JPH0458882A - Production of hybridoma cell for producing monoclonal antibody against human prolactin and production of same monoclonal antibody - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to hybridoma cells that produce monoclonal antibodies that react extremely specifically to human prolactin. The present invention also relates to a method of producing a monoclonal antibody that reacts extremely specifically to human prolactin by culturing the above hybridoma cells.

(Prior Art) Generally, mammalian prolactins have similar properties. For example, human prolactin and mouse prolactin are structurally similar. For this reason, it has been difficult to obtain antibodies that specifically react with these. That is,
Even if animals are immunized with natural human prolactin obtained from humans or natural mouse prolactin obtained from mice, the antibodies obtained are polyclonal, and monoclonal antibodies cannot be obtained. There wasn't.

Therefore, a monoclonal antibody that specifically reacts with human prolactin has not been obtained so far.

On the other hand, monoclonal antibodies against human prolactin are known as CI VANY IJ, a
n dDAVIES P,, Protides o
f Biological Fluids, 29.8
55-860. (1981) :)o The human prolactin treated here is natural human prolactin, and has been thought to be a polypeptide having the amino acid sequence shown in Table 2.

However, the monoclonal antibody against Dobhooktin, which consists of the amino acid sequence shown in Table 2, is made from natural human prolactin, and since it is difficult to obtain natural human prolactin, it is difficult to obtain it. The disadvantage was that it was not easy to produce.

(Problems to be Solved by the Invention) The present invention was made in an attempt to easily produce monoclonal antibodies that react extremely specifically to human prolactin.

(Means for solving the problem) When investigating monoclonal antibodies against human prolactin, until now only natural human prolactin has been considered. However, the inventor attempted to use recombinant human prolactin obtained through genetic recombination to produce a monoclonal antibody.

That is, an attempt was made to obtain a monoclonal antibody using human prolactin obtained by producing E. coli through genetic recombination. As a result, the inventor found that monoclonal antibodies with excellent antigen specificity can be easily obtained using recombinant human prolactin. This invention was made based on such knowledge.

Natural human prolactin has the amino acid sequence shown in Table 2, as described above. In contrast, recombinant human prolactin has the amino acid sequence shown in Table 1. The difference in the amino acid sequences between Table 1 and Table 2 is the presence or absence of Met, ie, methionine, at the beginning.

This invention is already known except that a recombinant type human prolactin is used instead of a natural type, and this is used for the production of clonal antibodies. We plan to follow the method. That is, immunizing mice with human prolactin and fusing mouse splenocytes thus obtained with myeloma cells to produce hybridoma cells, and culturing these hybridoma cells to produce clonal antibodies. There is no difference from the conventional method.

(Summary of the Invention) This invention relates to hybridoma cells in a monoclonal antibody-producing layer against human prolactin produced by fusing mouse splenocytes immunized with human prolactin having the amino acid sequence shown in Table 1 and myeloma cells. The main points are as follows.

The present invention also relates to a method for producing a monoclonal antibody against human prolactin by culturing the above hybridoma cells.

(Description of each requirement) In this invention, human prolactin (hereinafter simply referred to as 1st BP) consisting of the amino acid sequence shown in Table 1 is used. The first HP can be obtained for the first time by introducing DNA artificially produced by genetic recombination into, for example, E. coli and culturing the E. coli.

The first HP has a different structure from human prolactin obtained from the human body. Human floractin obtained from the human body has the amino acid sequence shown in Table 2, and compared to the first HP, it lacks MeL, methionine, at the initial position.

A method for obtaining the first HP is disclosed in Japanese Patent Application Laid-Open No. 2-445. Looking at Table 1 of this bulletin, 158-75
4 shows the ring group sequence of TTGOCCA...AACTGC, and immediately below it is shown the exact same amino acid sequence as shown in Table 2.

Looking at the example, it is clear that the first HP can be obtained.

The method for immunizing with the first UP follows a known method. That is, for example, 8-10 week old BALB/c mice are injected subcutaneously or intraperitoneally with an appropriate adjuvant (for example, complete Freund's adjuvant).
und's adj uvant)) with 1st
Administer HP 20-100 μ7/mouse. A further booster immunization is given two weeks later. Two days thereafter, the anti-prolactin antibody titer in the serum is examined by solid-phase enzyme immunoassay as described below.

