JPS60258128A - Rat monoclonal antibody - Google Patents

Abstract

PURPOSE:The titled antibody, produced by a fused cell formed by using a splenic cell of a rat immunized with human blood serum, unreactive with egg white albumin, bovine blood serum albumin, etc. and reactive specifically only with the human blood serum albumin. CONSTITUTION:A rat monoclonal antibody obtained by excising a splenic cell of a rat immunized with human blood serum albumin, and having an anthuman blood antibody value >=10<3> times that of the normal human blood serum, fusing the resultant cell with a myeomatous cell of a mouse by the ordinary method to form a hybridoma, selecting the monoclonal antibody specifically reactive with the human blood serum albumin, and preparing the aimed antibody from the above-mentioned hybridoma. The above-mentioned antibody is preferably used for an antihuman blood serum albumin column for removing the human blood serum albumin from a substance containing the human blood serum albumin or collecting the purified human blood serum albumin.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a rat monoclonal antibody that is useful for purifying trace substances in human serum and can be used as a pharmaceutical 1 diagnostic agent, reagent, etc., and a method for producing the same.

BACKGROUND OF THE INVENTION It has been known for a long time that when a foreign animal such as a mouse is immunized with human serum albumin, antiserum (polyclonal antibody) against human serum albumin is produced.

There is a report that a mouse was immunized with human serum albumin, and a hybridoma was created by fusing the lung cells of the immunized mouse with a mouse myeloma cell line, and a monoclonal antibody that reacted with human serum albumin was obtained [1mmuno
logic l, etters, 3.365-370 (
1981), Molecular Immunolog
y20. (5), 549-555 (1983)
].

Problems to be Solved by the Invention In recent years, trace substances contained in human serum have been attracting attention as pharmaceutical products 1 diagnostic agents, reagents, and the like. However, it is difficult to purify and isolate these substances from human serum, and in many cases, it is particularly difficult to completely remove human serum albumin, which is contained in large amounts in human serum.

Antibodies in antiserum can be collected and used to remove human serum albumin. However, such antiserum is not homogeneous and cannot be supplied in large quantities on an industrial scale, and its specificity is such that it reacts with serum albumin of non-human animals such as bovine serum albumin. There is also a problem.

Although there is no description of the reactivity between previously known monoclonal antibodies and serum albumin derived from other animals, the inventors conducted additional tests and found that when mice were immunized with human serum albumin, the resulting antiserum was It also reacted with ovalbumin, and therefore, all monoclonal antibodies prepared from the tile cells also reacted with human serum albumin as well as ovalbumin, and no monoclonal antibodies truly specific to human serum albumin could be detected.

(Table 1) Means for Solving the Problem The present inventor has developed an anti-human serum albumin monoclonal antibody that can be used in an anti-human serum albumin antibody column when purifying human serum albumin-containing substances on an industrial scale. We carefully considered the creation and production methods. the result,
ovalbumin by immunizing rats with human serum albumin.

We obtained an antiserum that reacts only with human serum albumin and does not react with bovine serum albumin at all. By fusing the tile cells of this immunized rat with a mouse myeloma cell line, we created a hybridoma strain that produces a significant amount of rat monoclonal antibodies that are specific for human serum albumin (does not react with ovalbumin or bovine serum albumin). .

The present invention will be explained in detail below.

The present invention provides rat monoclonal antibodies directed against human serum albumin. The monoclonal antibody of the present invention is produced as follows.

(1) Preparation of immunized animal cells □1 A rat is immunized with human serum albumin, and tile cells are prepared from the animal. The immunization method is to administer an appropriate adjuvant (for example, Complete Freun) subcutaneously, intravenously, or intraperitoneally to 8-10 week old rats.
d's Adjuvant or aluminum hydroxide gel and Bordetella pertussis vaccine, etc.), 20 to 4 ooμg/mouse of human serum albumin is administered. From then on, 1-2
Human serum albumin (20-400 μg/mouse) is administered 2-10 times every other week. On the 3rd to 7th day after each immunization, blood was collected from the fundus venous plexus, and the anti-human serum albumin antibody titer in the serum was measured using the enzyme immunoassay method (enzyme immunoassay method: Igaku Shoin series 1978) using the in-phase method shown below. investigate.

