JPH0266458A - Method for measuring vitronectin and vitronectin-thrombin-antithrombin iii complex - Google Patents

Abstract

PURPOSE:To obtain the simple and specific method for measuring vitronectin and VTAT complex by using two kinds of antivitronectin monoclonal antibodies which are different in antigen recognizing sections in the method of measuring the vitronectin in a sample by an immunological method. CONSTITUTION:The clone strains obtd. by dosing 0.5ml pristane into the abdominal cavity of BALB/c mice of 7-week old or above and culturing and propagating the strains after lapse of>=1 weeks are inoculated at 1 to 9X10<3> pieces/ mouse into the abdominal cavity at the time of producing the monoclonal antibodies. The ascites are taken after 10 to 14 days and are centrifugally separated for 10 minutes at 3,000rpm to obtain 5 to 15ml/piece monoclonal antibody-contg. ascites. The monoclonal antibodies are refined by using these ascites. Namely, the ascites are filtered with fatty cotton to remove the fats and are diluted twice with 50mM phosphoric acid buffer; thereafter, 100% satd. ammonium sulfate of a small volume is added thereto to fraction-collect the precipitated fraction. This fraction is dissolved in a small volume of the phosphoric acid buffer and is dialized against the buffer. The sample is processed with a DEAE- cellulose column, by which the monoclonal antibodies are obtd.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to vitronectin and vitronectin-thrombin, which are involved in blood coagulation and the complement system as well as cancer metastasis.
This invention relates to a novel method for measuring antithrombin complexes, and is particularly applicable in the field of clinical testing.

[Conventional technology]

Vitronectin is found in blood and tissue as a cell adhesion and spreading factor.
) also determined its entire nucleotide sequence and published it in Ile (Engineering/
yN C Y-S A/(EMBOJ, ) Vol. 4, pp. 2519-2524 (1984)). Vitronectin has a molecular weight of 75,000 and its physiological role is not completely clear.

However, it is associated with cancer cell metastasis (M
, L. Basaf (M.L., Ba5ara) et al.
Cancer Res, Vol. 45, pp. 2487-2494 (1985).
C, R, Ill et al., Journal of Physiology IV Chemistry (J.

Biol, Chem, ) Volume 260, No. 15610
15615) (1985)), and more recently, it has been shown that S-protein, known as a protein of the complement system, is identical to vitronectin (K, T. Preissner et al. Biochemical & Biophysic Research Communications (B10Che & Bio-physic
al Res, Comm, ) Volume 134, No. 951
956 (1986)), it has been desired to develop a method for measuring it as an important factor spanning cancer, blood coagulation, and complement.

The conventional method for measuring vitronectin is a beam.

W, Barnea et al. [Analytical Biochemistry (AnaLBiochem)
) Vol. 137, pp. 196-204 (1984))
method is known. That is, the sample was reacted with a polystyrene microtiter plate Ktlf,
The vitronectin in the sample was adsorbed onto a gray solid phase, and after washing, anti-vitronectin monoclonal antibodies (D, W
, Parness et al., Proceedings Op National μ
Academy of Saix7s Op the USA (
Proc, Natl.

Acad, Sci, USA) Volume 80, No. 136
2-1366 (f983)), and after washing, further reacting with peroxidase (POD) pJ recognition antibody XIg to develop color after washing.

On the other hand, as a method for measuring the biF-ronectin-F-thrombin-anti-F-thrombin (hereinafter abbreviated as VTAT) complex, an anti-thrombin polyclonal antibody was immobilized and a radioisotope-labeled anti-vitronectin polyclonal antibody was used to perform the San-Reiichi method. A method to measure υ has been reported (K, T, Pricener et al., The Biochemistry Research/L' (Biochem, J, )
Vol. 243, pp. 105-111 (1987)).

[Problem to be solved by the invention]

The above method for measuring vitronectin is susceptible to coexisting substances such as physical adsorption of vitronectin to the solid phase, and is a method with low specificity that is prone to errors and cannot be used in clinical testing. do not have. On the other hand, the above VTA
The T measurement method uses a polyclonal antibody, so there is a problem with specificity.Furthermore, it has been reported that vitronectin binds to thrombin but not to antithrombin.

E, R, POdPLck et al., Journal Op Phyologica μ Chemistry No. 26
1, pp. 7387-7392 (1986)). Therefore, there is a risk that not only VTAT but also the vitronectin-thrombin complex is being measured.

