JPS63230700A - Monoclonal antibody to c4b-binding protein - Google Patents

Abstract

NEW MATERIAL:Each monoclonal antibody having immunogen crossing reactivity to only one antigenic determinant among the antigenic determinants existing in a C4b-binding protein. USE:A reagent for measuring free protein S of blood coagulation factor. PREPARATION:For example, a fresh plasma of healthy person is treated with barium citrate to adsorb and elute a C4b-binding protein,which is then purified with a column chromatography, etc., and the purified protein is administered to a Balb/c mouse and immunized and an 3rd days after final immunization, the spleen is extirpated to provide a spleen cell, which is then cell-fused with a bone marrow cell and cultivated in a HAT culture medium to give a hybridoma. Further a clone specific to C4b-binding protein is selected from the hybridoma and cloned to afford the monoclone, which is then cultivated to provide the monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a monoclonal antibody against C4b binding protein useful for quantifying protein S, which is known as a blood coagulation regulator.

In recent years, it has been revealed that one of the causes of thrombosis such as cerebral infarction and myocardial infarction is quantitative and qualitative changes in blood coagulation control factors.

Protein C and protein S, a related factor thereof, have been studied as blood coagulation control factors.

Approximately half of this protein S exists in the blood as a complex bound to C4b binding protein (hereinafter referred to as C4bp), and this complex has no blood coagulation control effect, and free protein S It has been revealed that it has this effect (J, C11n, Tnvest, 74,
2082-2088 (1984)).

In addition, C4bp has a molecular weight of about 7 subunits with a molecular weight of 75,000 connected by S-8 bonds, sso, oo
This C4bp is a high molecular weight protein with a molecular weight of 160. It was separated into a fragment of Goo and a fragment with a molecular weight of 48,000, and the C4bp
The protein S binding site in this molecular weight 160
．． It has been revealed that it exists on the fragment side of Goo.

Conventionally, immunoassay methods or biological activity assay methods are known as methods for measuring protein S in blood. In immunoassays, protein S complexes and free protein S in blood are It only measures the total amount of
Furthermore, the biological activity measuring method only indirectly determines the activity of protein S through the measurement of protein C activity.

On the other hand, in the blood, there is C4bp that does not form a complex with protein S (C4bp that has a binding site for protein S).

Therefore, C4b that is not complexed with protein S
If it is possible to quantify each of p (uncomplexed C4bp) and total C4bp, it is possible to calculate the amount of free protein S from the measurement of the total amount of protein S complex and free protein S. becomes.

The present inventors succeeded in obtaining monoclonal antibodies having immunological cross-reactivity with only one of the antigenic determinants present in C4bp.

By using the monoclonal antibody according to the present invention,
Free prolein S in blood can be quantified as follows.

(2) Among the monoclonal antibodies according to the present invention, select those that have the ability to bind to the protein S binding site in C4bp.

Using the monoclonal antibody selected in ■■ and the monoclonal antibody that does not have the above binding ability, Il! Free @C4b in blood by LISA method, EXA method or IIIA method
Quantify p (this amount is defined as P).

■ Among monoclonal antibodies that do not have the above binding ability, C
Total C4bp in blood is quantified by ELISA, EIA, or RIA using two monoclonal antibodies with different binding sites at 4bp (this amount is defined as Q).

(2) The total amount of protein S complex and free protein S in the blood is determined by a known measurement method (this amount is designated as R).

(2) The amount of free protein S (X) can be calculated using the following formula.

X=R-(Q-P) The following is a method for obtaining monoclonal antibodies against C4bp according to the present invention, as well as the binding ability, specificity, etc. of these monoclonal antibodies.

A specific example will be described using an example.

Example 1 Preparation of anti-human C4bp monoclonal antibody (m) Antigen-
Preparation of human C4,bP Biocham, J, 209, 837-846 and 8
47-856 (1983), four breaths of fresh plasma from a healthy individual was adsorbed to barium citrate, and C4b p tt in the eluate was adsorbed to barium citrate.
DI! AH-8ephacal heparin-8aphar
osa (Kabi 11) and 5aphalosa
It was isolated and purified by CL-68 (Pharmacia) column chromatography. Purified human C4bp was purified from J, Mo1. Biol, 80.579-599 (
The molecular weight was approximately 70 when examined by sodium sulfate-polyacrylamide gel electrophoresis (SO5-PAGE) in the presence of a reducing agent according to the method of Lag-11 et al. (1973).
,000 Daltons (D).