Enzyme immunoassay: 96-well EIA plate (Flow Laborat
1% human serum albumin-phosphate buffer saline (BSA-PBS)
(Human serum albumin 10g, disodium phosphate 1.
83g, 1 potassium phosphate 0.21g 1 salt 7.65
g, distilled water II2 + pH 7,2) at 100-200μ
Dispense β/well and leave it at 4°C for 1-2 nights to coat the bottom of the plate hole with human serum albumin.
After discarding S and washing thoroughly with resin water, as the first antibody,
Sample diluted with BSA-PBS (mouse serum.

Hybridoma culture Tsuchiura, crude monoclonal antibody) was dispensed at 100 μβ/well and left overnight at 4°C. After washing once with resin water and six times with 2M NaCf1 solution, the second
Goat anti-rat immunocloprine oxidase conjugate (Kirkegaard & Perr) was used as the antibody.
10 of y Laboratories Inc.)
Dispense 100 μl/well of the 0-fold diluted solution and leave it at room temperature for 2 hours. 3 with phosphate buffer.

After washing twice, add peroxidase substrate solution [○-phenylenediamine 40 mg, phosphoric acid/citric acid buffer (citric acid 105 g and Na28 pH 358 g to 25 g of resin water)
Dissolve in Qml. pH5,0) 100ml, 30%
Dispense lI2024'Oml] 100 µi/well, leave it at room temperature for 10-30 minutes, then add 2MH2SO, 20 µl.
to stop the reaction. The antibody titer is calculated by colorimetric determination at 405 nm. Rats whose antibody titer against human serum albumin is 103 or higher than normal human serum are used as a source of immunized animal cells.

For cell fusion, immunized rats were given 20 to 400 μl of human serum albumin 3 to 4 days before the fusion treatment.
g/mouse is administered intraperitoneally, the lung cells are removed, and tile cells are prepared. The lungs were shredded in MEM (manufactured by Hinaga Pharmaceutical Co., Ltd.),
Loosen with tweezers, centrifuge at 120 rpm for 5 minutes, discard the supernatant, and treat with Tris-ammonium chloride buffer (pH 7,65) for 1 to 2 minutes to remove red blood cells.
The cells are washed three times with ME'M and provided as lung cells for fusion.

(2) Preparation of myeloma cells As myeloma cells, established cell lines obtained from mice are used. For example, 8-azaguanine resistant mice (BA
LB/c derived) myeloma cell line P3-X63Ag8-Ul
(P3-Ul) (:Current, topics
in Microbiology and Immunochemistry
nology81, ]-7 (1978)), P
3-NSI/1-8g 4.1 (NS-1) [Bur
open J, Immunology 6.511
-519 (1976): l, SP210-Ag14
(SP-2) (Nature 276, 269-27
0(1978)E, P3-X63-Ag 8.65
3 (653) (J, 1mmunology123,
154B-1550 (1979) ), P3-X63
-Ag8 (X63) (Nature 256.495
-497 (1975)) etc. are used.

These cell lines were grown in 8-azaguanine medium [RPMIi
640 medium with glutamine (1.5mM), 2-mercaptoethanol (5X10-5M), dientamycin (1.
0gg/ml) and fetal calf serum (FC3) (C3
(manufactured by Company L) (10%) and a medium containing 8~azaguanine (15 gg/ml)], but 3 to 4 days before cell fusion, the cells were subcultured in a normal medium.
On the day of the fusion, ensure a number of small moon capsules of 2 x 10' or more.

(3) Rats immunized with cell fusion (1) are intraperitoneally administered with 20 to 400 μg/mouse of human serum albumin, and 3 to 4 days later, the lungs are removed and tiles are prepared. Wash the myeloma cells obtained in (2) thoroughly with MEM medium or PBS,
Mix the cells so that the number of cells is 5 to 10:1, centrifuge (1.20 Or pm, 5 minutes), discard the supernatant, loosen the precipitated cells, and then While stirring, at 37°C, add polyethylene glycol 1,
000 (PEG-1,000, > 2 g.

