JPS63148994A - Monoclonal antibody, hybridoma, production of monoclonal antibody and separation of human protein s - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody as against human protein S, by cultivating a hybridoma capable of producing a monoclonal antibody specifically linking to the protein S and a monoclonal antibody to C4bp-protein S. CONSTITUTION:Protein S is isolated, purified from human blood plasma and used to immunize a mouse. Splenic cells of the mouse are then fused to mouse myelomatous cells to provide hybridoma cells, which are systematically tested for an antibody reactive with human protein S immobilized in a microtiter plate and selected. The obtained hybridoma is then cultivated to afford the aimed antibody.

Description

Detailed Description of the Invention a. Industrial Application Field The present invention is directed to a monoclonal antibody that specifically recognizes and binds to human protein S, a C4b binding protein (C4b bindi) of human protein S and human complement system regulator.
ng protein; C4bp) that specifically recognizes and binds to the complex with human protein S, hybridomas that produce these antibodies, 17'4 methods for these antibodies, and methods for separating human protein S. .

b. Prior art Protein S, like protein C, is a vitamin-dependent protein and was isolated from cows and humans by Dicipio et al. in 1977 [Di 5cipio, R.

G, total 1ermodson, M, A,, Yat
es, 3. G. andDavie, E.
W.: Biochenistry,,16:
698-706, (1977)].

Human protein S is present in human plasma at approximately 10rng7/
It is a single-chain glycoprotein with a molecular weight of approximately 69,000. In addition, its structure is very similar to that of other vitamin-dependent factors, and it has about 10 γ-ruboxyglutamic acid (Gla) groups at the NH2 terminal. Protein S exists in two forms in the blood. One of them is free protein S, and this free protein acts as a cofactor for activated protein C. The other exists in a non-covalent bond with the polymeric C4b-binding protein (hereinafter this protein may be abbreviated as "C4bp"), which is a regulator of the complement system. The protein S ratio is said to be approximately 1:1.

The importance of this function of human protein S is that it has a very strong affinity for the negatively charged surface of phospholipids [Ne1se
stuen, G., L., K15iel, W.
and[)i 3cipio, R,G, :
3iochemistry, 17; 2134-21
38. (1978)].

When cells are injured or activated, the QIa-, domain of protein S binds to phospholipids in the presence of Ca2, and C4bp also binds to this protein S.
It is thought that they form a complex and combine to exert their functions [[)ahlbjck, B,:Se
m1n, Thromb, HaelnO3taS,
, 10: 139-148° (1984)].

Regarding the functions of protein S and protein C related to coagulation and PJ lysis system, recent research has revealed that they play an extremely important role in the control mechanism, and their physiological significance has also been investigated in relation to thrombosis. Attention has been paid. It has been reported that congenital deficiency of protein S can cause thrombosis [Comp, P, C,,N
1xon, R, R1゜Cooper, M, R,
and Esmon, C.T.: J.

CIin, Invest, 74: 20
82-2088. (1984) ].

Therefore, the mechanism of action of protein S was clarified, and the antigen m of protein S in blood was clarified.

If we can measure the amount of activity and understand its trends,
This is considered to have a very important meaning in the fields of basic medicine and clinical medicine.

On the other hand, motuclonal antibodies are specific for a single antigenic determinant, and have the advantage of being able to stably produce antibodies with the same specificity. RIA) has recently become widely used in general. Especially functional analysis of antigen proteins.

A powerful means for molecular analysis is to find antibodies that recognize sites involved in the function of antigenic proteins or special structural sites.

The present inventors focused on the fact that protein S forms a complex with C4b binding protein (C4bp), which is a regulator of the complement system, and selected free protein S and a complex of protein S and C4bp. As a result of conducting research on monoclonal antibodies that recognize

Structure of the C8 Invention That is, the present invention involves culturing a hybridoma that produces a monoclonal antibody that specifically binds to protein S and a monoclonal antibody to C4bp-protein S, and extracting the human protein S from the resulting product.
Obtaining monoclonal antibodies against human protein S
This is a method for producing monoclonal antibodies against.

