JPH0212063A - Quantitative determination of pulmonary adenomatous carcinoma antigen - Google Patents

Abstract

PURPOSE:To make serum diagnosis of the pulmonary adenomatous carcinoma with high specificness by adopting an immunoassay of a sandwich system using an anti-human pulmonary adenomatous carcinoma monoclonal antibody ALC-864 as a 1st antibody and an enzyme- or radiation-labeled anti-human pulmonary squarmous cell carcinoma monochlonal antibody SLC-454 as a 2nd antibody. CONSTITUTION:The anti-human pulmonary adenomatous carcinoma monoclonal antibody ALC-864 is used as the 1st antibody and is made into a solid phase. A human serum specimen is brought into reaction therewith to conjugate the adenomatous carcinoma antibody. The enzyme- or radiation-labeled anti-human pulmonary squarmous cell carcinoma monoclonal antibody SLC-454 is then used as the 2nd antibody and is conjugated with the previous adenomatous carcinoma antigen conjugated to the 1st antibody. The quantity of the adenomatous carcinoma antigen in the serum specimen is determined by measuring the activity or the radiation doze of the enzyme labeled with the quantity of the conjugated 2nd antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides a method for quantifying &1i adenocarcinoma antigen, which is applicable to the clinical diagnosis of human lung adenocarcinoma.

Conventional technology anti-human lung cancer monoclonal antibody SLC-4
54 is a monoclonal antibody C1 produced by the present inventor.The positive rate for lung cancer using this antibody is 25% and the positive rate for lung adenocarcinoma is 30% (Japanese Patent Application Laid-Open No. 1988-195 (IL)).

Problems to be solved and attempted by the invention Serological diagnosis of lung adenocarcinoma using SLC-454 is currently B.
Although it has great clinical value, it is not necessarily specific to lung adenocarcinoma, and the false positive rate is particularly high in benign diseases such as the lungs. A diagnostic method is required.

Means for Solving the Problems The present inventor has developed anti-human lung adenocarcinoma monoclonal antibodies Δ and C
-1'lG4 as the first antibody, 2, enzyme or radiation,! According to a sandwich immunoassay using the anti-human I)Ii Q skin cancer monoclonal antibody SLC-454 as the second antibody, Le 1711! i! The inventors have discovered that it is possible to carry out serodiagnosis with high specificity for cancer, and have completed the present invention.

The present invention will be explained in detail below.

According to the present invention, anti-human lung adenocarcinoma monoclonal antibody A
LC-864 is used as the first antibody, it is immobilized, and a human blood 10 sample is reacted with it to bind the lung adenocarcinoma antigen,
Then enzyme-labeled or radiolabeled anti-human lung squamous.
- By binding the skin cancer monoclonal antibody S+, C454 as a second antibody to the lung adenocarcinoma antigen bound to the first antibody, and measuring the amount of bound second antibody by the activity or radioactivity of a labeled enzyme. Determination of lung adenocarcinoma antigen in serum samples
can do.

SLC-454 was first developed by the present inventors in JP-A-63-1.
The hybrid-7 strain S is a monoclonal antibody disclosed in No. 9561, and has the monoclonal antibody production residue,
r using C-454 (hereinafter referred to as SLC-454 strain)
! A can be built. St, C-454 strain is European
nCo11ection or^n1llalCel
It has been deposited with I Culture as ECACC86070306.

ΔLC-864 can be manufactured as follows.

(1) Animal immunity and antibody production details 11? ! Preparation of 1 3-1
A 0-week-old, preferably 8-week-old mouse is immunized with a polymeric fraction of fresh water from a lung adenocarcinoma patient, and antibody-producing cells in the animal's tiles, lymph nodes, and peripheral blood are prepared. It is preferable to use mice to be immunized that have been treated with human normal lung cells with 1111 to make them immune tolerant. The immunization method is to inject the animal subcutaneously, intravenously, or intraperitoneally with an appropriate air schwand.
For example, 70 India's Complete Adjuvant (Complete
te Freund's AdjuvanL) or
A polymeric fraction of fresh water (10 to 500 rats/animal) was administered to human lung adenocarcinoma patients along with aluminum hydroxide gel and Nagi-Hyushika vaccine. After that, take 1 and 2 antigens every 1 to 2 weeks.
Administer ~5 times. Blood was collected from the fundus venous plexus 3 to 70 days after each immunization, and the enzyme-linked immunosorbent assay (ELISA) was used to demonstrate that the serum reacts with human lung adenocarcinoma.
Igaku Shoin, 1976].

