JPS6258997A - Monoclonal antibody - Google Patents

Abstract

PURPOSE:A monoclonal antibody against human apolipoprotein A-I which can be obtained by isolating and purifying pure human apolipoprotein A-I belonging to the class IgG from human serum (or plasma). CONSTITUTION:Hybridoma 3 strain producing a large amount of a monoclonal antibody which has specificity to human apolipoprotein A-I and different recognition sites on the human apolipoprotein is prepared. A monoclonal antibody against anti-human apolipoprotein A-I produced by the hybridoma or an affinity column using it as an ligand can purify conveniently pure apolipoprotein A-I from human serum or plasma.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to human apolipoprotein 'j! LA-1 (32L
The present invention relates to a monoclonal antibody against apoA-I (hereinafter abbreviated as ApoA-I), a method for using the monoclonal antibody, and a method for producing the same.

ApoA-I is a lipobuton contained in the serum of supplemented mammals, e
HDL is a protein with a molecular weight of approximately 27,000 that constitutes protein, and exists as a main component of high-density buoy protein (hereinafter abbreviated as HDL). AyWA-1 is known as an activator of lecithin:cholesterol acyltransferase in the blood, and is attracting attention as a protective factor for arteriosclerosis because it esterifies peripheral cholesterol and incorporates it into HDL particles. Therefore, from the standpoint of clinical testing, it is of great significance to measure the A/A-I content, and several kits are commercially available. The method for measuring apoA-I is the single radial immunodiffusion method (Single rad immunodiffusion method).
ial immunodiffusion), laser nephelomet IJ-, rocket immunoelectrophoresis, enzyme immunoassay, and radioimmunoassay, all of which are based on antigen-antibody reactions to generate antiserum specific to riabo A-I. It is necessary to collect it. For this purpose, it is essential to obtain immunologically pure A4h-1 from human blood as an antigen and standard.

On the other hand, due to the clinical significance as described above, ffA-I is also expected to be used as a preventive or therapeutic agent for arteriosclerosis. In this case as well, it is necessary to highly purify apoA-I from human blood.

Conventionally, the method for purifying apoA-■ from human serum or blood plasma involves adjusting the density of serum (blood plasma) and delipidating HDL obtained by ultracentrifugation with an appropriate solvent (e.g., ethanol:ether mixture). Then, this was dissolved in a buffer containing 6 to 8 M urea, guanidine hydrochloride, or sodium dodecyl sulfate (for example, Tris-hydrochloric acid buffer), and subjected to gel filtration chromatography, ion exchange chromatography, etc. to obtain 'N4g! It was common to do so. (e.g. Biochim Biophys Acta 285
36 (1972), J. Biol, Ohem.
247(8)5842(]972), etc.).

In addition, in order to obtain high-purity DL, HDL separated by ultracentrifugation is purified with Sepharose 6B, delipidated, and then subjected to column chromatography (Clinical Chemistry (2)) 26 (1982)).

However, these methods have the disadvantage that operations are complicated and time-consuming, and pure apoA-I may not always be obtained.

ApoA-1f: A report on the purification of ribonosec particles containing apoA-I from human serum by affinity chromatography using a polyclonal antiserum with a % heterogeneity (
J. Biol Chem, 259(19)1220
1 (1984), Proc, Natl, Aca.
d, Sci, USA 811356 (1984), but it was not used as a purification method for pure apoA-I.

The present inventors have discovered that anti-human apoA-1 can be isolated and purified from human serum (or blood plasma).
We conducted extensive studies on the preparation and use of anti-human ffA-1 monoclonal antibodies that can be used in I antibody columns, and found that they are specific to human yfA-i and are mutually apoA-1.
-Development of monoclonal antibodies with different recognition sites on
The present invention has been completed by successfully creating three hybridoma strains that produce the following.