Still another feature of the present invention is that a human protein S-containing mixture is brought into contact with an adsorbent in which a monoclonal antibody against human protein S is bound to an insoluble carrier, so that human protein S is bound to the adsorbent. This is a method for separating human protein S from a human protein S-containing mixture.

Hybridoma cells producing the monoclonal antibodies of the present invention can be prepared by the method of Köhler and Milstein [K6hle
r&M 1lstein; N atu
re: Yu-sei.

495-497 (1975)] by a method known per se. After immunizing a mouse with Hi-Protein S, the spleen cells of the mouse were fused with mouse myeloma cells, and the resulting hybridoma cells were immobilized on a microtiter plate.
Systematically tested for antibodies that react with protein S,
selected.

The monoclonal antibodies of the present invention are obtained from products produced by such novel hybridoma cells, and act specifically against human prodin-S.

The monoclonal antibody of the present invention has the property of highly specific binding to human protein S.
It is very useful when used for the separation and purification of Protein S. That is, one type of monoclonal antibody according to the present invention is immobilized on an insoluble carrier, and human protein S is adsorbed from crystals, other human protein S@containing mixtures, or crude extracts, crude purified products, and solutions thereof.

Separate and add in a suitable buffer (e.g. 20mM Tris
-HCa, 0.5M Na CR, pH 7,4)
After washing, transform the solution into solution 3 containing chaotropic ions.
Human Protein S can be eluted by replacing M Na5CN I)87.0).

Next, a specific method for producing a monoclonal antibody according to the present invention will be explained in detail.

Isolation and purification of A, JJ: Human protein S used as an antigen was prepared by the method of Bajaj et al. [Bajaj SP, Rapaport Sr.
.

Maki SL, Brown SF: Pr
ep, Biochem.

13, 191. (1983)] was isolated and purified from human plasma.

B. Immunization of mice with human protein S: 11Ba
Although lb/C mice can be used, other strains (Str
ain) mouse may be used. The immunization regimen and the concentration of human protein S should then be chosen so that lymphocytes with sufficient antigenic stimulation are formed. For example, 50 μ3 of human protein S was given to mice for 2 days.
After three intraperitoneal immunizations at weekly intervals, an additional 30 μl of the vaccine is administered intravenously.

A few days after the final immunization, spleen cells are removed for fusion.

Ω-"L1 屡"L2 The spleen of the mouse immunized as above was removed aseptically.
From there, prepare a single cell suspension. These spleen cells are fused with mouse myeloma cells from an appropriate line using an appropriate fusion promoter. A preferred ratio of splenic cells to myeloma cells ranges from about 20:1 to about 2:1. For approximately 108 spleen cells, use of 0.5-1.5 fusion media is suitable.

Mouse myeloma cells used for cell fusion are well known, but in the present invention, P3-X63-"Ag8-Ul
Cell (P3-U1) [Yelton, D,
E.

et al, Current, Topics in
Microbiology and l mmu
nology, 81. 1 (1978)].

Preferred fusion promoters include, for example, an average molecular weight of 1+o
Polyethylene glycols from 0 to 4000 can be advantageously used, although other fusion promoters known in the art can also be used. In the present invention, the average molecule ff
i 1540 polyethylene glycol was used.

D, fused cells; mix the unfused spleen cells, unfused mouse myeloma cells, and fused hybridoma cells in a separate container (e.g., microtiter plate) without supporting the unfused mouse myeloma cells. Dilute with selective medium and culture for a sufficient time (about 1 week) to kill unfused cells. The medium should be one that is drug resistant (e.g. 8-azaguanine resistant) and does not support unfused mouse myeloma cells (e.g. HA
T medium) is used. In this selective medium, unfused myeloma cells die. Since these unfused spleen cells are non-tumor cells, they die after a certain period of time (one week).

