JPS6042400A - Monoclonal antibody to human igm - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody having specificity to human IgM, and specifically linking ability to chain on the surface of B cell. USE:Capable of dyeing specifically B cell clearly without dyeing background by making it with a fluorescent substance or an enzyme, dyeing tissue. Useful for classification and diagnosis of leukemia such as B cell chronic lymphocytic leukemia of cancerated B cell, differential diagnosis for glomerulonepheritis. PREPARATION:A different animal is immunized against IgM purified from human serum, an antibody-producing cell having the immunization is collected, it is fused with a myeloma cell (preferably cell strain of 8-azaguanine resistance) of different animal. An antibody-producing hybridoma having human IgM resistance (e.g., 2C12 strain, or 3F1 strain) is selected from the prepared hydridoma by enzyme linked immuno sorbent assay, and it is cultivated to give the desired antibody.

Description

[Detailed description of the invention] The present invention relates to a monoclonal antibody against m.m.

In recent years, a method to produce a single clone antibody by fusing antibody-producing cells with bone marrow hepatocytes to create hybridomas (fused cells) and culturing them has become known as a method for obtaining large-scale highly specific antibodies. Ure (Milstein et al., N.
attire, Vol, 256 +p495 (197
5]), a large number of monoclonal antibodies have been obtained.

Antibody-producing cells differentiate into stem cells, pre-B cells, B cells, activated B cells, and antibody-producing cells (plasma cells). During this process, in pre-B cells, IgM II 1M (μ chain)
is synthesized in the cytoplasm, and when it becomes a B cell, 1 is deposited on the cell surface.
Comes to possess gM molecules. B cells that receive antigen stimulation become activated B cells, which further differentiate into antibody-producing cells, such as IgM, IgG, and IgA.

■ Secretes antibodies such as g-star. Therefore, anti-IgM labeled with a fluorescent substance or an enzyme can be used. Furthermore, in patients with glomerulonephritis, immunoglobulins are deposited as immune complexes in the kidneys, and cases of IgA nephropathy in which IgA is deposited, as well as cases in which phylum IgG and Igil are deposited, are also known.

Anti-1gM antibodies can also be used in the differential diagnosis of such glomerulonephritis.

However, antiserum obtained by immunizing a foreign animal with human IgM has a problem with its specificity for IgM, and
It has been difficult to specifically stain cells.

0it, T for monoclonal antibodies against human IgM
Kuri'tani was reported to Nana (YuEXp,
Mjd,, VO1155, p839 (1982)),
It is commercially available from several companies. However, although these monoclonal antibodies have excellent IgM specificity, they have drawbacks in tissue staining of B cells, such as nonspecific binding and background staining. , was not satisfactory.

The present invention was completed based on this technical background, and is a single clone antibody that has specificity for IgM and has the ability to specifically bind to the μ chain on the surface of B cells. This is what we provide.

The antibody of the present invention has specificity for IgM and
It has the property of having specific binding ability to μ chain on the cell surface. Therefore, if the antibody of the present invention is labeled with a fluorescent substance or an enzyme and used for tissue staining, it will not cause non-specific binding on the surface of B cells and will not stain the cells. Specific staining is possible. As described above, since the antibody of the present invention specifically binds to the μ chain of B cells, it is possible to specifically and clearly stain B cell regions in tissues such as lymph nodes. This achieves an extraordinary effect on the utility of antibodies.

The preparation method and preparation form of the i-antibody of the present invention are not particularly limited, and should be appropriately selected depending on the purpose. Hybridomas producing the antibodies of the present invention can be obtained by applying general cell fusion methods.

This cell fusion method will be explained as follows.