Add 0.2-11'I11/106 tile cells to a mixture of 2 ml of MEM and 0.7 ml of dimethyl sulfoxide, and add 1-2
Add 1-2 ml of MEM several times every minute, then add MEM to bring the total volume to 5 Qml. After centrifugation (900
After discarding the supernatant and gently loosening the cells, add 10 Qml of normal medium and gently suspend the cells by suctioning or blowing with a measuring pipette.

This suspension is dispensed into a 24-well culture plate at 1 ml/well and cultured at 37° C. for 24 hours in a 5% CO2 incubator. 1 ml/well HAT medium in culture plate [
Hyboxanthin (10-'M) and thymidine (
1,5X 10-5M) and aminopterin (4X1
Add 0-'M) and culture medium for another 24 hours.After that, add 1 ml of culture supernatant every 24 hours for 2 days.
1 is discarded, the same amount of new HAT medium is added, and cultured at 37°C in a CO2 incubator for 10 to 14 days.

Discard 1 ml of supernatant from holes where fused cells that have grown in colonies are observed, add the same amount of HT medium (HAT medium minus aminopterin), and continue to culture for 2 days.
Conversion to HT medium is performed every 24 hours.

After culturing in HT medium for 3 to 4 days, a portion of the culture supernatant is taken and the anti-human serum albumin antibody titer is measured by the enzyme immunoassay method described above. At this time, the reactivity with bovine serum albumin, ovalbumin, etc. is also measured using the same method, and those that specifically react with human serum albumin are selected. Cloning was repeated twice using the ocular dilution method for holes that strongly reacted with human serum albumin but did not react with bovine serum albumin, ovalbumin, etc., and those that showed a stable antibody titer strongly against human serum albumin were used as anti-human Select as a hybridoma strain that produces serum albumin monoclonal antibodies.

1) Preparation of monoclonal antibodies 8- to 10-week-old nude mice treated with Bristan (intraperitoneal administration of 0.5 ml of 2,6,1,0,14-tetramethylpencudecane and reared for 2 weeks) were given (3 ) The anti-human serum albumin monoclonal antibody-producing hybridoma cells obtained in 2 to 4×10 6 cells/mouse are injected intraperitoneally. The hybridoma turns into ascites cancer in 10 to 21 days. Ascitic fluid was collected from this mouse and centrifuged (3,00 Or pm).
, 5 minutes) to remove solids, salted out with 50% ammonium sulfate and 40% ammonium sulfate, and added PBS (disodium phosphate 1.83 g
, monopotassium phosphate 0.21g, salt 7.65g.

Dialyze with distilled water (1β, pH 7,2) for 1 to 2 days.

This dialyzed fraction is passed through a column such as a heptaphryl S-200 (Pharmacia Fine Chemicals), protein A-Sepharose column, etc., and the IgM fraction is collected and used as a purified monoclonal antibody.

The specificity of a monoclonal antibody is determined by the above-mentioned binding assay and inhibition assay using enzyme immunoassay.

Determination of antibody isotype is performed using Ouchter Iony
(double immunodiffusion method) (Introduction to Immunology Experiments, Biochemistry Experiment Methods 15, Gakkai Publishing Center, P, 741981).

The amount of protein was quantified using the Folin method and absorbance at 280 nm [1,4 (OD2+, o ) ===, calculated from immunoglobulin 1 mg/mD.

Thus, the monoclonal antibody K1150 obtained from the hybridoma strain designated Klvl-50 was identified to belong to the IgM class isotype.

The antigen specificity of KM-50 is as shown in Example 1.

The method for removing human serum albumin from a proteinaceous substance containing human serum albumin using the monoclonal antibody of the present invention is as follows.

20 mg of the monoclonal antibody KM-5'0 of the present invention is dissolved in 1 ml of PBS' and reacted with 1 ml of a carrier such as CNBr-activated Sepharose 4B (manufactured by Pharmacia Fine Chemicals) to obtain an immobilized monoclonal antibody. After filling a column with this immobilized monoclonal antibody, a solution containing human serum albumin (5 mg or less) is passed through the column. With this operation, 99% to 100% of human serum albumin is adsorbed onto the column. If this passed sample contains proteins other than human serum albumin, those proteins will pass through the column and come out in the non-adsorbed fraction. If the ratio of monoclonal antibody to human serum albumin is within the range of 5:1, human serum albumin can be completely removed from the sample in -1 passes.