Enzyme immunoassay: In a 96-well EIA plate (manufactured by Flow Laboratories),
Membrane components of normal or tumor cells9 tissues (l as protein content)
1,000 μg/all membrane fragments)
Dispense 0 to 200 μm/hole, leave the field at 4t for 1 to 2 nights, remove the supernatant, and add resin water or PBS.
(Disodium phosphate 1.83g, potassium phosphate 0
．． 21g, salt? , 05g.

After washing thoroughly with distilled water 1j', pH 7,2), 1% S^
PBS solution (IIS^-P) containing (bovine serum albumin)
Dispense 100-200 μm/well of 100-200 μm/well and incubate 1-2 μm at 4°C.
The plate was left to stand overnight to block (profking) residues binding to the protein remaining on the plate.

After that, discard l3SA-PBS and use resin water or PBS.
[After thorough washing with 3S, use BSA-r as the first antibody.
Sample diluted with '13s (mouse serum).

Pipette 100 μl/well of Hybrid-7 culture supernatant, purified monoclonal antibody) and leave overnight at 4T. 2M NaCj once with resin water! After washing six times with the solution, rabbit anti-mouse immunoglobulin [gG
- Peroxidase conjugate [manufactured by Dako (i) AKO),
100ml of 100 times diluted solution from Kyowa Medex (seller: Kyowa Medex)
Dispense into wells and leave at room temperature for 2 hours.

After washing well with PBS, ABTS substrate solution [2゜2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium 55011g was added to 0.1M citric acid
Using i2 buffer (a solution prepared by adding 1 d/ml of hydrogen peroxide to a solution dissolved at pH 4, 2>11 immediately before use),
Color development is measured by absorbance at OD s + Sn*. At this time, mice that strongly react to lung adenocarcinoma cells, tissues, or their membrane components are used as human lung adenocarcinoma-immunized mice as a source of antibody-producing cells for the production of Hybrid-7.

When using cells themselves as antigens for enzyme immunoassay, Falcon
Culture target cells in 3072 plates, 0.25%
Add geltaraldehyde-PBS, leave at room temperature for 1 to 2 hours, wash thoroughly with PBS, and add 1% BSΔ-PBS.
100-21) Add 0 μg, leave for 2 hours, wash thoroughly with resin water or l'fls, and use the plate to measure antibody titer in the same manner as using general antigen-coated plates. I did it.

For cell fusion, 10 to 400 μg/mouse of the breast macromolecular fraction of human cancer patients was intraperitoneally administered to immunized mice 3 to 4 days before the fusion treatment.
IQ! Whole visceral cells and lung cells are prepared.

The ff1tl viscera was shredded in MEM (manufactured by Hinaga Pharmaceutical Co., Ltd.),
Loosen with a bottle set, centrifuge for 5 minutes at 20 Orpm, discard the supernatant, and dilute with Tris-ammonium chloride.
The cells are treated with i solution (pH 7, (i5) for 1 to 2 minutes to remove red blood cells, washed three times with MEM, and provided as a stock cell for fusion.

A polymer fraction of fresh water from a lung adenocarcinoma patient to be used as an immunogen is prepared as follows.

That is, after thawing the pleural effusion of a lung adenocarcinoma patient stored at -80°C, it was centrifuged at 3.00 rpm for 10 minutes, and the supernatant after removing the solid content was purified using Cellulofine GCL-2000S.
F (Seikagaku Kogyo Co., Ltd. I!JJ) column, and collect the high molecular fractions with molecules m+, ooo, ooo or more, and use them as the high molecular fractions of fresh water from patients with lung adenocarcinoma.