■ Preparation of antibody-producing cells Antibody-producing cells are prepared using human Ig purified from human serum.
- immunizing xenobiotic animals such as mice, rats, rabbits, sheep, horses, and cows, and obtaining antibody-producing cells such as lung cells, thymocytes, lymph node cells, and/or peripheral blood cells that have acquired the immunity. This is done by

■ Preparation of myeloma cells As myeloma cells, cell lines of various animals such as mouse, rat, rabbit, and human can be used. The cell line used is preferably one that is resistant to drug paper, and has the property that it cannot survive in the selective medium in an unfused state, but can survive in a fused state. The most commonly used are 8-azaguanine-resistant cell lines, which are treated with hyboxanthin phosphoribomel 5.
hypoxanthi, ne
phospboribosyl transferas
e), and has the property of not being able to grow on hypoxanthine aminopterin thymine (HAT) medium. In addition, it is preferable that the cell line be a ``Yuru-1 non-secreting type'' cell line.A specific example of such a cell line is PB derived from the mouse myeloma MOP C-2,1 line.
/X 68-Ag'8 Ul (PBUI), Pg/
X 68. Ag8 6 5 ・ 3 (X 68 6
・58), P8/NSI-1-Ag4-NSl
-1-A, S, 210-Ag14 (SF3), rat myeloma 21°RCY8 Ag1・2・8 (Y8
・Ag 1.23), human myeloma [1-266,-API.

GM1500 can be mentioned.

D Cell fusion Cell fusion is carried out in Eagle's minimal essential medium (MEM), RP
107- in animal cell culture medium such as MI 1640.
108 myeloma cells and antibody-producing cells at a mixing ratio of 1:4
Mix for ~10 minutes. As a fusion promoter, the average molecule m
1000-6000 polyethylene glycol (P
EG), polyvinyl alcohol, and viruses are used.

Selection of hybridomas in a selective medium Hybridomas are selected from cells after cell fusion treatment by selective growth in a selective medium. For example, 15 cells
% Uno Fetal Serum Contains TRP MI' 164047
: Properly dilute and use microtiter plate 1
- Sprinkle the cells at a density of about 10 5 to 10 6 per well, add a selective medium (for example, HAT medium) to each well, and then culture by replacing the selective medium appropriately. For example, if an 8-azaguanine-resistant strain is used as myeloma cells and HAT medium is used as the selective medium, unfused myeloma cells will die within about 10 days of culture, and antibody-producing cells, which are normal cells, will also die for a long time in vitro. It cannot grow for a period of time.

Therefore, 1. All cells that grow from day 10 to 14 of culture become hybridomas.

6) Search for anti-human IgM antibody-producing hybridomas Search for anti-human IgM antibody-producing hybridomas by enzyme immunoassay (Enzyme Linked 1nnnn).

3prbe'nt As5ay, E L I S A
).

'IgM purified from human blood liV, or
IgM purified from N-pulmonary lung patient blood in advance was 10 to 10
0μ9/ml phosphate buffered saline (PBS),
Dissolve sodium bicarbonate (pll 8.0) in buffer solution Iζ and place on a soft plate (96 wells) with polyvinyl chloride (PVC).
Add 0 IJ in increments and incubate at 4°C for ~night. Next, the above antigen was discarded, and after washing with PBS, PBS containing 196 bovine serum albumin (BSA) was added and left at room temperature for 1 hour.
Block the non-antigen binding sites with BSA. Add 50μγ of the upper half of the hybridoma and let it cool at room temperature.
Leave for an hour and wash 8 times with PBS. Next, biotinylated anti-mouse immunoglobulin antiserum (second antibody) is added and left at room temperature for 1 hour. After washing 8 times with PBS,
Add avidin D-enzyme conjugate and let stand at room temperature for 15 minutes. After washing four times with PE3S, an enzyme substrate is added to develop color.

Wells to which culture supernatant producing antibodies with antigen-binding ability have been added can be easily identified by the following two operations, and hybridomas can be searched for.

(2) Cloning Since there is a possibility that two or more types of hybridomas are growing in each well, cloning is performed by a limiting dilution method or the like to obtain a single clone antibody-producing hybridoma.

■ Production of antibodies The purest single-clonal antibodies are produced by hybridomas containing 10-15% fetal bovine serum.
It can be obtained from the culture supernatant by culturing it in an animal cell culture medium such as 1640 medium. The cell culture method and conditions may be appropriately adapted from the animal cell culture method of Tsuyoshi Hayabusa.