[Means to solve the problem]

To summarize the present invention, the first invention of the present invention relates to a method for measuring vitronectin, and the first invention relates to a method for measuring vitronectin in a sample using an immunological method. It is characterized by using

The second invention of the present invention relates to a method for measuring a VTAT complex, which includes an anti-vitronectin monoclonal antibody, an anti-antithrombin monoclonal antibody, and a method for measuring a VTAT complex in a sample by an immunological method. It is characterized by the use of a specific antibody.

In view of the above circumstances, the present inventors conducted intensive research to develop a method for specifically and easily measuring vitronectin or vTAT complexes, and as a result, they developed two monoclonal antibodies with different antigen confirmation sites for vironectin. We also confirmed that by using a monoclonal antibody against vitronectin and a monoclonal antibody against antithrombin, it is possible to specifically and easily measure vitronectin and the VTAT complex, respectively. Completed the invention.

The outline of the method for producing the monoclonal antibody used in the present invention is as follows. When a foreign animal is immunized with vitronectin or antithrombin (III) as an antigen, antibodies against a large number of the antigenic determinants are produced by antibody-producing cell groups.

These antibody-producing cells are fused with myeloma cells that have unlimited proliferation potential, and the fused cells are cloned to screen for cells that produce the target antibody. The antibodies produced by each of these cell groups are antibodies specific for vitronectin or antithrombin. In the present invention, two anti-vitronectin monoclonal antibodies, such as VN-47 and VN-58, suitable for the vitronectin antibody method were selected from among them. On the other hand, VTAT
An anti-vitronectin monoclonal antibody such as VN-5 and an anti-antithrombin monoclonal antibody such as AT-2 were selected which were suitable for the two-antibody sandwich method of complex.

The method for measuring vitronectin of the present invention involves simultaneous measurement of a first anti-vitronectin monoclonal antibody immobilized on a solid phase, a sample such as human blood plasma body fluid or a vitronectin-containing solution, and a second enzyme-labeled anti-vitronectin monoclonal antibody. It can be carried out by reacting.

On the other hand, the VTAT complex measurement method uses a solid-phase antibody obtained by contacting an anti-antithrombin monoclonal antibody immobilized on a solid phase with a sample such as human blood plasma, body fluid, or a solution containing the VTAT complex. combined VT
This can be carried out by allowing an enzyme-labeled anti-vitronectin monoclonal antibody to act on the AT complex.

When a monoclonal antibody is used as a solid-phase antibody in the present invention, known methods can be used for immobilization, and as a carrier, for example, ball beads or microplates using polystyrene, polyethylene, polypropylene, etc. are used. On the other hand, enzymes used as labeling compounds include horseradish-derived be-oxidase, β-D-gawoctosidase,
Enzymes such as alkaline phosphatase are used. Furthermore,
Substrates are used to measure the activity of these enzymes.

For example, in the case of horseradish-derived μ oxidase, the substrate is 0-phenyl diamine-H20! ,2,2'
-Ajino! /-[! 5-Ethylbenzthiazolinesulfonic acid (6)] ammonium salt (ABTS)-H,O, etc. are used.

〔Example〕

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these Examples.

Example 1 Preparation of monoclonal antibodies (1) The antigen antithrombin (■) manufactured by Midori Juji Co., Ltd. and the hydronectin manufactured by Tora Shuzo Co., Ltd. were used.

(2) Immunization of mice with antithrombin■ and vitronectin at 0 and 15%, respectively.
10 mM phosphate buffer (p
H7,4) and mixed well with Freund's complete adjuvant at a ratio of 1:1 (v/v) to give an amount of antithrombin or vitronectin of 50% per mouse.
The cells were immunized intraperitoneally so as to be μt. 21 days after first immunization
50 μt of antithrombin or vitronectin after 1 day.
was mixed with Freund's incomplete adjuvant and administered intraperitoneally, and 14 days later, antithrombin ■ or vitronectin 20μ? was administered through the tail vein.

(3) Cell fusion and cloning Three days after the final infusion, the pancreas of the mouse was taken out, and its KW cells and mouse myeloma P3U1 were mixed at a ratio of 10=1. Keller (G., 1975)
Cell fusion was performed according to the method of ) et al. Next, it was implanted into a 96-well microplate and HAT (hyboxanthin 1X10M, aminopterin 4X10M, thymidine 1
．． T)MEM-I G%FC containing 6XIQ-'M)
After culturing in 8 medium (HAT medium) for 10 to 17 days, HT (
Hyboxanthin 1×10M, Thymidine 1.6×10M
) was transferred to DMEM-10% FC8 (HT medium), and after it became possible to culture in a flask (25d), culture was continued in DMEM-10% FC8 medium. The antibody titer in the culture supernatant of wells in which proliferation was observed was measured by enzyme antibody method, and hybridomas were cloned from appropriate sea urchin p by limiting dilution method. That is,
Plant 2.5 x 104 mouse thymocytes per well in a microplate, then incubate with DMEM medium for 5.1
(L5 hybridomas/α1-) were diluted and cultured by inoculating each α1-/well into the above-mentioned microplate. Colonies visible to the naked eye were formed 10 to 14 days after the start of culture, and a clone strain was established. I got it.