In addition, human serum albumin adsorbed on the column was 0.0
Almost 100% elution can be achieved with a 1-0.1 M glycine-hydrochloric acid buffer (pH 2, 3). By this operation, the present antibody column can be reused 5 to 10 times.

Examples of the present invention are shown below.

Example 1゜(1) Preparation of immunized rat tile cells Five 6-week-old C0B5 female rats (Shizuoka Prefecture Laboratory Animal Agricultural Cooperative Association) were given 2 mg/rat of aluminum hydroxide gel as an adjuvant and killed Bordetella pertussis vaccine (Chiba). Prefectural Serum Research Institute) 1 x 10 I' cells/animal, antigen good rW) Serum a/l
/-j=7 (Human'Serum Albu
min, manufactured by Kabi, Sweden) 10
Immunization was performed by intraperitoneally administering 0 μg/mouse.

Thereafter, 100 μg/mouse of human serum albumin was intraperitoneally administered every week for second and subsequent immunizations. After the third immunization, blood was collected 5 to 7 days after the immunization, and the anti-human serum albumin antibody titer in the blood was examined by enzyme immunoassay using the same phase method described above.

Antibody titers were observed in all 5 animals after the 3rd immunization, but immunization was performed 2 more times, making 5 immunizations.

On the third day after the fifth immunization, the lungs were removed from the rats, lung cells were prepared, and the cells were subjected to cell fusion.

(2) Preparation of Mouse Myeloma Cells The 8-azaguanine-resistant mouse myeloma cell line P3-Ul was cultured in a normal medium, and at the time of cell fusion, 2×10 7 or more cells were obtained and used as a parent strain for cell fusion.

[3] Hybridoma production The lung cells and myeloma cells obtained in steps (1) and (2) were mixed at a ratio of 5:1.
were used at a ratio of Measure the antibody titer, select active holes, change to normal medium, repeat cloning twice,
A hybridoma strain, KM-50, which produces a monoclonal antibody specific for human serum albumin was selected.

(4) Preparation of purified monoclonal antibodies The hybridoma strain KM-50 obtained in (3) was added to 8-week-old nude female mice treated with pristane at 4 x 106 cells/
The animals were injected intraperitoneally. After 10-21 days, the hybridoma turns into ascites cancer. Ascites was collected from mice with accumulated ascites (5 to 10 ml/mouse) and centrifuged (3,000 rpm).
pm, 5 minutes) to remove solids. The supernatant was subjected to 50% ammonium sulfate salting out and 40% ammonium sulfate salting out, and was dialyzed for 2 days against pBS (pH 7.2). 10.2 ml of this dialyzed product (10 mg/m
1) was passed through a column of heptaphryl S-2005001111 (1.6 x 60 cm), subjected to gel filtration, fractionated into 1 ml portions, and the IgM fraction was collected to obtain a purified monoclonal antibody. This antibody preparation was electrophoretically confirmed to be a single protein.

(5) Antigen specificity of purified monoclonal antibodies The antigen specificity of purified monoclonal antibodies was examined by in-phase enzyme immunoassay. As an antigen, bovine serum albumin (BS
A) (manufactured by Sigma), human serum albumin (
H3A) (manufactured by Kabi), ovalbumin (OVA)
) (manufactured by Sigma) was used. The results are shown in Table 2.

Table 1: When a mouse is immunized with human serum albumin, the resulting antiserum also reacts with ovalbumin, as shown in Table 1. Therefore, all monoclonal antibodies prepared from the lung cells also react with human serum albumin. It also reacted with ovalbumin, and it was not possible to detect a truly human serum albumin-specific monoclonal antibody.

On the other hand, when rats were immunized with human serum albumin, the antiserum obtained was specific to human serum albumin.

