kd. This sample was mixed with 0.05M) Lis-HCl buffer (p) 8.
Pharmacia FPLC (Fa@
tProtein, p@ptld*, Polynu
cleotide, LlquldChromatog
raphy) system. Elution is 0.0
Dalasiendo elution of common salt from 5M to 0.1M was performed. BUF-3 activity was eluted at around 0.1M sodium chloride. The purification ratio in this step was approximately 5 times, and the protein was purified to a nearly single protein. Next, add this sample to Highbore RP3
04 (manufactured by Bio-Rad, C-4 reverse phase column) to perform reverse phase high performance liquid chromatography. Ta. Condition is 0
．． 11) Using lifluoroacetic acid as a developing solution, elute by changing the concentration of n,-gulononol linearly from Os to 5O1s.

The elution pattern is shown in FIG. The protein peak shown in FIG. 3 and the activity completely matched. The activity peaks were collected to obtain approximately 100 μg of a purified sample. 5D8-polyacrylamide gel electrophoresis for this sample (concentration: IS, O, i, o* in the presence of mercaptoethanol)
I did it. As a result, a single band (silver staining method) was observed at 16 kd, and no other protein bands were detected. In addition, in the absence of mercaptoethanol, 25 kd
Met.

The specific activity of the sample purified in this way is approximately 2X1
It was 0'U/da protein.

Sample 30 purified single electrophoretically in this way
0μ9 was reduced with 600 μl of dithiothreitol for 6 hours, then pyridylethylated, and 10 μl of the resultant was subjected to Edman degradation sequentially from the N-terminus using an Applied Biosystems gas phase amino acid sequencing analyzer (model 470A).

The liberated phenylthiohydantoin-amino acid,
Analysis was carried out using high performance liquid chromatography (HPLC device 5P8100 manufactured by Spectrophysics, column: Solpax 008 manufactured by DuPont).

As a result, the amino acid sequence from the amine end was as follows.

Gly-L*u-Glu-Cys-Asp-Gly-L
ye-Val-Asn-I 1e-Cys-Cys-L
ya-Lys-Gln-Pha-Pha-Va
1-ao r-Phe-Lye-Asp-II@-Gl
y-Trp-Asn-Asp-Trp-IIs-IIs
-Alm -Pro-8@r-G 1 y-Tyr-M
l 5-Ala-As n-Tyr-Cys-Gl
u-Glya Based on these results, it was confirmed that the poly(peptide) thus obtained was the same substance as the known BUF-3.

Thereafter, BUF-3 purified in this manner was used as an antigen in experiments.

Note that THP-1 used in the present invention is Int, J, Can
c@r.

26.171-176, (1980) K, and was provided by the author of this miscellaneous text. THp-x also supports Proc, Natl, Acad,
Sci, :U,S,A 80 pp 5397-
5401 (1983), Cancer Research
42, 484-489 (1982), and has been distributed to these research institutions.

Furthermore, the inventors are willing to distribute THP-1 to any third parties that may have the rights.

<Example 2> Equal amounts of the purified solution of reduced BUF-3 obtained in Example 1 and Freund's condelete adeppant were mixed and emulsified to form BA.
LB/cAnNcrj? A mouse (female, 4 weeks old, 2 Japan Charles River ■) was immunized by subcutaneously administering BUF-3 at 30 μl/mouse in three doses at one week intervals. After another week, 5μI BUF
A final immunization was carried out by injecting only the -3 solution. Four days after the final immunization, the mice were sacrificed and the pancreas was removed. After shredding this, it was suspended in stainless steel mesh 'IIL Eagle's Minimum Essential Medium (MEM).
A tile cell suspension was obtained.

These tile cells and mouse myeloma cells (X 63-Agg
-6, 5, 3) three times with MEM, mix tile cells and myeloma cells at a ratio of 4:1, and centrifuge (14
00 rpm for 8 minutes). 404 polyethylene glycol 4000, 1096 dimethyl sulfoxide (abbreviated as DMSO) p) 17.2.10μj9 to the obtained precipitate
1 d/ml poly-L-arginine/PBS solution was gradually added and left at room temperature for 1 minute to perform cell fusion.

MEM 2− was then added for 2 minutes, followed by MEM 7
d was added for 3 minutes to make a total of 10ml, and then centrifuged (800ml).
rpm 5 minutes). This precipitate was added to RPMI 1640 medium containing 1OL4 tallow serum (Fe2) 2 x 1.
The cells were suspended at a density of 0' cells/d and planted in a 96-well microplate, each well having an area of 0.2 m. The next day HAT (
Hypoxanthine I X 10-'M, aminopterin 4 X 10-'M.