(4) Screening method The culture supernatant of sea urchin μ in which hybridomas and clones have proliferated is separated, and enzyme-linked immunoturben F
Assay (lli;zyme LinkedImmu
nosorbent As5ay: ELIS
A) The ability to produce antibodies against civitronectin or antithrombin ■ was examined by method. Microtiter Delay) K vitronectin or antithrombin ■ α5μ
Dispense at 2150μt/μt of sea urchin and incubate at 4°C for 18
Vitronectin or antithrombin (■) was allowed to adsorb onto the solid phase by allowing it to stand for a period of time. 10 Emit phosphate buffered saline (P
Bs) (1) Hy, 4) After washing each well 5 times with 200 μt, 1% bovine serum μ pmin (BsA
) containing 100 μt/well of pBse at 37°C.
The mixture was allowed to stand for a period of time, and the unadsorbed portion of each well was blocked. Next, add 50 μL/well of the sample culture solution at 37°
The reaction was carried out at C for 1 hour. After washing three times with PBS, 10
Oxidase-labeled anti-mouse I9G diluted 1:00
(manufactured by Cavell) was added at 50 μm/well and reacted at 37° C. for 1 hour. Wash with PBS, add 0.01% hydrogen peroxide, o, s s my/1nt ABTs (2,2'
-Azinodi(3-ethylbenzothiazoline-sulfone)) (manufactured by Boehringer Mannheim) (LI
Add M citrate buffer (pHa, o), wavelength 40
Absorbance was measured at 5 nm. Intense color development was observed when antibodies against vitronectin or antitog/bi/IIT were present in the sample.

Among the clones with high antibody production ability obtained as a result, monoclonal antibodies suitable for vitronectin two-antibody sandwich EIA method were produced.
N-58, also the sandwich EI of the VTAT complex
Clone V producing the monoclonal antibody identified in method A
N-5 and AT-2 were obtained.

Each of the clone strains is Hybridoma VN-
47 and FERMP No. 10228 (FERMP
-10228), Hybridoma V N -58
, and imitation engineering laboratory No. 10229 (FEBM P-
10229), labeled as Hybridoma V N-5, and published as FEIBM P-102 No. 10227 (FEBM P-102
27), designated as Hybridoma A'I'-2 and published as Microtechnological Research Institute No. 10230 (FEBM P-10250).
) has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

(5) Preparation of monoclonal antibodies Pristane (Aldrich Co. 1.
Clone strain 1 cultured and propagated after one week or more
˜9×10 M mice/mouse were inoculated intraperitoneally. 10～
Mice were sacrificed after 14 days and ascites fluid was collected. This is S,
Centrifuge at OOOrpm for 10 minutes, then separate the layers at 5-15 fnt.
Monoclonal antibody-containing ascites was obtained from each animal.

(6) Purification of monoclonal antibodies The ascites obtained in (5) above was filtered through a defatted filter to remove fat - 50mM phosphate buffer (pH 7:3)
After diluting the mixture twice with -, an equal volume of 100% saturated ammonium sulfate was added, and the precipitate fraction was collected. This fraction was dissolved in as small a volume as possible of the above phosphate buffer and dialyzed against the same buffer. This sample was applied to DEAE-cellulose column K to obtain a monoclonal antibody.

Example 2 (1) Preparation of peroxidase-labeled anti-vitronectin monoclonal antibody Anti-vitronectin monoclonal antibody VN-58 or VN,5 was prepared using the Nakane method (P, Nakane et al., Journal of Histochemistry and Cytochemistry (J, Histochem, Cytochem,
) Vol. 22, p. 1084 (1974)].

2) Immobilization of anti-vitronectin monoclonal antibody on microplate Anti-vitronectin monoclonal antibody VN-47 was adjusted to a protein concentration of 20 μm/- with phosphate buffer saline [water (pBs% pH 7,4), and this was E
Add 100 μt/well to each IA microplate,
- Leave at 4"C day and night, wash with PBS, add 200 μt/μ of each sea urchin of PBS containing 1% BSA, and block.
VN-47 immobilized microbes V-) were obtained.