On the other hand, as a method for producing antibodies in even larger amounts, a mineral containing bristan (2,6,10,l 4-tetramethylbentatecan) was intraperitoneally administered to an animal syngeneic with the animal from which the parent myeloma cells of the hybridoma were derived. ! After that, the hybridoma was injected intraperitoneally! j. It is possible to adopt a method of multiplying them in large quantities. High fritoma is 10-18
1 forms ascites lung cancer, and high concentrations of antibodies (approximately 1 to 20 mg/rn) are produced in serum and ascites. When purification is necessary, ascites is fractionated with ammonium sulfate. After, D
The purpose can be achieved by purification by EAE cellulose ion exchange chromatography, affinity column chromatography using Sepharose 4B bound with human I/IgM, single molecule sieve column chromatography, and the like.

Examples of hybridomas suitable for producing the antibody of the present invention include the 2012 strain, the 8F1 strain, and the 4! (7 shares etc. have been acquired.

More specific explanations will be given below regarding the preparation method of these hybridomas and the properties of the antibodies of the present invention produced therefrom.

! , Obtaining hybridomas Human IgM (11+1 g/*/) was dissolved in physiological saline and mixed 1:1 with complete Freund's adjuvant to form an emulsion, which was given to BALB/C mice (female, 6 weeks old). Inject 50μ9/bead intraperitoneally several times every two weeks, and finally inject 80μg intravenously.

Three days after the final immunization, mouse tile cells were removed and ME
Washed with M. Wash mouse myeloma P3tJl with MEM, mix tile cells and pBul at 10:1, centrifuge, and add 50% PEG1000 MEM solution III to the pellet.
Cell fusion by gradually adding t? I made it. More MEM
The solution was added gradually to a final volume of 10 ml. Centrifuge again and transform the pellet into RPM I1 containing 15% fetal bovine serum.
640 medium as p8u, 1 x 105cell11
The suspension was suspended at a concentration of 0.1 m/l and spread onto a 96-well microplate in 0.1 l portions.

One day later, 0.1 mt of HAT medium was added, and then half the amount of the medium was added every 3 to 4 days to a new HAT well, and growth of one hybridoma was observed.

Above the well where hybridomas have grown
gssg+a'ssg+sasigsggs*awsa
Five microliters of m1111WIWIIIWII were each added to a 96-well soft plate previously coated with Human Summer gM. From the ELISA method ζ described above using avidin D-peroxidase (manufactured by Bactane 1) as the avidin D-enzyme conjugate and hydrogen peroxide, 4-aminoantipyrine, and phenol as the substrate and coloring agent, what is human IgM? I will react though.

One that did not react with human IgG was selected and cloned using the limiting dilution method.

As a result, hybridomas 2 C1lid and 8 Fla produced antibodies with specificity for human IgM.

4H7 strain was obtained.

③ Production of single clone antibodies Hybrid strains 72012 strain, 8F1 strain, and 4H7 strain were each cultured in RPM11640 medium containing 15% fetal bovine serum in a 96-well plate, 25 cm flask, and 75c4 flask without being scaled up. Supernatants were collected and monoclonal antibodies were prepared.

m Determination of antibody subclass After blocking each single clone antibody in a 96-well soft microplate with PBS containing codon, 1% BS, and A, MONOAB ID EIAKIT (ZYMED
anti-1gA antibody, anti-1gG1 antibody, anti-1gG
2a antibody, anti-IgG2b antibody, anti-IgG3 antibody, anti-Ig
The subclass of each single clone antibody was determined by observing the reaction with M antibody.

The type was determined by examining the reactivity of the chain with anti-sail and anti-λ antibodies.

As a result, the antibody produced by the 2C12 strain was G21)'/'
The antibodies produced by the 3F1 strain and 4H7 strain are IgG.
It turned out to be 1/.