(2) Preparation of myeloma cells As bone mill cells, established cell lines obtained from mice are used. For example, 8-azaguanine-resistant mice (
BALB/c Yamaki) myeloma cell line P3-X638g8-
Ul (P3-Ul) [Current topics in microbiology and immunology-1 (C
currentTopics Dose Microbiol
ogy and 1mmunology -1)] [European Journal Ogy 12 Nology 4 (f
lturopean J, Immunology)
6, 511-519 (1976)], SP21
0-Ag 14 (SP-2) [Nature
re) 276, 269-270 (1978)), P
3-X 63-A g8653 (653) C')+
-f-Ru of Immunology 4 (J, 1mmuno
123.1548-1550 (1979
] , P 3-X 63-Δg8(X63) [Nature 256.495-49
7 (1975)] etc. are used. These cell lines were grown in 8-azaguanine medium (RPMI-16/lo medium with glutamine (1.5 mM) and 2-mercaptoethanol (
5X10-'M), gentamicin (10 μg/ll
1) and fetal bovine serum (F'C3) (manufactured by C5L, 10
8-abguanine (1%) was added to the normal medium containing 8-abguanine (1%).
5 μs/ml), but 3-4 days before cell fusion, subculture in normal medium, and on the day of fusion, 2×10'
Ensure a sufficient number of cells.

(3) The antibody-producing cells immunized in cell fusion (1) and the myeloma cells obtained in (2) were thoroughly washed with MEM medium or PBS, and the number of cells was determined as follows: antibody-producing cells: myeloma cells = 5 to IO After centrifugation (1,200 rpm, 5 minutes), the supernatant was discarded, and the precipitated cells were thoroughly loosened.
While stirring, at 37°C, polyethylene glycol
1,000 (I'EG-1,000)2 g, MEM2
Mixture of mi and dimethyl sulfoxide 0.7011 0
．． Add 2-1 ml/I O' antibody producing cells, 1-2
After adding MEM1-21 several times every minute, add MEM to make a total volume of 501. Centrifugation (900r
ρm, 5 minutes), the supernatant was poured over the bridge, the cells were gently loosened, and normal medium (RPMI-16 containing 10% Fe2) was added.
Add 100+ nl of culture medium) and gently suspend the cells by suctioning and blowing with a volumetric pipette.

This suspension was dispensed into a 96-well culture plate at 100/well, and kept at 37°C for 24 hours in a 5% CO□ incubator.
Incubate for hours. 1ooALI/well of HA in culture plate
T medium [hypoxanthine (10-'M) in normal medium,
Thymidine (1,5X10-'M) and aminobutyryl 7
(4 x 10-'M)] was added, and a further 24
Incubate for hours. For the next 2 days, every 24 hours, culture supernatant 1
Boil oou1, add the same amount of new HAT medium, and add 5%
Culture for ~14 days at 37°C in a COt incubator.

For holes in which fused cells that had grown in colonies were observed, 100 tQ of supernatant was discarded, and the same amount of o'r Jr'; Conversion to To1'F medium is performed every 24 hours during the day.

After culturing in IIT medium for 3 to 4 days, a portion of the culture supernatant was taken and subjected to the above enzyme immunoassay or immunohistological determination method (AB
C method) (enzyme antibody method, Interdisciplinary Planning Publishing, 106 pages, 1985
(2013) to measure antibody titers against human lung adenocarcinoma. At this time, the reactivity with normal human cells, tissues, or membrane components thereof is also measured using the same method, and those that specifically react with human lung adenocarcinoma cells 1 tissue or membrane components thereof are selected. Strongly reacts with human lung adenocarcinoma cells 1 tissue or its membrane components, and human normal cells.

Regarding holes that do not react with tissues or their membrane components,
Cloning is repeated twice by the limiting dilution method, and a hybridoma strain that stably exhibits a strong antibody titer against one human lung adenocarcinoma cell tissue or its membrane component is selected as an anti-human lung adenocarcinoma monoclonal antibody-producing hybridoma line. A specific example of the hybridoma strain is LC864 to the hyperidoma strain.
(hereinafter referred to as ALC-864 strain).

The ΔLC-8611 strain was transferred to the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERMB P-1783 on March 9, 1986.
It has been deposited as.

