JPS6067860A - Diagnosing/examination agent - Google Patents

Abstract

PURPOSE:To enable the diagnosing and examination of cancer cells by using a monoclonal antibody for recognizing alpha-fetoprotein existing on the surface of a cell membrane. CONSTITUTION:A monoclonal antibody for recognizing alpha-fetoprotein (AFP) as marker of cancer existing on the membrane surface of a cancer cell used to be a diagnosing/inspecting agent for cancers. To obtain the monoclonal antibody, human AFP is immunized to a mouse. Thereafter, the spleen is extracted and fused with mouse's myeloma cell using polyethylene glycol to obtain a hydridoma. Then, a monoclonal antibody is obtained from the supernate. The antibodies thus obtained are used to dye a liver cancer cell line and an antibody indicating a positive value is selected. The massproduction of monoclonal antibodies can be done by cultivating a large amount of hybridoma in a cultivation tank.

Description

[Detailed description of the invention]
well known. CEA is said to exist on the membrane surface of cancer cells in the digestive system. On the other hand, although it has been recognized that AFP is produced in many liver cancers and cell lines, it has only been confirmed that it exists in the cytoplasm, and it is not necessarily clear whether it exists on the membrane surface or not. .

The present inventors conducted various studies in order to find a monoclonal antibody against AFP suitable as a diagnostic/testing agent, and discovered a monoclonal antibody A that binds to the AFP angle 1 present on the membrane surface. Reached.

That is, the gist of the present invention is that A' present on the cell membrane surface
It is a diagnostic/testing agent that uses a monoclonal antibody that recognizes FP.

The present invention will be explained in detail below.

First, the monoclonal antibody used in the present invention can be obtained by the following method.

That is, first, after immunizing AFP'i derived from human placenta, such as BAL B/C mice, the lungs were removed and polyethylene glycol Y was used.

Fuse with mouse myeloma cells such as P3-vl and obtain hybridomas by standard methods. Monoclonal antibodies are then obtained from the hybridoma supernatant.

Next, using these antibodies obtained by conventional methods, a liver cancer cell line (eg, PLO.

KN, NuE) 7 immunohistochemically stained and select antibodies showing positive. This detection is based on the enzymatic activity of horseradish peroxidase using the apiocincopyotinated horseradish peroxidase kelp/mouse (ABC) kit.

It is carried out using diaminobenzidine as a substrate.

In order to obtain a large amount of antibodies that show positive results, hybridomas producing the selected antibodies can be intraperitoneally injected into BAT, B10 mice, allowed to proliferate, and ascites fluid collected.

Alternatively, the above hybridoma may be cultured in large quantities in a culture tank.

The monoclonal antibody thus obtained has the following properties.

j) Using this I40-labeled antibody, placenta-derived A
When examining whether binding to the membrane of FPl:J''-PLC liver cancer cell line is inhibited, it is found that AFP competitively inhibits the binding of antibodies to the cell line membrane.

II) Label the pLc cell line with 140-leucine, solubilize it with its membrane component '&'')IJ)' and examine its binding to the antibody. Clear binding is observed.

In addition, binding to secreted proteins in the culture medium was also confirmed.

The solubilized membrane components of the Machi pLa cell line and antibody reactants in the culture medium were collected using O'Farrel.
When two-dimensional electrophoresis is performed according to the method described above, membrane-derived,
It can be seen that all the bound substances derived from the culture supernatant have the properties of AFP (isoelectricity 1 point, molecular weight).

When the membrane fraction of the IVJPLO cell line is digested with a proteolytic enzyme such as trypsin, protease, or thymotrypsin, the binding property with the present antibody decreases or tends to decrease.

In addition, the binding property showed a decreasing tendency with the "l-IJ)" treatment, and the binding property increased with the lipase treatment.

The diagnostic/testing agent according to the present invention uses the above monoclonal antibody.

This diagnostic/testing agent can be widely used for immunological measurement of AFP. For example, double diffusion method in agar gel [
M, 0 method], - original immunodiffusion method, immunoelectrophoresis method (Lau
rell method], latex agglutination reaction, red blood cell agglutination reaction,
Radio immunoassay/f: CRI A method], enzyme immunoassay method, and the like.

Although these methods can measure the AFP content in serum, the diagnostic/testing agent according to the present invention uses a monoclonal antibody that recognizes AFP present on the cell membrane surface, so it is particularly difficult to use for cytodiagnosis. suitable.

That is, it is applied to, for example, detection of free 9774 spheres, detection of tissue sections, and the like.

Even in these cases, conventional detection methods can be used, such as the EL:5SA method, RIA method, fluorescent antibody method, enzyme antibody method, biotin-avidin-horseradish peroxidase method, etc. It will be done.

For example, to explain the case using the ELISA method, first, target cells are immobilized on a plate, each well is
Add the monoclonal antibody to 1), incubate at room temperature, and then wash with buffer. Next, add anti-mouse labeled with -peroxidase, incubate at room temperature, wash, add o-phenyldiamine to react, add NaF to stop the reaction, and use a microscope or spectrophotometer. Judge by.

In this method, peroxidase mouse antiperoxidase (PAP J complex) is used instead of an enzyme-labeled antibody, and after reacting with a target cell t monoclonal antibody and an anti-mouse egg, this is further reacted and colored with a substrate. You can also do it.

In addition, the diagnostic/testing agent according to the present invention contains a radioisotope (
By administering a monoclonal antibody labeled with RI) into the body, it can also be used for so-called "imaging," which measures the localization and dynamics of RI with a gamma camera.