The anti-human apoA-1, tE'A-) monoclonal antibody obtained by the present invention and the affinity column using it as a ligand can purify apoA-1 from human serum (or blood plasma) more easily and more easily than conventional purification methods. Since it uses a monoclonal antibody produced by a hybridoma as a ligand, it has high specificity and the same monoclonal antibody can be obtained semi-permanently since a high-cylidoma strain has been established.

The present invention will be explained in detail below.

The monoclonal antibody of the present invention is produced as follows.

(1) Anti-W, sensitized flh cells in mice)
) Prepare splenocytes by immunizing IDL. Immunization is performed by administering an appropriate adjunocent (e.g. Pr
eunds complete 50 ~] OOμ2/mouse is administered several times with appropriate adjunocent. Blood was collected from the fundus venous plexus of mice at appropriate times, and the serum anti-human)
The IDL antibody titer is determined by the Ouchterlnny method (double immunodiffusion method) (Introduction to Immunology Experiments, Biochemistry Experiment Methods 15, published by Gakkai Publishing Center, p. 74, 1981). Mouse splenocytes in which sedimentation lines were clearly observed by this method were subjected to fusion.

3-4 days before cell fusion 1: Human HILL 50/100μ
2/mouse is administered intraperitoneally or intravenously, the pancreas is imaged, and splenocytes are prepared. RPM11640 (Irvine)
5 scientific company), centrifuged with tweezers at 1500 rpm for 5 minutes, and discarded the supernatant. Wash the cell pellet two more times with RPM11640;
It was centrifuged and subjected to fusion.

(2) Preparation of myeloid cells The myeloid cells used are 8-azaguanine resistant strain P3-NSI/] -Ag3-1 (
MS-1), P3-Xfi3-Ag8-Ul (P3
U1), 5P210-Ayl4(Sp2),
P3-X63-Ag8-6.5.3. (X63-6.5
．． 3. ) etc. are used. These cells were grown in PM11640 medium containing 8-azaguanine (15 Mg/l) [Glutamine (295 Mg/l) in PM11640 medium.
, sodium pyruvate (110 ■/l).

Sodium bicarbonate (29/l), penicillin G potassium (xoo, ooou/z), strezotomycin sulfate
(50”t/l), fetal bovine serum (FoS) (
(manufactured by GIBOO) (Medium with 1o% (v/v) added)
Culture cells at logarithmic growth phase to ensure a cell density of 2X]O' or more.

(3) Mouse splenocytes prepared in cell fusion (1) and myeloid cells obtained in (2) were washed several times with RPMI medium (FoS-free), and the ratio of the number of cells between the two was determined. Cells = 5-10:
] After mixing, centrifuge at 1500 rpm for 5 minutes and discard the supernatant. So % (v/v) in cell pellet
/diethylene glycol 4000 (PEG4000
) (in RPMI1640 not including FoS) l- to 1
Gradually add dropwise while stirring for 1 minute. RP after mixing for 1 minute
MI] 640 medium (without POS) 1-1
Gradually add dropwise while stirring for minutes. Further RPMI]
640 medium 8- was gradually added dropwise and centrifuged for 1500 rpm, the supernatant was discarded and suspended in RPM 11640 medium 30-. 100 µt thereof was dispensed into a 96-well microplate and cultured at 37°C for 24 hours in a 54002 incubator. After 24 hours, OOμt (medium supplemented with haminopterin (0.4 μM) and I-thymidine (16 μM)) is added. Afterwards, discard 00 pL of the culture supernatant every 24 hours for 3 days, add 100 pt of fresh EHAT medium, and culture for about 2 weeks.

Wells in which hybridoma growth was observed were screened by the enzyme immunoassay (hereinafter abbreviated as ELISA) described below.