(4) Preparation of monoclonal antibodies Bristan treatment [2゜6. 10. 14-Butramethylbentadecane (I'risLane) 0.5 m
The ΔL obtained in (3) was applied to 8-IO week old BΔLB/cfl mice (intraluminal administration of 1 ml of BΔLB/cfl and reared for 2 weeks).
2-4 x 10' cells/mouse of C-864 strain are injected intraperitoneally. The hybridoma turns into ascites cancer in 10 to 21 days. Ascites was collected from this mouse and centrifuged (3,000
Orρm, 5 minutes) to remove solids, salted out with 50% ammonium φ acid, and added with 0.04 M phosphate buffer (
pH8,0,0,03M NaCj! After dialysis with D E 52 (Whata+an; Bet Polycomb 50o+I) at a flow rate of 20 to 3011
The column was passed through the column at a rate of 11/hour, the IgG fraction was collected, and the purified monotalo-
- Null antibody ALC-8 (referred to as i4).

Determination of antibody isotypes was performed by Ouchterlony (Oucht.
er 1ony) method (double immunodiffusion method) (Introduction to immunological experiments, Biochemical experimental methods 15, published by Gakkai Publishing Center, 7)
4, 1981).

The amount of protein was quantified using the Folin method and absorbance at 280 nm (1,4 (OD2-,)ζ immunoglobulin 1 mg
/ml].

The specificity of the obtained monoclonal antibody was determined by its reactivity with various types of human normal or tumor tissue or its membrane components obtained from multiple specimens, and with the reactivity with 8 types of human normal or tumor cells. Reactivity with culture stocks or human fetal cell cultures or their membrane components, reactivity with conventionally known carcinoembryonic antigens (eg CEΔ), center of gravity.

The reactivity with various patient sera is measured by enzyme immunoassay, fluorescent antibody method, immunohistochemistry (ABC method), etc.

Enzyme labeling of monoclonal antibody SLC-454 was carried out using peroxidase, urease, alkaline phosphatase, β-galactosidase, etc. as enzymes, and the periodic acid crosslinking method [Journal of Biohistochemistry and Cytochemistry]. I (J, IIistoc
hegl, Cytochem, ) 22.1084-10
91. (1971) ).

The periodate crosslinking method using peroxidase is carried out, for example, as follows.

Peroxidase was dissolved in 0.3M sodium bicarbonate buffer (pH
8,1) Dissolve in In+1, add 0.1 ml of 1% ethanol solution of fluoro-2,4-dinitrobenzene (DNP), add 1 ml of 0.06 Ma iodic acid, and react for 30 minutes at room temperature. This was adjusted to pH 9.5 to 0.
Dialyze with 01M carbonate buffer to obtain a dialysate containing peroxidase with an aldehyde group converted to DNP. Add 5 mg of antibody as protein amount to this dialyzed solution, react at room temperature for 2 hours, dialyze against 0.01M phosphate buffer, pH 7.2, and add 5 mg of antibody as protein amount to the dialyzed solution.
G4.100 column chromatography and gel plating.

The active fraction is used as an enzyme-labeled antibody.

When peroxidase is used as the enzyme, a ΔBTS substrate solution is used, and color development is measured by absorbance at OD*+sn+a. The substrate for urease was 8i+g bromcresol purple, 100 mglR and 0.2mM.
Using a solution containing EDTA in 100ml (r+) (4,8), measurements were made using a Journal
ds) 53, 187-194 (1982)
Perform according to the method described in . As a substrate for alkaline phosphatase, 1 mg/ml p-nitrophenyl phosphate dissolved in 0.1 M jetanolamine (Φ1) is used.

The enzyme 'tFAJ can also be performed via biotin-avidin binding. Biotin 4+Wl was published in the Journal of Histochemistry and Cytochemistry (J, llistochem, Cytochem,
) 27, 11.31-1139 (1979).

Serological diagnosis is performed as follows.

In a 96-well ElΔ plate, ΔF, C was added as the first antibody.
Dispense -864In~lonμg/ml into 4 holes of 50~200d and leave at 4°C~room temperature for 2 hours~2 nights. P
After washing with 13S, 1% [35△PBS 200μ
Add Q/hole and leave at 4"C to room temperature for 2 hours to overnight. After washing the plate well with PBS, add 50 to 100 ρ of serum sample at 1 to 100 times dilution to each formula. After leaving at 4°C to room temperature for 2 hours to overnight, wash thoroughly with P[3S. Next, biotinylated SLC-454 (10 to
100 q/ml) as the second antibody and 50-100 m/ml
Add holes and leave for 2 hours to overnight at 4°C to room temperature. After washing the plate thoroughly with PBS, avidin-biotin-peroxidase [manufactured by Vector]
(10m/ml) was added 50-100μ/well, left at room temperature for 10-30 minutes, and 50-100μ of 5% SDS solution was added.
Add m/hole and stop the reaction. The OD value of each formula is measured, and the amount of antigen in the serum sample is calculated from the degree of color development.