(b) Preparation of antibody-producing cells Two 6-week-old Ha Lb/c female mice are first immunized with the purified human C4bp described in (8) above in Freund's complete adjuvant. 5 for each of those mice
0 μg of human C4bp is administered intraperitoneally as a solution of 0.4 a jl. Furthermore, on the 15th day, a booster dose of 50 μg of human C4bp dissolved in physiological saline is given. As an initial and final immunization, supplementary immunization is performed by intravenous administration (45 μg, 7100 μg of physiological saline) on the 52nd day, and 3 days later, the mouse lungs are removed and lung cells are harvested.

(C) Cell fusion (1) Use the following materials and methods.

RPMI 1640 medium: RPMI &1640 (
Difco I, abo-ratorlai), sodium bicarbonate (12mM), sodium pyruvate (1w
M), L-glutamine (2mM), penicillin G potassium (500/-Ω), streptomycin sulfate (50u
g/-m), and amikacin sulfate (100 μg/-m)
m Q ) to 1, adjust the P1 soda to 7.2 with dry ice, and sterilize and filter with a 0.2 μ Soon Toyo membrane filter.

-MS-1 medium: sterile-filtered calf fat serum (M,
) to a concentration of 15% (V/V).

PE64,000 solution: Polyethylene gelicol 4,000 (PEG 4,0
00, Merck Is Co.

Tnc,) 50% (v/w)! a. Prepare serum solution.

8-azaguanine-resistant myeloma cells MS-1 (P3-
Fusion with NSI-1) is 5alectad Metho
d 1nCallular Itsunology (
ad, B, B, Mighall and S, M, Shi.
igi), V, Cao 1. Freeman and
Coa+pany (1080)351-372
The method of Ol et al., described in , was performed with some modifications.

(2) Nucleated lung cells prepared in (b) above (viable cell rate 1
00%) and myeloma cells (viable cell rate 100%) at 5
: Fuses at a ratio of 1. The lung cells and myeloma cells were washed separately with the above-mentioned RPMT 164G medium and then suspended in the same medium for fusion.

Mix in the above proportions. A conical styrene resin test tube (Iwaki Glass+) with a capacity of 50 Ω was used.

39 ■ Sad (F) 400 in RPMI 1640 medium
Centrifuge at Xg for 10 minutes and aspirate the supernatant completely. The precipitated cells were heated at 37°C P[! G 4,00G solution 4.5aQ
was added dropwise over 1 minute with gentle stirring, and stirred for another 1 minute to resuspend and disperse the cells. 6. Next, heat at 37°C.
Drop 4.5 sQ of I'MI 1640 medium over 1 minute.

After repeating this operation] and pJ, the same medium 32
Add mfi dropwise for 2 to 3 minutes with constant stirring to disperse the cells. This is centrifuged at 400 x g for 10 minutes, and the supernatant is completely removed by suction.

Next, this precipitate 1III cell was added to 37°C heated MS-1 medium 4.
Immediately add 5-Q and carefully pipette to disperse large clumps of cells using a 10-inch pipette. Furthermore, 90ml of the same medium was diluted, and the polystyrene 1196-well microwell (Ivaki Gla
ss), add 6.0 x 10' cells per well at 10,1 mA. Note 1. For the 966 microwells used at this time, 0.2-0 MS-1 medium was added as a pretreatment.
Incubate overnight in a carbon dioxide gas incubator (37°C), and remove the medium by suction before use.The above microwell containing the a-cells was incubated in 7% carbon dioxide, 793% air at a temperature of 37°C and a humidity of 100°C. Subject to culture below %.

(d) Selective growth and growth of hybridomas using selective media (1) The culture medium used is as follows.

HAT medium: Hypoxanthine (100 μM), aminopterin (0,
4 μM), and thymidine (16 μN).

11T medium 11 above except that niaminopterin was removed.
It has the same composition as AT medium.