In addition, the monoclonal antibody used in the present invention can be labeled by a conventional method (RI, enzyme, fluorophore, metal sol, etc.) depending on the purpose at the time of measurement.

Since the diagnostic/testing agent according to the present invention uses a monoclonal antibody that recognizes AFP'% present not only in the cytoplasm but also on the surface of the cell membrane, it is possible to increase the analytical ability and improve the accuracy of diagnosis, especially in the liver. It is useful for early detection of liver diseases such as cancer.

Hereinafter, the present invention will be further explained in detail with reference to Examples.

Reference example/ (1) Production of mouse monoclonal antibody: human AFP
A substance purified from placenta with a purity of qq% or higher and which does not immunochemically react with human albumin (H8A) (■
(manufactured by Morinaga Seikaken) was used.

10μji of this human AFP was administered to BALB/C mice.
The animals were sensitized once with Freund's complete adjuvant, the final immunization was given by intravenous injection, and the lungs were removed 3 days later.
P3-TI using polyethylene glycol + 00
'/mouse myeloma was fused with the mouse myeloma, and the IbriM-ma was produced by a conventional method. Cloning was carried out using the limiting dilution method, which was repeated 11 times or more. In addition, a large amount of antibodies are BALB/C! Collected by mouse ascites system.

About yoo clones of hybridomas producing anti-AFP antibodies were selected through six fusions. One of these days, /
The antibody titer IgG amount of the culture supernatant of /clone is shown in Table 1 (
Measure after removing the supernatant and concentrating it 10 times. ).

Table / Clonal Antibodies (2) Antibody Selection a) Cell line: - weeks or more, Eagle MEM
After culturing in 7% basic medium, incubate with 7% phosphate buffer.
1) Immediately react with hybridoma culture supernatant; and 1i) After fixation with y% paraformaldehyde, inactivate the endogenous enzyme with methanol and H2O2 solution and react with antibody. there was.

Antibody detection is done using Vectastain.
The enzyme activity of horseradish peroxidase was carried out using diaminobenzidine Y as a substrate using the ABC kit manufactured by Kasei.

b) Cell line and maintenance human liver cancer cell line &i,KNl,' PL
O, also a cancer cell line NuE4 derived from fetal liver cells, and a human gastric cancer culture line KAT as a control.
Cell lines of O, MKN, and 31 colon cancer CI were used. The cultured cells were cultured in RPM/A'IO culture medium containing 0% tallow serum at 37.0°C and s%CO2.95.
The cells were maintained and grown under conditions of % Air.

Culture supernatant containing the anti-AFP monoclonal antibody in (1) above and its dilution (up to 28 times) immunohistochemically reacted with 7 liver cancer cell lines P, LC!, KN and fetal liver cells NuK A strong reaction was observed for , /9F/, 2. This reaction was the same for unfixed specimens, fixation, methanol, and H2O2 treatment methods.

In addition, as a control, a commercially available anti-AFP monoclonal antibody (manufactured by Hybritic) was used with an antibody titer of 103.
However, as with other hybridoma clones, no positive reaction was observed.

/9F/u does not have a particularly strong antibody titer, nor does it have a large amount of IgG, as shown in Table/.
As mentioned above, unlike the others, it was positive.

On the other hand, cell line MKN in which AFP production is not observed.
+h, KATO■, C-/Nibako/9F12 did not react.

VOI 3 using the monoclonal antibody obtained in Example/Reference Example
'7.30, 79g/).

θ μl of purified commercially available anti-AFP antibody (rabbit) was added to a 9-well immunoplate and incubated at 1.77° C. for 1 hour. Next, after covering the excess binding site with 5% bovine serum albumin, 4 so μg of samples y containing various concentrations of AFP were added and allowed to react at 37° C. for 1 hour.

After thorough washing with physiological saline, the above peroxidase-labeled anti-AFP monoclonal antibody was added and a time reaction was performed. I did it.

Competing peroxidase activity was detected using the sea aminoantipyrine phenol method using hydrogen peroxide 7 as a substrate.
The calibration curve shown in FIG. 1 was obtained.Using this calibration curve 7, the AF and P concentrations of a sample with unknown concentrations can be determined.

In FIG. 7, the vertical axis represents the absorbance at soonm, and the horizontal axis represents the AFP concentration.

[Brief explanation of drawings]

Figure / shows an example of a calibration curve for measuring the AFp concentration in a sample using the test/diagnostic agent according to the present invention. Applicant Mitsubishi Chemical Industries, Ltd. Agent Patent Attorney Hase - (and 7 others)
1 AFP (/Lt97ml) Procedure Neni Original Book (Method) 1 Indication of the case 1982 Patent Application No. 176774 2 Name of the invention Diagnosis/Testing agent 3 Relationship with the case to be amended Patent applicant (596) Mitsubishi Kasei Kogyo Co., Ltd. 4 agents Mitsubishi Kasei Kogyo Co., Ltd., 2-5-2 Marunouchi, Chiyoda-ku, Tokyo 100 (1 idiot) 5? Date of official order: January 31, 1980 (shipment date) Procedural order: February 20, 1980 1 Case description: 1982 Patent Application No. 176774 2
Title of the invention Diagnostic/Testing agent 3 Relationship with the case of the person making the amendment Applicant (596) Mitsubishi Chemical Industries, Ltd. 4 Column 6 of ``Detailed explanation of the invention'' in the attorney's specification 4 Contents of the amendment

Claims (1)

[Claims] (1) α-fetoprotein (A) present on the cell membrane surface
FP)'i monoclonal antibody that recognizes α-fetoprotein as a cancer marker.