A normal value is determined by comparing the antigen amount in the serum of a healthy person and that in the serum of a cancer patient obtained in this manner, and those exceeding the normal value are determined to be positive for lung adenocarcinoma. Lung adenocarcinoma antigen can be similarly quantified using SLC-154 as the first antibody and biotinylated ALC-864 as the second antibody.

Examples of the present invention are shown below. 1゜Example 1゜Plate for 96 holes E I [Flow Laboratories]
Reference Example 1. A1 manufactured by A1. , C-864 (IOJ
1g/ml) 1004/well was added as the first antibody, 4
After standing overnight at ℃, PI35'T! Precipitate and 1% T3S
Added Δ-I'BS 200#/hole and left it at 4℃ overnight.
Healthy human serum (144
samples), lung cancer patient serum (92 samples), gastric cancer patient serum (33 samples), breast IFiS faithful serum (27 samples), human I
l! Cancer II patient serum (25 samples), pancreatic cancer patient serum (IO sample), gallbladder/cholangiocarcinoma patient serum (5 samples), pure benign disease patient serum (+6 samples), gastric ulcer patient serum (l-0 sample),
Liver cirrhosis patient blood l# (3 samples), pancreatitis patient blood l? A 10-fold dilution of J (3 samples) was added to each well in 504 volumes, and the wells were left at 4°C overnight and then thoroughly washed with PBS. Biotinylated SLC-4
54 (10q/ml) l 00 q/ml was added as a second antibody, left overnight at 4°C, thoroughly washed with PBS, and avidin-biotin-peroxidase (10 q/ml) was added as a second antibody.
Add 11) at 100 per hole and leave it at room temperature for 1 hour.
Washed thoroughly with PBS.

Next, 100 d/well of ABTS substrate solution was added, the reaction was allowed to proceed for 30 minutes at room temperature, and 1 oad/well of 5% SDS solution was added to stop the reaction. The color development of each formula was measured with an absorbance meter (OD, S). The results are shown in Figure 1.

As shown in Figure 1, in this serodiagnostic method, the positive rate for lung adenocarcinoma was high, and many cases had a large amount of antigen.

Positive cases were also found in squamous cell lung cancer, large cell lung cancer, gastric cancer, colon cancer, pancreatic cancer, and gallbladder/cholangiocarcinoma, but the antigen amount was relatively low. No positive cases were observed in healthy subjects or in benign diseases such as the lungs, stomach, liver, and pancreas.

The above results demonstrate that the serodiagnostic method of the present invention can be used to treat lung cancer, especially lung cancer.
1. It has been shown that this method provides an effective means for clinical examination of adenocarcinoma.

Reference example ■, anti-human lung adenocarcinoma monoclonal antibody ^LC-
Preparation of -864 (1) Preparation of immunogen Preparation of a polymer fraction of lung adenocarcinoma patient wash water as an immunogen.
I went as follows.

- After thawing the lung adenocarcinoma patient's water that had been stored at 80℃,
．． Centrifugation was performed at 00 rpm for IO minutes, and 8 ml of the supernatant after removing the solids was added with 0.5 M NaCj! including 10
mm! Cellulofine GCL-20 with a bed volume of 750 ml equilibrated with J acid buffer (1117,2)
00SF C Seikagaku Kogyo Co., Ltd.) column, and
A polymer fraction with an fi of 1,000,000 or more (void fraction; fraction numbers 41 to 50) was collected and used as a polymer fraction of purified water from patients with lung adenocarcinoma. The elution pattern of Cellulofine GCL-2000SF is shown in FIG.