50 μt of human HDL prepared at 250 μ2 as a titanate was added to each well of a 96'7 L microtiter plate for ELISA (manufactured by Dynatech), and after standing at room temperature for 2 hours, the HDLfd solution was collected. Each well was diluted with phosphate buffered saline (PBS, disodium phosphate, 12H 0.02.9%) containing 0.05% Tween20.
f, potassium phosphate 0.22, sodium chloride 8.0
? , add potassium chloride 0.2 F distilled water,
After washing with PBS containing 1% (W/v) bovine serum albumin (pH 7.4), each well was filled to the brim with PBS containing 1% (W/v) bovine serum albumin, and after standing at room temperature for 1 hour, Pf33 (pH 7.4) containing 0.05 Tween 20 was filled.
Wash thoroughly with washing solution). To the well coated with the antigen in this manner, 50 µt of a culture supernatant of a hybridoma in which proliferation has been observed is added, and the well is allowed to stand at room temperature for 2 hours, and then washed with the above-mentioned washing solution. Next, piotin-conjugated anti-Macx Ig
G (10pf/ml) (Vector) 100
Add μt and let stand at room temperature for 1 hour. After washing the wells with washing solution, avidin-conjugated alkaline phosphatase (Vecto
Add 50 μt of a diluted solution of R company type), leave it to stand for 15 minutes, wash thoroughly with washing solution, add 1 drop of substrate (p-nitrophenyl phosphate, manufactured by Sigma), and add 100 μt of diethanolamine buffer (pH 9,8). After adding and reacting at room temperature for 30 minutes, 40
Measure at 5 nm.

For culture supernatants determined to be positive for antibody production by this method, purified apoA-
ELISA is also performed on plates using 1. Cloning was performed by repeating the limiting dilution method twice for wells that strongly reacted with either HDL or purified apoA-1. Stable anti-humanity even after cloning, 1! A hybridoma strain producing the A-1 antibody was selected.

(4) Preparation of monoclonal antibody 1 (ALB, 107 ri;
Approximately 2 weeks after administering pristane (2,6,10,14-tetramethylpentadecane) of vnVV5, 1 to 2 x 10'/mouse of anti-human arfA (monoclonal antibody producing hybridoma cells) obtained in (3) were transplanted into the peritoneal cavity. Ascites will form in about 2 weeks, so collect shallow water and add 3.00 ml.
Orpm Centrifuge for 10 minutes, collect the supernatant, and add an equal volume of saturated ammonium sulfate (50 ammonium sulfate) for salting out. The resulting precipitate is centrifuged (s, oo).

rpm 5 min) and collect PBSI: After dissolving PB8
- Night dialysis. The dialyzed fraction was applied to an Affinili column bound with 0 NBr-activated Sepharose 4B (Pharmacia ff) l2WDL to adsorb the monoclonal antibody, and 0.2 M glycine-Tsuka acid buffer (p) (2,3
) to obtain purified monoclonal antibodies.

The sensitivity of monoclonal antibodies is determined by the so-called Western blotting method in addition to the above-mentioned ELISA. The class of the antibody is determined from the H chain of the antibody by dodecyl sulfate (Jll Lamb SDS) electrophoresis in the presence of mercaptoethanol.

The amount of protein is quantified by the Folin method.

Whether the recognition sites on ApoA-1 are the same or different (the two are estimated by a competitive method by labeling the obtained antibodies with respective enzymes).

In this way Hl 5g9. H3Dsu0,1(14
The monoclonal antibody obtained from the hybridoma strain named A9 belongs to the IgG class and has a mutual 1:apoA-1
These antibodies have different recognition sites on the top.

The method for purifying apoA-1 from human blood plasma using the monoclonal antibody of the present invention is as follows.

Monoclonal antibodies purified as described above were added to 0N
The immunoadsorbent bound to Br-activated Cephalone 4B is exploded. In this case, each of the three monoclonal antibodies can be bound to ONBr-activated Sepharose 4B, and one of them, or a mixture of two or three of them can be used as an immunoadsorbent. Lifotan/4 lithium particles containing apoA-i can be obtained by adsorbing human blood as is to this immunoadsorbent, thoroughly washing with PBS, etc., and eluting with glycine-hydrochloric acid buffer (pH 2, 3).