Cells fused to these cells have both the tumor properties of the parent cells of myeloma and the properties of the parent flimsy cells, so they can survive in the selective medium.

E. Confirmation of antibodies against human protein S in each container; thus, after hybridoma cells are detected, their culture supernatants are collected and analyzed for antibodies against human protein S by enzyme immunoassay (FzymeL 1nked).
1 mmunosobent A 5Say).

Once a hybridoma cell producing the antibody of interest is cloned by an appropriate method (eg, limiting dilution), the antibody can be produced in two different ways. According to the first method, monoclonal antibodies produced by hybridoma cells can be obtained from the culture supernatant by culturing hybridoma cells in an appropriate medium for a certain period of time.

According to a second method, hybridoma cells can be injected into the peritoneal cavity of isogenic or semi-isogenic mice. Monoclonal antibodies produced by the hybridoma cells can be obtained from the blood and ascites of the host animal after a certain period of time.

The monoclonal antibody against human protein S is immobilized or bound to an insoluble carrier to obtain an adsorbent. The insoluble carrier used in this case may be one that is commonly used as a base material for measurement reagents or measurement kits using monoclonal antibodies. For example, Sepharose, polyacrylamide, cellulose, dextran, maleic acid polymer, or a mixture thereof is preferably used as the material. These inert carriers may be in the form of two powders, pellets, or beads.

It can be in various forms such as fibrous. Furthermore, it is advantageous to use a plate (well) having a large number of concave depressions, which is generally used for measuring or separating plasma or its fractionated components.

When the human protein S-containing mixture is brought into contact with the adsorbent, the monoclonal antibody immobilized on the adsorbent binds to human protein S, and as a result, human protein S is attached to the adsorbent. Join. In this manner, human protein S can be separated and removed.

In addition, human protein S is bound to the adsorbent as described above, the residual mixture is washed away if possible, and then the human protein S bound to the adsorbent is treated with a suitable solution containing chaotropic ions (SCN-), etc. When contacted with or washed with a suitable solution, human protein S detaches from the adsorbent, and by isolating it, human protein S can be isolated.

Thus, according to the separation method of the present invention, removal of human protein S from a mixture containing human protein S, separation and purification of human protein S from the mixture,
Measurement of the content of human protein S in the mixture can be accomplished with extremely simple operations.

The present invention will be described in detail below with reference to Examples. In the following examples, protein S may be abbreviated as PS.

Example 1 Two female Balb/C mice (4 weeks old) were immunized four times at 14-day intervals with purified bunny PS.

The first immunization was dissolved in PBS. 50 μ9 Hi]~・P
S with an equal amount of complete Freund's adjuvant (Compl.
ete Freund's adjuvant
) and the emulsion was administered intraperitoneally (
0.5Rg/head), the second and third times,
The same <50μ3 of human PS was added to Freund's incomplete adjuvant (Freund's incomplete adjuvant).
te adjuvant) and similarly administered intraperitoneally. For the final immunization, 30 μ7 of human PS was additionally administered as a PBS solution through the mouse tail vein. Spleen cells from immunized mice 3 days after the final immunization were used for cell fusion.

Mixing immunized mouse milia cells and syngeneic mouse myeloma cells (PIJl) at a ratio of about 2:1 to about 15:1,
50% polyester glycol 1540 (manufactured by Wako Hyakuyaku Co., Ltd.) as a fusion promoter and K0hler and M1lste.
Cell fusion was performed according to the method of In. The cells after fusion are
Suspend in 10% FC3-RI)MI-1640 medium to a cell concentration of 1x10' cells/adl, and place in 96 wells. Microplate (Cost)
er) at a rate of 100 μρ per well.

The fused cells were incubated in a CO2 incubator (5% C○2,3
Hyboxanthin, aminopterin:
Replace the medium with a medium containing thymidine (HAT medium, and
Hybridomas consisting of spleen cells and myeloma cells were screened by growing them in AT medium.