(3) Immobilization of anti-antithrombin monoclonal μ antibody on microplate Anti-antethrombin monoclonal p antibody AT-2 was immobilized on a microplate in the same manner as in Example 2-12), and AT-2-immobilized microplate I got it.

(4) Anti-vitronectin monoclonal antibody Measurement of vitronectin in healthy human blood plasma by one-step sandwich EIA method A 1000-fold dilution of healthy human blood plasma was placed on the VN-47 immobilized microplate obtained in (2) above.
μt and the POD-VN-58 solution obtained in (1) above
opt was added and reacted at 37°C for 30 minutes. P.B.
After washing with S, 1200 μt of POD substrate (ABTS (
cL1M including L5++v/- and (LO05%H,O!
Add citric acid, phosphate buffer pH 4,5) and
The color was developed at °C for 30 minutes, and the absorbance at 405 nm was measured. Vitronectin in human blood plasma was measured using the blood plasma of 3 (1 person) healthy volunteers as a standard material for the calibration curve using the standard method.The calibration curve is shown in FIG. 1, and the measured values of 10 people are shown in Table 1. FIG. 1 is a graph showing a calibration curve obtained by measuring diluted pool blood from healthy individuals using the vitronectin measurement method of the present invention, and the horizontal axis represents the dilution rate of pool blood from healthy individuals vertically. The axis is absorbance oD (4osnm
) is shown.

Table 1 *%: The vitronectin level in the blood plasma of a healthy person is 1
Example 5 Measurement of VTAT complex in the blood plasma of healthy individuals and in the blood of 8 cases of cerebral infarction and myocardial infarction Blood samples μ were placed on the AT-2-immobilized microplate obtained in Example 2-+2) above. 2-fold dilution of 50
After adding μt and reacting at 37°C for 30 minutes, the POD-VN-5 obtained in Example 2-(1) was washed with PBS.
50 μt of the solution was added and reacted at 37°C for 30 minutes, followed by washing with PBS. 100 μt of POD substrate solution was added to this, color was developed at 25° C. for 30 minutes, and absorbance at 405 nm was measured. 8 cases of healthy persons, 8 cases of cerebral infarction using pooled blood as a standard material for the calibration curve.
For example, the VTAT complex in the blood of 8 patients with myocardial infarction was measured. The calibration curve is shown in FIG. 2, and the measured values of blood from healthy subjects and patients are shown in Table 2. The patient's blood sample showed high levels.

In addition, Fig. 2 is a graph showing a calibration curve when measuring multiple dilutions of pooled plasma from healthy individuals using the VTAT complex measurement method of the present invention υ, and the horizontal axis represents the dilution of pooled plasma from healthy individuals. The vertical axis indicates the absorbance OD (405 nm).

Table 2 Human infarction*%: VTAT value in healthy healthy people swimming at 100%
Fourth, the cross-reactivity of the VTAT complex and the vitronectin-thrombin complex was investigated. The VTAT complex and the vitronectin-thrombin complex were prepared by the method of E. R. Podack et al. (Journal of Biological Chemistry, supra). In other words, vitronectin 50μ? and thrombin 40μ2 in TBS 100μ
was added and reacted at 37"C for 30 minutes to prepare a vitronectin-thrombin complex. On the other hand, 80μt of vitronectin, 80!tf of thrombin, and 140μt of antithrombin I were added to 100μt of TBS and reacted at 37°C for 30 minutes. When these complexes were measured using the VTAT complex measuring method according to the present invention, no cross-reactivity with the biFronectin-thrombin complex was observed.

〔Effect of the invention〕

As explained in detail above, according to the present invention, vitronectin and VTAT? A simple and specific method for measuring J coalescence has been provided, and has been shown to be particularly useful for diagnosing diseases related to blood coagulation and fibrinolysis.

[Brief explanation of the drawing]

FIG. 1 is a graph showing a calibration curve obtained by measuring multiple dilutions of pooled blood from healthy individuals using the method for measuring vitronectin of the present invention, and FIG. It is a graph showing the test line 1 when measuring diluted sardines.

Claims (1)

[Scope of Claims] 1. A method for measuring vitronectin in a sample using an immunological method, which comprises using two types of anti-vitronectin monoclonal antibodies having different antigen recognition sites. 2. A vitronectin-thrombin-antithrombin method characterized by using an anti-vitronectin monoclonal antibody and an anti-antithrombin III monoclonal antibody in a method for measuring the vitronectin-thrombin-antithrombin III complex in a sample by an immunological method.
Method for measuring the III complex.