(2) Preparation of antibody-producing cells Human normal lung tissue membrane components (100 μl protein/mouse) were intravenously administered to newborn BALB/c mice within 24 hours after birth. After 8 weeks, mice were treated with 100 μg of the polymer fraction (in terms of protein) of lung adenocarcinoma patient wash water, 2 mg of aluminum hydroxide gel/mouse, and killed Bordetella pertussis vaccine lXl0'/7 mice.
It was administered intraperitoneally with the rats. Thereafter, 7 animals were immunized with 100 μg (in terms of protein) of the same antigen 3 to 5 times every 1 to 2 weeks. Among these immunized mice, mice whose anti-blood i11 strongly reacted with human lung adenocarcinoma cells or tissues or their membrane fragments were used as immunized mice. Subjected to fusion.

(3) Preparation of Mouse Myeloma Cells The 8-azaguanine resistant mouse myeloma cell line P3-Ul was cultured in a normal medium, and at the time of cell fusion, cells with a size of 2xio' or more () were used as a parent strain for cell fusion.

(4) Preparation of hybridomas The tile cells and myeloma cells obtained in (2) and (3) were combined into 5:
The cells were fused using the method described above, and cultured in H to T medium for 14 days at 37°C under 5% CO□. After selecting the fused cells and further culturing them in HT medium, measuring the antibody titer against anti-human lung lIl cancer, selecting active wells, changing to normal medium, repeating cloning twice, and incubating the enzyme. A hybridoma strain that produces a monoclonal antibody that specifically reacts with human lung adenocarcinoma without reacting with normal human cells or tissues or other cancers, as determined by immunoassay and immunohistochemical determination (BC method). ALC-864 was selected.

(5) Purification of monoclonal antibodies The hybridoma strain ALC-864 obtained from 8-week-old Brystane-treated B1, B/c female mice (Sy) was 4x
Seven 10" cells were injected intraperitoneally.

After 10-21 days, the hybridoma turned into ascites cancer. Collect ascites from mice with ascites (5-10ml/
Then, centrifuge RI! (3,000 rpm.

5 minutes) to remove solids. Salting out with 50% ammonium sulfate, 0.04 M phosphoric acid tjt solution (pH 8
, containing 0,0,03M NaCl), then DE
52 (manufactured by Whatg+an) (Betpoly 5-
L501) 0) Force 51-2 flow M20 ~ 301/hour, collect IgG fraction, purify monoclonal antibody formula LC
-864. In this way (humiliated eight LC-8
64 was found to belong to [gG,l] by the Ouchterlony method.

(G) Specificity of ALC-864 The reaction specificity of the anti-human lung adenocarcinoma type clonal antibody AL (-864) is shown in Table 1.

Table 1 Two cultured uterine cancer cell lines and reactions Effects of the invention According to the invention, human Ijli 11! Ill11 serum diagnosis specific to i11F6 can be performed easily and efficiently.

[Brief explanation of the drawing]

FIG. 1 shows the results of serodiagnosis in Example 1. Figure 2 is for reference 49 Cellulofine G CL-200
The elution pattern of 0SF is shown. Figure Patent Applicant (1 (12) Kyowa Hakko Kogyo Co., Ltd. Figure Procedure Amendment S (Method) Case Indication 1985 Patent Application No. 163434 Name of the Invention Person Amending the Quantification Method for Lung Adenocarcinoma Antigen Case and Relationship Patent distributor postal code 100 Address 6-1 Chome, Otemachi, Chiyoda-ku, Tokyo Name
(102) Kyowa Hakko Kogyo Co., Ltd. Su0 60 Wa o g
. Fura 7 Tame 7 Kogo (9 敞／11釋p) Figure 1 is clearly written in sufficiently rich black.

Claims (1)

[Claims] The anti-human lung adenocarcinoma monoclonal antibody ALC-864 was used as the first antibody, which was immobilized on a solid phase, which was reacted with a human serum sample to bind the lung adenocarcinoma antibody, and then the anti-human lung adenocarcinoma antibody labeled with an enzyme or a radioactive compound. The squamous cell carcinoma monoclonal antibody SLC-454 is used as a second antibody to bind to the lung gland antigen bound to the first antibody, and the amount of bound second antibody is determined by measuring the activity of a labeled enzyme or measuring the radiation dose. A method for quantifying lung adenocarcinoma antigen, which comprises quantifying the amount of lung adenocarcinoma antigen in a specimen.