Table 2 Normal rat serum 10-' 0.005 0.002
0.01510-' (1,0150,000Q, QO
Olo-' 0.0140.0000.005H3A immune rat serum 10-'' 1.482 0.000 0
．． 06210-31.1740.0000.00010
-30.9470. G(1(1(1,(1G(110-
' From the results of 0.7060.0000.000 or more,
It can be seen that this monoclonal antibody has extremely high specificity for human serum albumin. (6) Classification of monoclonal antibodies When the KM-50 incubator was examined using the uchterlony method, it was identified as a monoclonal antibody belonging to the 1gM class.

Example 2 20 mg of anti-human serum albumin monoclonal antibody KM-50 obtained in Example 1/carbonate buffer (0,I
M NaHCO2, 0.5M NaCj! .

p)(8,3) on CNBr-activated 70-phase 4B
(manufactured by Pharmacia Fine Chemicals) IITl
1 at room temperature for 2 hours to obtain an immobilized monoclonal antibody. This immobilized monoclonal antibody was applied to a column (1 m
1) and the column was loaded with 0.5 mg/ml human serum albumin/carbonate buffer (0,IM NaH
CO2, 0,5M Na (1!, pH 8,3) 6.7
Pass through the column at a flow rate of ml/hour and collect 1 ml of the effluent fraction in 2.
The absorbance at 80 nm is shown in FIG.

As is clear from Figure 1, the total amount of human serum albumin is 4 mg (0,511 g/ml x 3n+I) on a column immobilized with 20 mg of monoclonal antibody.
It can be seen that human serum albumin is completely adsorbed on the antibody column until .

In addition, this once adsorbed human serum albumin was transferred to an antibody column using a 0.04M glycine/hydrochloric acid buffer (pH 2, 3).
), it was possible to elute almost 100% of the antibody column, and the antibody column could be reused.

Example 3 1 mg of human serum albumin and 1 mg of ribonuclease were added to the same KM-50 antibody column as in Example 2 under the same conditions.
, chymotrypsinogen 1+ng, ovalbumin l
mg to carbonate buffer y (0,IM NaHCO2゜0.
5M NaCjl! , pH 8, 3) was passed through the column, and the effluent was analyzed by electrophoresis. As a result, ribonuclease, chymotrypsinogen, and ovalbumin passed through as non-adsorbed fractions, and the entire amount was recovered. but,
It was found that all human serum albumin was adsorbed to the column and was not observed in the effluent until the total amount reached 4 mg. That is, only human serum albumin could be completely removed from a mixture of various proteins using this antibody column.

Effects of the Invention The anti-human serum albumin rat monoclonal antibody obtained by the present invention and the affinity column prepared by binding it to a carrier are useful for purifying trace substances present in human serum, such as erythropoietin, and for purifying human serum albumin. It is extremely versatile and has high application value, as it can be used for the purification of albumin.

[Brief explanation of the drawing]

FIG. 1 shows human serum albumin adsorption in Example 2. Patent applicant (102) Kyowa Hakko Kogyo Co., Ltd.

Claims (1)

[Claims] (]) Rat monoclonal antibody against anti-human serum albumin. (2) The monoclonal antibody according to claim 1, wherein the monoclonal antibody belongs to the IgM isotype. (3) A method for purifying a substance comprising human serum albumin, which comprises removing human serum albumin from the substance by subjecting the substance to affinity chromatography using a rat monoclonal antibody against human serum albumin. (4) By subjecting a substance containing human serum albumin to affinity chromatography using a rat monoclonal antibody against human serum albumin,
A method for purifying human serum albumin, which comprises collecting human serum albumin from the substance. (5) A hybridoma obtained by fusing mouse myeloma cells with lung cells obtained by immunizing a rat with human serum albumin using a conventional method, and which has the ability to produce rat monoclonal antibodies against human serum albumin. The monoclonal antibody is produced and accumulated in the culture solution or ascites by culturing the hybridoma containing the antibody in a medium or transplanting it into the peritoneal cavity of a nude mouse to cause ascites cancer, and the monoclonal antibody is extracted from the culture solution or ascites. A method for producing a rat monoclonal antibody against human serum albumin, which comprises collecting.