Antibodies in the culture supernatant of hybridomas were extracted using a microtiter plate coated with antigen human PS.
Detected by LISA method. For the second antibody, an alkaline phosphatase-labeled rabbit antibody and mouse IgG antibody was used,
The binding to antigen PS was examined. Out of a total of 494 wells in which the fused cells were seeded, colony formation was observed in 487 wells, and among these, 94 wells were positive for producing antibodies that showed binding to the antigen PS.

Out of these antibody-producing positive wells, four wells were subjected to cloning tests using the limiting dilution method twice, and six clones were obtained. 90% clones obtained
was suspended in Fe2-10% DMS○ and stored in liquid nitrogen.

The monoclonal antibodies produced by each clone were grown intraperitoneally in 3alb/C mice and purified from the ascites using a protein A-Sepharose 4 B column.

Table 1 (Cell fusion) Example 2 (Properties of purified monoclonal antibodies) ICIG of each clone purified from mouse ascites was examined for class and binding to human protein 5 (PS).

The class of mouse monoclonal antibodies was determined by the Ophthalony method using anti-mouse antiserum specific to each class.

The results are shown in Table 2 below.

The binding property to human protein S is suitable for human protein S immobilized on a microtiter plate!
Evaluation was made by reacting with a monoclonal antibody diluted once and detecting with goat anti-mouse IQG labeled with alkaline phosfactorase.

As a result, the binding strength of six types of monoclonal antibodies to human protein S was 289F12 to 239C1.
0>3C3G8>3C4G4>289G3>>2E12
It turned out to be C7.

The results are shown in attached Figure 1.

Table 2 Monoclonal antibody class example 3 [Reactivity towards human C4b binding protein and protein S complex (C4bp-PS complex)]
Coat Microtiter Plate 1 at a concentration of -, add healthy human plasma diluted with 1% BS8 to a concentration of Blocking 111, iM,
The C4bp-Protein S complex in plasma was reacted with a monoclonal antibody. Next, an alkaline phosphatase-labeled anti-C4bp antibody was added to detect and examine the binding properties of the six monoclonal antibodies to the C4bp-brotin S complex.

As a result, monoclonal antibody 2E12C7 has very weak binding to free prodin S, but
It was found that it showed highly responsible binding to the bp-protein S complex.

The results are shown in FIG. 2.

These various antibodies were analyzed using immunological 3111 assay methods (,E
I△, RI△), free human protein S and C4 in solution state (e.g. human plasma)
It is thought that it can be applied to the measurement of bp-protein S complex.

Example 4 (Separation method of human protein S) (1) Immobilization of antibody on insoluble carrier; Bromcyan-activated Sepharose 4B (Pharmacia)
0.5 g of dry gel (manufactured by Fine Chemino J's),
1001d on G3 glass filter! 11+1M
Swell and wash using HCI, and further add camping buffer (0.1M NaCl containing 0.5M NaCl).
a Washed with HCO3DI-18,3>. After removing the coupling buffer 1- by suction, immediately remove the gel from the antibody (6).
Coupling buffer solution of H2> (3IffL/m
1) Add the solution to 2 d of water, suspend it, and slowly photograph it at 4 degrees Celsius overnight. The gel was then diluted with 1 M ethanolamine-Hap.
(II 8,0.:2Id) and shaken at room temperature for 2 hours to block remaining active groups. After blocking, run the antibody-bound Sepharose gel on a glass filter with 0.1Ml! containing 0.5M NaC9! ! Acid buffer pH 4.0, and 0.1M boric acid buffer p containing 0.5M NaC2 (8.0) were used alternately for washing.The absorbance of the filtrate at 280 nm was 0.0.
When it becomes less than 1, add 0 containing 1mM benzamidine.
．． The column was equilibrated with 05 M Tris/HCf pH 7.4 and packed into a column. Affinity chromatography was performed using the thus prepared anti-human PS monoclonal antibody (289C10)-coupled Sepharose 4B column.