Further, this is delipidated with a solvent (ethanol: ether, etc.), and the resulting abutononitrium fraction is dissolved in PH1, adsorbed again to the monoclonal antibody-binding immunoadsorbent described above, and eluted in the same manner, resulting in electrophoretically simple First appointment A-1
is refined. According to this method, blood serum can be applied as is, and purification can be performed without ultracentrifugation.

This column can be used repeatedly.

Examples of the present invention will be shown below.

Example 1 (1) Collection of antigen Blood from five healthy individuals was collected using sodium citrate as an anticoagulant. Immediately centrifuge (]0.OO
Orpm 10 minutes) to separate the blood sputum. Some of this was carried out using monoclonal antibody columns.
It was stored frozen at -80°C for purification. Disodium ethylenediaminetetraacetic acid (EDTA)
) and sodium azide 0.05% each (W/v')
The density was adjusted to 1.063 (Ri1) by adding sodium chloride (solid). Using an ultracentrifuge (Hitachi 55P73), 000 x 9
After removing chylomicrons, very low density lipoproteins (VLDL), and low density lipoproteins (LDL) that separated and floated at 0°C, brominated (JllM solid) was added to the residue to increase the density to t21 ("m ) was also 160
,000 x 248 hours at ]0°C. Collect the floating HDL fraction, mix it with a solution with a density of 1.21 ('/7) prepared separately with sodium chloride and sodium bromide, and remove tangent substances such as realzomine by ultracentrifuging it several times. did.

The HDL thus obtained was 0.05% E
D.T.A.

The HDL fraction was obtained by dialysis against 0.09% sodium chloride solution overnight.

(2) Preparation of antigen-sensitized mouse splenocytes The HDL prepared in (1) and Freund's were injected into the peritoneal cavity of five BALB/C mice 'jiji; 5 weeks old (Japanese Charles No. ζ-).
complete adjuvant (Dlfco)
HDL] 00μ was mixed in equal amounts and administered to 7 mice for immunization.

Immunization was repeated in the same manner with HDL 100 pf/mouse at approximately 2-week intervals, and the above-mentioned Ouchte was immunized for 4 to 5 times.
A clear sedimentation line was observed by the rtnnny method.

Three days before the fusion, 50 μt of HDL was administered through the mouse tail vein, and on the day of the fusion, splenocytes were prepared and subjected to the fusion.

(3) Preparation of Max myeloid cells 8-Azaguanine-resistant mouse myeloid cells N5-1 were
- Prepared in RPM11640 medium containing azaguanine to ensure cells of 2×10 7 or more at the time of fusion and used for fusion.

(4) Preparation of hybridomas +21 The splenocytes (IXIO' or higher) obtained in +3) and mouse myeloid cells were fused at a mixing ratio of 5:1 by the method described above. Hybridomas were selected in HAT medium, antithermic wells were selected using ELIsA described above, and limiting dilution was performed twice using HT@field (HA'f' medium in which only aminopterin was removed). By culturing a hybridoma strain that stably produces antibodies in PM11640 medium, l-115E9. H3D10. H14A9
Three hybridoma strains were obtained.

(5) Purification of monoclonal antibodies in ascites The three hybridoma strains obtained in (4) were each transplanted into the peritoneal cavity of 8-week-old pristane-treated BALB10 males. After about 2 weeks, the ascites produced was collected and 3000r each was collected.
The supernatant obtained by centrifugation for pmlO was fractionated with 50% ammonium sulfate, and P
Dialyzed against BS.

A column (I
cm) were prepared, and the previous dialysis solution was separately poured into the three types of antibodies. Wash with PBS containing 0.5 M sodium chloride, and when the absorbance at 280 nm becomes 0.02 or less, add 0.2 M glycine-hydrochloric acid buffer (pH 2,3).
) was eluted. Immediately after elution, an appropriate amount of IM) lyshydroxyaminomethane solution was added to neutralize. Both fractions eluted with 0.2M glycine-hydrochloric acid buffer were collected at 0.05% ('
/V) A purified monoclonal antibody was obtained after dialysis against PBS containing sodium azide. The three purified monoclonal antibodies thus obtained were found to be single by 5ns-[electrophoresis. In addition, 5DS-electrophoresis under mercaptoethanol reduction revealed that the three purified monoclonal antibodies gave the same H chain (H chain) chain corresponding to the mouse γ chain, and all were found to be IfG class antibodies.