RPM1164 containing thymidine (1.6 x 10 M)
One drop of 0-101 FC8 medium () EAT medium) was added to each well in 5 gopipets at 0.1 mj. Thereafter, half of the medium was replaced with HAT medium every 3 to 4 days, and growth of hybridoma cells was observed from the whites of the 7th eyes.

System screening was performed using ELISA for antibodies against BUF-3 in the culture supernatant of Hyturidoma cells. The antigen used for screening was purified BUF
-3, the second antibody was alkaline phosphatase-labeled sheep anti-mouse.

7 out of 165 wells were tested by ELISA)
It was positive and produced antibodies against BUF-3.

Next, after the seven strains of antibody-producing cells were grown to approximately 104 cells or more, KH/T medium was added. Then, the medium was replaced with HJT medium three times in total every 3 to 4 days, and thereafter culture was performed using normal RPMI 1640-104FC8 medium. Next, in order to obtain a single cell colony, we added 7 hybridoma cells that showed positive results in the antibody activity test against BUF-3 mentioned above, 3 cells per well of a 96-well microplate (flat bottom), and 1 cell colony. , diluted to 0.5 cells, added BALB/e mouse thymocytes as feeder cells, distributed into plates,
Cultured in RPMI 1640-104 FC8 medium.

Wells that were confirmed to be single cell colonies were selected by observation under a microscope, and hybridoma cells with the highest activity were screened by ELISA and cloned.

A total of 10 clones from 7 Heituridoma cell lines
of the wells were positive by ELISA and BUF
-3 was produced. For example, 2F6 is an example of the Hytridoma obtained in this way.
There is 2G12 (FERMt P-9631).

Next, this 2F62G12 (FEBM P-
9631) was expanded to approximately 10 7 cells and then injected intraperitoneally into BALB/e mice pretreated with pristane.

Centrifuge the ascites fluid collected on the 98th day, and store the supernatant at -80°C.
The monoclonal antibody was obtained by freezing the mixture, thawing it, and centrifuging it for 10,000 rpm 1 () minutes.

The class of the monoclonal antibodies thus obtained was determined by an octarol diffusion test using a class-specific anti-mouse antiserum. the result.

The class of monoclonal antibodies against BUP-3 is Ig.
It was M.

<Example 3> Bur-3 using a monoclonal antibody obtained using BUF-3 as an antigen in the solid phase competition method of ELISA
The quantification of is shown below.

BUF-3 capacitor 5μ7 on 96-hole microtiter grate
50μ of 77MI PBS solution! and left at room temperature for 2 hours. Next K 14 FC8-containing PBS solution 100 μl
Blocked with. Example 2 was placed in a microtiter plate that was separately blocked with a PBS solution containing 1% FC8.
The production culture supernatant of the monoclonal antibody shown in Tve@
BUF-3 diluted 100 times with a phosphate buffer containing n-20 (hereinafter abbreviated as PBS'r solution) and BUF-3 were mixed with PH1Tween to a final concentration of 0.5 to 10all-.
The prepared solution was mixed and allowed to stand at room temperature for 1 hour.

Put this into the above BUF-3 coating plate,
After incubating for 1 hour at room temperature, PBSTv...n
Wash with Psroxidase-labeled sheep anti-mouse antibody (Cappe1, Psroxida
ugated GoatAntl Mous@Immu
noglobal inn(IgA+IgG+IgM
)] was added and reacted for 1 hour. Wash this with a PBS solution and add peroxidase coloring agent (orthophenylenediamine 341119/100mg PBS, 30μ!
3) was reacted with H2O2). After 30 minutes, add 2 4N sulfuric acid.
0μ! In addition, the reaction was stopped, and the absorbance at 492 nm was measured using TiterTetta Multiscan. Reaction is with unlabeled BU
There is a correlation with the concentration of F-3, and the concentration of BUF-3 is 1.25.
Linearity was obtained between ~10 μI/- (Figure 4) <Example 4> Quantification of BUF-3 using a monoclonal antibody obtained using BUF-3 as an antigen in the RIA method is shown below.