The enzyme immunoassay method is as follows. First, 96 holes EI
PBS (7,
2mM> 71! Dispense 100 μl/well of prolactin dissolved in 2.8 mM monosodium phosphate, pH 7.2) and leave it at room temperature for 1 hour to adsorb the antigen. After washing 3 times with PBS, .5%BSA
, 200μ of 0.05% sodium azide/PBS solution
Dispense 1/well and leave at room temperature for 1 hour to coat the plate. After washing three times with 0.05% Tween-20/PBS, the antibody appropriately diluted with 0.05% Tw6en-20/PBS is dispensed at 100 μj/well and left at room temperature for 1 hour. 0.05% Tween-20/PB
After washing three times with S, 0.05% Tween-20/P
Dispense 100 #/well of peroxidase-labeled antibody diluted appropriately with BS and leave at room temperature for 1 hour. 0
After washing 3 times with 405% Tween-20/PBS, OP
D substrate solution (25mM of O-phenylenediamine 10m2)
Citric acid, dissolved in 25rnl of 50mM sodium phosphate, to which 10μj of hydrogen peroxide was added) was added to 100μl/
Dispense into each hole, leave at room temperature for 30 minutes, and add 2M sulfuric acid to 50%
+! ! Dispense the solution into each hole to stop the reaction, and measure the absorbance at 492 nm.

Known myeloma cells are used. It is e.g. P 3-X 63-Ag 8-u obtained from mouse
ICP3-Ul) (Curr, Top, Microbi
ol, Immunol, 81, 1.1978) and P
3-NSI-1-Ag4-ICNS-1) (Europ
ean J, Immunol, 6,511°197
6). These cell lines are cultured in a medium containing 8-azaguanine and returned to normal medium 2 to 3 days before cell fusion.

Cell fusion is also performed according to known methods. First, the spleen is removed from the immunized mouse described above, and splenocytes are prepared. The splenocytes and myeloma cells were mixed at a ratio of 10:1,
Fuse with polyethylene glycol, etc. After cell fusion, H containing hyboxanthin, aminopterin, and thymidine
Cultivate in AT medium to select fused cells. The medium is changed every 2 to 3 days, and when cell proliferation is observed after 10 to 14 days, the medium is changed to HT medium containing hypoxanthine and aminopterin.

Thereafter, culture supernatants are taken from wells in which hybridomas have grown in the form of colonies, and the antibody titer against human prolactin is examined by the enzyme immunoassay method described above. Cells in wells with high antibody titers are cloned by limiting dilution to obtain hybridoma cell lines for producing anti-human prolactin monoclonal antibodies.

According to the present invention, monoclonal antibodies against human prolactin can be obtained by culturing and proliferating the hybridoma cells obtained above and secreting them from the m-cells. In vitro for culture.

There are two methods.

The in vitro method uses R containing fetal bovine serum.
A novel hybridoma for producing an anti-human prolactin monoclonal antibody is cultured in a medium such as PMI 1640 or DMEM at a cell concentration of approximately 1×10 Ce11S/lne at 87°C in a CO2 incubator. 2. After culturing for 3 days and when the cells have multiplied 5-10 times, the culture solution is centrifuged to obtain monoclonal antibodies.

In the in vivo method, B
The anti-human prolactin monoclonal antibody-producing hybridoma cells obtained by the above method were intraperitoneally injected into ALB/c mice at 2-5×10 cells/mouse, and 1-
Two weeks later, ascites fluid containing monoclonal antibodies is collected.

This ascites is centrifuged to remove solid matter and obtain monoclonal antibodies.

When a highly pure monoclonal antibody is required, for example, in the case of immunoglobulin IgG class, a crude monoclonal antibody can be obtained by salting out ammonium sulfate, collecting the precipitate, and dialysis. For further purification, DEAE-
Apply to a cellulose column, 5ephacry IS-300 column, etc.

(Effects of the Invention) According to the present invention, since mouse splenocytes immunized with human prolactin having the amino acid sequence shown in Table 1 are used, hybridoma cells obtained by fusing these with myeloma cells are human It has the ability to produce monoclonal antibodies that are highly specific for prolactin. Therefore, these hybridoma cells are useful for producing highly specific monoclonal antibodies.

Further, according to the present invention, a method for obtaining a monoclonal antibody by culturing the above-mentioned hybridoma cells is provided, thereby making it possible to obtain a monoclonal antibody with strong specificity for human prolactin. Therefore, it is useful in detecting human prolactin.

Examples are given below to explain the advantages of this invention.

Example 1 In this example, a hybridoma cell line for producing an anti-human prolactin monoclonal antibody was obtained as described below.

1) Preparation of immunized mouse splenocytes
Adjustments were made according to the description in the publication. The human prolactin thus obtained was mixed with Freund's complete adjuvant at 8%
It was administered subcutaneously to week-old female BALB/C mice. Two weeks later, 100 horses of human prolactin shown in Table 1 were administered subcutaneously together with Freund's incomplete adjuvant. Three weeks later, 100n of human prolactin shown in Table 1 was further administered intravenously.