(6) Specificity of purified monoclonal antibodies The specificity of the antibodies obtained was confirmed by Western blotting. After the human HDL and blood serum described above were run by 5-20% gradient polyacrylic acid bielectrophoresis containing 0.1% SDS, the gel and nitrocellulose paper (Bio-Rad Inc. I!) were superimposed. Scissors in gel holder, 25mM trishydroxyaminomethane,
o (v/v) in transfer buffer (pH 8,3) containing 1g 2mM glycine, 20% (v/v) methanol.
The gel was transferred at 4° C. for 4 hours, and the protein and protein separated in the gel were transferred to nitrocellulose gel. Nitrocellulose penoso after transfer
I cut it out and part of it is Comazie Brilliant Blue 250.
The remainder was immersed in PBS containing 1% comb serum albumin for 1 hour, and then reacted overnight at 4°C with bath solutions of each of the three purified monoclonal antibodies. 0.05% Twee
After washing with PBS containing n20, horseradish peroxidase-conjugated anti-mouse IgG antibody (manufactured by Bio-Rad)
After reacting at room temperature for 2 hours, 0.05 tween
20 in PBS. The positions of spots appearing after 30 minutes of reaction with a substrate solution (Bio-Rad) containing 4-chloro-1-naphthol were compared with bands already stained with Comasi-Brilliant Sol-R250. The results are shown in Figure 1.

That is, Figure 1 shows the specificity of purified monoclonal antibodies.
After separation by gradient polyacrylamide gel electrophoresis containing 5-20% S, the cells were transferred to a nitrocellulose membrane by Western blotting. In the figure, A shows Comassie Brilliant Blue R250 staining of the protein transferred by Western blotting, and B to D show the purified monoclonal antibody H1sE9. after being transferred by Questan blotting. H3
D10. The staining diagram obtained by reacting with each solution of Hl 4A9 at 4°C overnight, reacting with peroxidase-conjugated anti-mouse IPG antibody, and then contacting with a substrate solution containing 4-chloro-1-naphthol is shown. .

As is clear from FIG.
T? It was confirmed that the antibody did not react with any protein other than h-■ and showed specificity only for apoA-7.

(Differences in the recognition sites on apoA-I of the 3 types of monoclonal antibodies 3f1 to investigate whether the antigenic determinants on apoA-I that each monoclonal antibody recognizes are different from each other.) In the enzyme labeling method, monoclonal antibody molecules were digested with porcine gastric juice pepsin (Sigwa) to obtain F(ab').

Fab' obtained by reducing with mercaptoethylamine
and horseradish peroxidase into which a maleimide group has been introduced (Boahringer Manheim & Rito (Immunology Experimental Procedures 'iJ, I) 34
97゜Japan Society of Immunology, 198).

The difference in recognition sites between these enzyme-labeled antibodies and non-enzyme-labeled antibodies was estimated by measuring their ability to compete with HDL.

ELISA coated with HDL as described above
Add the above enzyme-labeled antibody (0.2p//I/) and enzyme-unlabeled antibody (0 to
Add 50 µl of isostatic mixture of 1000 µP/nt) and leave it to stand at room temperature for 2 hours, then wash with PBS containing 0.05% Tween 20 and use 2.2'-azinody (3-
Diammonium salt (A
BTS (manufactured by Sigma) and R20 were reacted (at room temperature for 30 minutes), and the reaction was stopped with 6 succinic acid.
The absorbance at was measured.

The results are shown in Figure 2.