The IgM solution of the monoclonal antibody against mouse BUF-3 obtained in Example 2 was appropriately diluted with PBS and dispensed into 96-well microtiter plates in 50 μm portions. After leaving this at room temperature for 2 hours, it was washed four times each with distilled water and physiological saline. Next, 50 µm of a PBS solution containing 196vcA was added and allowed to stand for 1 hour. PBS after reaction
The plate was washed 6 times with 125I-labeled BUF-3 and a certain amount of 1/l of 125I-labeled BUF-3 and unlabeled BUF-30, 3 to 30 μm, and reacted for 1 hour at room temperature. After washing at least 5 times each with physiological saline and distilled water, the 6 holes were dried, cut out with scissors, and placed in a gamma counter test tube to measure radioactivity. The response was correlated with the unlabeled BUF-3 concentration;
Linearity was obtained between F-38 degrees and 0-1250 nl Apl (Figure 5) <Example 5> The monoclonal antibody obtained in Example 2 neutralized the differentiation-inducing activity of BUF-3 against Friend leukemia cells. did.

Friend leukemia cells F5-5 were diluted in Ham'ml 2-104 FC8 medium to 6 x 10' cells/- and plated in 150 μm portions in microtiter plates. Add 50 μl of BUF-3 and 25 μl of the above-mentioned monoclonal antibody appropriately diluted to this, and
The cells were cultured at 7°C and 51 CO2 for 6 days. Staining is 20μ! Dianisidine solution (Dianisidine 600+11910.9m
The reaction was carried out by adding 29.93 Ij acetic acid (29.93 Ij water + 34 H2O2). Cells are stained when they differentiate, so the degree of staining is an indicator of cell differentiation. Now, when the concentration of the monoclonal antibody is 23.4 μm1/ltl or less, Friend leukemia cells F5-5 are sufficiently differentiated and the cells become stained.
With the above, this differentiation-inducing activity was sufficiently neutralized (Fig. 6). <Example 6> Using the monoclonal antibody against mouse BUF-3 obtained in Example 2, BUF-3 was purified as follows. I did it like this.

Afui Glu 10 (BioRad) 2- was placed on a glass filter set in a Puchner funnel and poured with cold DW2 (
After washing three times with double-distilled water), transfer to a test tube,
Add 9 monoclonal antibodies against BUF-3 obtained in 0.1 M NaHC and mix for 4 h at 4 °C.
Wash the glue twice with O3/0.15 M NaCL solution and add 2 TILl of 0.1 M ethanolamine HCL (p
H8) was added and reacted for 1 hour at room temperature. This was packed in a column (2.5 x 3.5 cm) and washed well with rIW2, and then eluted with 77- (0.2N acetic acid, 0.15M NaC2
, 0.1% Trlton X-100 pH 2.5). Furthermore, the start buffer (0,15MNaCL, 0
．． 11TrltonX-100) and BUF-
The 3 mixtures were layered on a column, and after flowing the starting buffer in an amount 5 times the volume of the column, it was eluted with the elution buffer, and the eluted fractions were collected.

Through the above operations, BUF-3Jfi was successfully purified and recovered.

[Brief explanation of the drawing]

FIG. 1 shows four turns of elution of the human differentiation-inducing factor BUF-3 of the present invention in hydrophobic chromatography using butyltoyol. The shaded area indicates the active fraction. FIG. 2 shows the elution sequence of the human differentiation-inducing factor BUF-3 of the present invention in filter filtration chromatography. The shaded area indicates the active fraction. FIG. 3 shows the elution sequence of the human differentiation-inducing factor BUF-3 of the present invention in reversed phase high performance liquid chromatography. Figure 4 shows the quantification of BUF-3 by the solid-phase competition method of ELISA using a mouse monoclonal antibody against the human differentiation factor BUF-3 of the present invention. BU by RIA method using mouse monoclonal antibody against -3
Quantification of F-3. FIG. 6 shows the neutralization rate of the differentiation-inducing activity of BUF-3 by the mouse monoclonal antibody against the human differentiation-inducing factor BUF-3K of the present invention. 'Figure 3 Elution time (min)

Claims (2)

[Claims] (1) Mouse monoclonal antibody against human differentiation factor BUF-3, which has the activity of causing mouse leukemia cells to differentiate and mature into normal cells. (2) A method for quantifying follicle-stimulating hormone-releasing protein, which is characterized in that an isotope-labeled immunoassay method uses a mouse monoclonal antibody against human differentiation-inducing factor BUF-3, which has the activity of causing mouse leukemia cells to differentiate and mature into normal cells.