1. Specificity of Antibody Human J1 myeloma protein was purified and coated in 50111 microtiter plates at 100 ml/we each. Next, the antibody of the present invention (1/1g/
It) 501t (l) was added, and after washing, the binding to each antigen was examined by ELISA method.

As a result, as shown in Table 1, the antibody of the present invention
It was confirmed that it binds only to M and does not react at all with other classes of immunocropurins and immunoglobulin complex chains.

Table 1 V Antibody titer The antibody titer in the culture soil of strain 2012 was measured by ELISA and was found to be 8,000.

In addition, the titer is calculated from the stock solution of the antibody sample that exceeds 50% of the absorbance obtained by ELISA method for a sample in which there is a sufficient amount of antibody against the immobilized antigen as 100%. It is expressed as a dilution ratio.

The nozzle value measurement curve at this time was as shown in FIG. As a control, a commercially available single clone antibody against human IgM was compared. As is clear from FIG. 1, the antibody of the present invention had a higher absorbance value in the antibody-excess region than the commercially available product.

This indicates that the amount of monoclonal antibody bound per 1 gM molecule is large.

(2) Tissue staining with the antibody of the present invention Human lungs were fixed using Ti5sue-TEK Il, and sections were prepared using a cryostat.

These section specimens were stained with the biotin-avidino system using the antibody of the present invention.

That is, section specimens were soaked in PBS (0, OIM, pH 7,
At 2y, the cells were incubated with the antibody of the present invention (derived from 2C12 strain) as a secondary antibody for 40 minutes at 87°C. After that, after washing again, anti-mouse IgG was used as a secondary antibody.
A biotin-labeled antibody (TAGO) was reacted in the same manner.

Furthermore, after washing again, -15iiiaii-fluorescein-labeled avidin (E, Y Laboratories, hereinafter 1-
F, I 'I' CJ).
The washed specimen was sealed in glycerin to prepare a stained specimen.

These specimens were examined using a fluorescence microscope.

Next, tissue staining of the kidney of a patient with glomerulonephritis was performed using the same method. As a result, as shown in Photo IX2, IgM antibodies were deposited and 4 areas were stained.

Furthermore, it is also possible to separate and identify fluorescently labeled B cells by cytoflowmetry. Specifically, a lymphocyte fraction was prepared from human peripheral blood by the Ficoll method, incubated with the antibody of the present invention (produced from 2C12 strain I) for 45 minutes, washed 3 times, and incubated with anti-mouse immunoglobulin FITC for 45 minutes. Inkyuhenyon L
, FAC3IV (manufactured by Baekhyun Dickinson).

As a result, ll]-cells decrease the value by 5-20%,
A good correlation was observed with the proportion of B cells measured with 213 cells Martuno-9LEU 111 (manufactured by Becton Detskinsonnej).

[Brief explanation of the drawing]

゛Figure 1 is a titration curve of the antibody of the present invention. Photo 1 is a micrograph of a human lung specimen tissue-stained using the antibody of the present invention. Photo 2 is a microscopic photograph of a kidney specimen from a patient with glomerulonephritis. Patent applicant (677) Yamasa Shoura t1, ceremony month ¥ Wt
Amended Procedures for the Lease of the Wild Ox House (Method) August 23, 19801, Case Description 1982 Patent Application No. 1521562, Name of the Invention Single Clone Antibody against Hili-1gM 8, Relationship with the case of the person making the amendment Patent applicant address: 14-2-10 Shinseicho, Choshi City, Chiba Prefecture Date of amendment order: 1 July 11, 1980 (Shipped on July 81, 1980) 5 , the column for a brief explanation of the drawings of the specification to be amended, and Drawing 6, Contents of the amendment l) In the column for the brief explanation of the drawings on page 17, lines 16 to 19 of the specification, ``Photo 1 is... ...(omitted)...
This is a microscopic photograph/diagram. ” will be deleted. -2) Delete photos 1 and 2 from the drawing. 8) “Submit a reference photo as shown in the attached sheet.

Claims (1)

[Scope of Claims] A single clone antibody that has specificity for human IgM and has the ability to specifically bind to μ chain on the surface of B cells. .