This figure 2 shows the competitive ability of enzyme-labeled and unlabeled monoclonal antibodies against HDL. Figure A is H3D] O).

(1114A9 in Figure 2B)] and enzyme-unlabeled antibody (0-
1000 μr/d: H35E9. l-114A9. Or add 50 μt of isostatic mixture of H3D10), and
After standing for an hour, it was washed and further used as a substrate with 2.2'
-Azinodi-(3-ethylbenzthiazoline sulfate)-diammonium salt and H2O2 are reacted at room temperature for 30 minutes, and after the reaction is stopped with 6-thioxylic acid, the absorbance at 405 nm is measured. In the figure, Bo=absorbance in the absence of an enzyme-unlabeled antibody, B=absorbance in the presence of an enzyme-unlabeled antibody.

The above results revealed that the three types of monoclonal antibodies do not compete with each other and have different recognition sites.

Example 2 Of the three monoclonal antibodies obtained in Example
14A9 was added to 0NBr f & sexized Sepharose 4B.
The immunoadsorbent was coupled in 0.1 M hexatrichum bicarbonate buffer containing 5 M sodium chloride, pH 8.3, and 1.81 q of monoclonal antibody was bound per gel (3 mA was prepared as a gel).

This was packed into a glass column with an inner diameter of 1 cln and equilibrated with PBS containing 0.5M Jll rum (chloride). The aforementioned human plasma l- was applied to this column, washed with PBS containing 0.5 M sodium chloride, and when the absorbance of the effluent at 280 nm became 002 or less, it was eluted with 0.2 M glycine-hydrochloric acid buffer.

The eluate was immediately neutralized. This fraction is mostly ayl
IJJ with fA-1 and some apoA-11 and apo0 group
They were tan and lithium particles.

After dialysis of this fraction against PBS, ethanol:ether = 3:2
The apoprotein was then degreased with ether and dried with nitrogen gas. This was dissolved in PBS, applied to the same column again, and eluted in the same manner. The fraction was dialyzed against 0.1 M ammonium carbonate and lyophilized. This fraction is 5DS
- Electrophoresis showed only a single node of ApoA-■.

By this method, apoA-I can be isolated and purified without ultracentrifuging blood plasma.

[Brief explanation of the drawing]

Figure 1 A, B, O, D shows the specificity of the purified monoclonal antibody of the present invention.
The samples were separated by 5-20% gradient polyacrylamide gel electrophoresis containing 1% SDS and then transferred to a nitrocellulose membrane by Western blotting. Figures 2 A and B show the competitive ability of enzyme-labeled and unlabeled monoclonal antibodies against HDL.
/mt:C Figure 2 A is H3D10), (Figure 2 B is H
14A9)) and enzyme-unlabeled antibody (0-1000 μf/m
/ : H15E9. H14A9 or H3D10
) was added to 50 μt of isotopic solution, left to stand at room temperature for 2 hours, washed, and further added with 2,2′-azinozyme (
3-Ethylbenzthiazoline sulfate)-diammonium salt and H2O are reacted at room temperature for 30 minutes, and after the reaction is stopped with 6% oxalic acid, the absorbance at 405 nm is measured.

Claims (1)

[Scope of Claims] 1. A monoclonal antibody against human apolipoprotein A-I, characterized in that the monoclonal antibody belongs to the IgG class. 2. Three types of monoclonal antibodies according to claim 1, in which the recognition sites on apolipoprotein of the monoclonal antibodies are different from each other. 3. A method for purifying highly pure apolipoprotein A-I from human serum or blood plasma by affinity chromatography using one, two, or three monoclonal antibodies with different recognition sites on human apolipoprotein as ligands. . 4. Hybridomas obtained by fusing splenocytes obtained by immunizing mice with human high-density lipoproteins and mouse myeloid cells in a conventional manner to produce human apolipotank chambers A-I.
Hybridomas that produce monoclonal antibodies specific only for A method for producing a monoclonal antibody against human apolipoprotein A-I, which comprises collecting the antibody.