(2) The next day (1st day) after starting the culture in (e) above, apply 2 drops of IIAT medium (approximately 0.1aQ) to the cells using a Pasteur pipette.
) is added. 2.3.5.8.11 Half of the 100-day medium (
0, beQ) was replaced with fresh HAT medium once, and the culture medium was incubated for 14 days.
Replace half of the medium with fresh HT medium. From then on; 3
Replace half of the medium with fresh HT medium every ~4 days.
Usually, sufficient hybridoma growth is observed within 2 to 3 weeks (fusion rate 62%), and the solid phase-antibody binding test method described in the next section (e) is performed for all hybridoma-growing wells ([!
Next, check the positive wells by LISA) containing 107 mouse thymocytes as feeders.
1sfi of the medium was placed in a polystyrene 24-well cell well (Tw
akl Glass) was used to transfer the entire contents of each of the positive hybridomas detected above, and in the same manner as in (c) above, in the presence of 7% carbon dioxide, 37
Culture at ℃ for about 1 week. In the meantime 1~2Ii! 1. Replace 0.5 μl of supernatant in each well with 0.5 μl of fresh HT medium. Once the hybridoma has grown sufficiently [! L
Reconfirm the positivity using the ISA method, and perform cloning for each sample using the limiting dilution method described in the next section (f).

In addition, the remaining liquid after being used for cloning was transferred to polystyrene 11.
25-Transfer to tissue culture flasks (Tvaki Glass) and prepare samples for cryopreservation.

(6) Search for anti-human C4bp antibody-producing hybridomas by solid phase-antibody binding test (R1, TSA)Ana1.
Biochew, 104, 205-214 (198G
A slightly modified method of Rannard et al. described in ) is used. This method is suitable for detecting hybridoma antibodies. 96-well microtight region plate (F
low Lahoratorias, Inc.) to 0.
Coat with 5-1.0 μg of human C4bP and then block the uncoated areas with 1% bovine serum albumin (BSA). A portion of the supernatant of the hybridoma growth well obtained in (d) above is added to this and incubated at room temperature for about 1 hour. Horseradish peroxidase-labeled goat anti-mouse immunoglobulin (Cappal L) was used as a secondary antibody.
ab,) and further incubate for approximately 1 hour at room temperature.

Add hydrogen peroxide and the substrate 0-phenylenediamine, and qualitatively judge the degree of brown color produced by in-house staff, or
Alternatively, the corona two-wavelength microplate photometer (HTP)
-22, Corona Denki Co., Ltd.) to measure the absorbance at 500 nm.

(f) Cloning After the operation in (d) above, since there is a possibility that two or more types of hybridomas are growing in each well, cloning is performed by the limiting dilution method to obtain monoclonal antibody-producing hybridomas. A cloning medium containing 107 mouse thymocytes as feeders per all MS-1 medium was prepared, and 36 wells of 96 six microwells were prepared.

5 per well for 36 wells and 24 wells.

Add 1 and 0.5 hybridomas.

On days 5 and 122, add approximately 0.1 mjl of N5 to all wells.
-1 medium is added. Sufficient hybridoma growth was observed 14 to 15 days after the start of cloning, and for groups with 50% or more of wells negative for colony formation [! LIS
Perform method A. If all wells tested are not positive, check the number of colonies in the antibody-positive wells, select 4 to 6 wells in which one colony was confirmed, and re-clon.

Finally, 20 hybridoma strains producing monoclonal antibodies against human C4bp were obtained.

(g) Propagation of monoclonal antibodies in vitro and in vivo Monoclonal antibodies are propagated by conventional methods. That is, the monoclonal antibody targets each hybridoma obtained with N5
-1 culture medium or other suitable culture medium (in vitro expansion M),
Monoclonal antibody protein can be obtained from the culture supernatant (monoclonal antibody protein concentration is 10-1009 g/■). On the other hand, in order to obtain antibodies in large quantities, it is necessary to obtain antibodies from animals syngeneic to the animals from which the lung cells and myeloma cells were derived ( Ba1b/c, mice) were treated with the tumorigenic agent bristane (2,6,10,14-tetramethylpentadecane, ^1drich Chamica
Co, Inc.) at 0.5 ml per mouse.
After 1 to 3 weeks, each hybridoma was administered 1xt.
o' pieces are also administered intraperitoneally. After 1 to 2 weeks, the monoclonal antibody protein concentration is 4 to 7 ■g/■Ω.
Ascites fluid obtained in (g) above can be obtained (in vitro propagation (h) heavy chain, light chain, and isotype of monoclonal antibody. Each of the ascites fluid obtained in (g) above is first transferred to a microtitrecimin plate coated with human C4bp as described above. Combine according to the RLISA method.