8 d of IM 5acf2 solution was added to 100 ml of plasma in 13 mouthfuls and stirred at 4°C for 1 hour. Centrifuge and collect the precipitate for 5 m
M8a CJz, after washing with 0.1~jNacf containing 5 mM benzamidine, 15d 0.2M' EDTA containing 5 mM benzamidine I) H7
, 4 to obtain a barium adsorption fraction. This barium-adsorbed fraction was mixed with 0,
0.05M Tris/HCj DI-17,4 and 0.05M Tri containing 1 mM benzamidine.
Antibody 289C10 binding column equilibrated with S/H (J.
0,05 MTris/l-1 (J l) including fi
Wash with H7,4, 3M Na5CN I) l-17
, 0 solution, a single beak containing PS was obtained. The recovery rate was about 75.4%.

[Brief explanation of the drawing]

Attached Figure 1 shows the strength of the binding of the monoclonal antibody of the present invention to human protein S, and Figure 2 shows the strength of the binding of the monoclonal antibody of the present invention to the C4bp-Protein S complex. It is something that (Procedure NERT) J-E Book 1986/Mon) kto1 'F4J'i; , Agent (1) Correct "Basic Medicine" on page 5, line 5 of the detailed letter to "Basic Medicine." (2) On page 6, lines 5-6, "monoclonal antibody against rc4bp-protein S" is corrected to "monoclonal antibody that selectively recognizes rc4bl)-protein S complex". (3) "Crystal" on page 7, line 15 is corrected to "human self-plasma." (4) At the end of the same page 7, replace [Solution 3MJ with "Solution (e.g. 3
Correct MJ. (5) l' (Fnzyme L) on page 11, lines 1-2.
inkedImmunosobent As5ay)
J to r (Enzyme LinkedImmunos
Orbent As5ay) Correct as J. (6) 1 Polyester Glycol, page 14, line 14”
is corrected to "polyethylene glycol". (7) On page 14, line 15, [to ohlerJ to 1 to 6h
Correct lerJ. (8) [Rabbit antibody mouse 1] on page 15, lines 10-11
gG J is corrected as [Rabbit anti-mouse I (IG J). (9) 90% of the clones are Fe
2-10% D) "Clone in fsO" in 90% FC
3-10% DI-130 medium” is corrected. (10) "Colony format" in the left column of Table 1 on page 16
is corrected to "formation of one colony." (11) "(Purified monoclonal antibody" on page 17, line 1) should be corrected to "(purified monoclonal antibody"). (12) 12B9F12~ on page 17, lines 4-3 from the bottom.
239C10" is corrected to 12B9F12-2B9C10J. (13) "Composite" in line 1 of page 19 is corrected to "complex". (14) "Antibody (6112) J" on page 20, lines 1-2 is corrected and reversed to "monoclonal antibody (6N2) J." (15) "4 degrees" on page 20, line 3 of the same page is changed to "4 degrees". C,” he corrected. (16) On page 20, lines 16-17, "Affinity Croc" is corrected to "Affinity Chroma 1-". (17) On page 21, line 7, [“stoning”] is corrected to “dialysis.” (18) Change “Tris/To 102” on page 21, line 8 to [
Corrected as pH 7,4J. that's all

Claims (1)

[Claims] 1. Human protein S and human complement system regulator C4b
A monoclonal antibody against human protein S that specifically recognizes and binds to a complex with a binding protein. 2. A monoclonal antibody against human protein S that selectively recognizes free human protein S but does not recognize the complex of human protein S and the C4b binding protein of human complement system regulator. 3. A hybridoma that produces a monoclonal antibody against human protein S according to items 1 and 2 above. 4. A method for producing a monoclonal antibody against human protein S, which comprises culturing the hybridoma according to item 3 above and obtaining a monoclonal antibody against human protein S from the product. 5. A monoclonal antibody against human protein S was added to an adsorbent bound to an insoluble carrier.
Human Protein S, characterized in that human Protein S is bonded to the adsorbent by contacting a mixture containing the same.
A method for separating human protein S from a containing mixture.