Thereafter, two days later, the antibody titer against human prolactin in the mouse serum was examined by the enzyme immunoassay method described above, and it was found that the antibody titer was sufficiently high.

One day later, lung cells were removed from the mice. The excised lung cells were ground with a glass slide, passed through a stainless steel mesh to form single cells, and then placed in Trrs buffer.
Lyse the red blood cells with (p)I 7.2), then HANK
It was washed with S solution (manufactured by Tamizu Seiyaku Co., Ltd.).

2) Preparation of mouse myeloma cells (P3-Ul) 8-Azaguanine-resistant mouse myeloma cells (P3-Ul) were added to a growth medium (RP old 1640 (manufactured by Tamizu Pharmaceutical Co., Ltd.) with Na)Ic.
O:+28/j! , sodium pyruvate 0.11
g#! , penicillin 31.25B/jL streptomycin 50.0 mg/j2-gentamicin 154. O
mg/j2.2-mercaptoethanol 2.5X10
-'M and 10.0% fetal bovine serum] (37°C, CO, incubator), HANKS
Mouse myeloma cells were obtained by washing with solution.

3) 1X10' immunized mouse splenocytes obtained in the hybridoma production and 1X10' mouse myeloma cells were mixed, centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed.

After thoroughly loosening the mixture of immunized mouse splenocytes and mouse myeloma cells obtained as a precipitate, 45% polyethylene glycol 1d was gradually added over 1 minute.
Mix well. Thereafter, 20 ml of HANKS solution was gradually added over 5 minutes, centrifuged at 1500 rpm for 5 minutes to remove the supernatant, and a growth medium was added to suspend the cells so that the cell concentration was 5 x 10' cells/d.

96-well culture plate 1- (manufactured by FALCON) 20
Dispense 0uf/well and culture (37°C, Co.

After 1 and 3 days of using HAT medium (I
100μ! added. After 2 days, add 100μ of culture solution.
! was discarded from each well and 100 μl of HT medium (growth medium containing IXIO-'M hypoxanthin, 1.6 x 10 M thymidine) was added to each well. added. After 2 days, culture solution 10
0 μp was discarded from each well and 100 pl of growth medium was added. In this way, hybridoma cells were obtained.

Three days later, the antibody titer against human prolactin in the medium supernatant was examined by the enzyme immunoassay described above. Cells in wells containing antibody-producing hybridoma cells were cloned twice by limiting dilution method to obtain two types of cell lines A31-ASCL-1 as anti-human prolactin monoclonal antibody-producing hybridoma cells.

Example 2 In this example, the hybridoma cells obtained in Example 1 were
The monoclonal antibodies produced in the culture supernatant were cultured in vitro, and the properties of the monoclonal antibodies produced in the culture supernatant were determined as follows.

Each type of mouse immunoglobulin (IgA, 1gM,
The isotype was determined by enzyme immunoassay using a peroxidase-labeled second antibody (manufactured by ZymeL) specific to rg (, IgGza, IgGzb, TgG2). As a result, it was found that both A31-ASCl-1 produced antibodies classified as IgG.

Example 3 In this example, the antigen specificity of the obtained monoclonal antibody was investigated.

The measurement was performed by the enzyme immunoassay method using the above-mentioned phase method. As antigens, rrPRL produced by Escherichia coli according to the method described in JP-A-2-445) and mouse prolactin (rmPRL) (manufactured by Shikishimabo Co., Ltd.)
Natural human prolactin (hPRL) (CALBI
OChem) and natural ovine prolactin (oP
RL) (manufactured by Sigma) was used.

Examples of antibodies include monoclonal antibodies produced according to the present invention, human prolactin antiserum (manufactured by Oriental Yeast Co., Ltd.), and rat prolactin antiserum (UCB Bio
products, Inc.) was used. Note that the human prolactin antiserum and raft prolactin antiserum are polyclonal antibodies.

The results were as shown in Table 3 below.

Here, + indicates positive, that is, reacting, and - indicates negative, that is, not reacting.

According to Table 1, A31 and 0 were obtained by this invention.
1-1 was found to have superior antigen specificity compared to other antisera.

Claims (1)

[Claims] 1. A hybridoma cell for producing a monoclonal antibody against human prolactin produced by fusing myeloma cells with mouse splenocytes immunized with human prolactin having the amino acid sequence shown in Table 1. 2. Production of a monoclonal antibody against human prolactin, which is characterized by culturing hybridoma cells produced by fusing mouse splenocytes immunized with human prolactin having the amino acid sequence shown in Table 1 and myeloma cells. Method.