After washing with PnS, the isotype-specific ~heron anti-mouse rg antibody (Zymad Labcsratorias
) is added. After washing with P[lS, horseradish peroxidase-labeled goat anti-rabbit IgG (H+I,) antibody was added and detected using 2,2'-azinodi(3-ethylbenzothiazoline sulfate-6) and hydrogen peroxide as J[. did. The results are summarized in Table 1 below.

Of the obtained monoclonal antibodies against human C4bp, 14 were directed against the immunoglobulin chain γ11 and 5 were directed against the 12a/12a/
and one had γ2b/.

(i) Purification of monoclonal antibodies Ammonium sulfate fractionation of each ascites obtained in (g) above (40% saturation)
After that, IgG, type sodium chloride 0.06M,
IgG, a and IgG, b types are sodium chloride 0.
The non-adsorbed fraction of HAR-8ephacal equilibrated with phosphate buffer (pHa, o) in 40s containing IM was collected, and these IgGg fractions were further phosphated in 50s containing 0.4211 sodium chloride. 5aphacryl S-3005superfi equilibrated with acid buffer, pH 7.4
Gel filtration was performed using a ne (Pharmaeia) column to separate and remove FCS and mouse-derived proteins in the medium.

Example 2 Effect of anti-human C4bp monoclonal antibody on complex formation between protein S and C4bp (a) Search for effect on complex formation 96-well microtitration plate (Flow L
aboratorias, Inc.), 10 μg/
Coat with 50 μa of C4bp of mfi and then block the uncoated areas with 10% bovine serum albumin (BSA).

To this, 80 μa of ascites diluted to 1710 containing anti-human C4, bp monoclonal antibodies shown in Table 1, respectively, was added.
Incubate for approximately 1 hour at room temperature. These are each washed with Tris-HCl buffer PH7.5 containing 0.1% BSA.

Next, 850 μa of protein dissolved at 5 μg/sojl in the above buffer is added and further incubated at room temperature for about 1 hour. After washing this with the above buffer solution, a horseradish peroxidase-labeled rabbit anti-human protein SIgO antibody dissolved in the above buffer solution as a dihydrochloride was added at 100%
Add μΩ and wash further.

Next, hydrogen peroxide and the substrate O-phenylenediamine were added to produce the product. The degree of browning was determined by measuring the absorbance at 500 nm using a Corona two-wavelength microplate photometer (NTP-22, Corona Electric Co., Ltd.).

Bound protein S is quantified. Based on these quantitative values, the ability of each monoclonal antibody to bind to the protein S binding site in C4bp was determined.

The results are shown in FIG. Among the obtained monoclonal antibodies, No. 16 (clone number 1l-23H
10) strongly inhibited the binding of protein S to C,4bp.

(b) Confirmation of binding ability of loan number 1l-23H10 C4bp molecule M 160,000 fragment and molecular weight 4
8.000 fragments, respectively, as described in (a)
After coating by SA method, clone number 1l of m
-23H10 anti-human 1-C4bp monoclonal antibody was added, incubated, washed, and then dihydrated.
Horse and rust peroxidase-labeled goat anti-mouse immunoglobulin (Cappel Lab) was added and incubated, followed by the addition of hydrogen peroxide and the substrate O-7 22-diamine.

Absorbance at 500 nm was measured to examine the binding ability of the monoclonal antibody (No. 16) to each fragment.

As a result, clone number 11-23H10, which strongly inhibited complex formation in (a), bound to a fragment with a molecular weight of 180,000. This results in clone number 1l-23H10
The monoclonal antibody of protein S at C4bp
It was confirmed that it has the ability to bind to the binding site of .

Table 1

[Brief explanation of drawings]

Figure 1 shows C4bp obtained according to one embodiment of the present invention.
Protein S in C4bp of monoclonal antibody
This is a graph showing the results of investigating the binding ability to the binding site of , where the horizontal axis shows the monoclonal antibody sample numbers shown in Table 1, and the vertical axis shows the results of Example 2 for each monoclonal antibody sample. Among the amounts of bound protein S quantified by the method shown in (a), the one showing the maximum value is 10
0, the amount of bound protein S in each sample is %
is shown. Patent applicant: Fuji Pharmaceutical Co., Ltd. No. 1 IIl α) The shame of C4bP monoclonal antibodies.

Claims (1)

[Scope of Claims] Each monoclonal antibody has immunological cross-reactivity with only one of the antigenic determinants present in the C4b binding protein.