JPS61234358A - Assay of human lung cancer antigen - Google Patents

Abstract

PURPOSE:To discover lung cancer patients, by a method wherein an anti-human lung cancer specific monoclonal antibody ALC-1 is labelled with oxygen to be bonded to a lung cancer antigen and the amount of the second antibody bonded is assayed by measuring the activity of the label oxygen. CONSTITUTION:An ALC-1 (10mug/ml) is applied to an EIA plate with 96 holes at the rate of 100mul per hole, allowed to stand at 4 deg.C overnight and the plate is washed by PBS. Then, 1% BSA-PBS is applied to the plate at the rate of 200mul per hole allowed to stand overnight and the plate is washed thoroughly by PBS. Thereafter, normal human serum (25 samples) and serum of lung cancer patients (34 samples) are applied to the plate at the rate of 100mul per hole, allowed to stand at 4 deg.C overnight and then, the plate is washed thoroughly by PBS. Then, biotinated anti-lung cancer monoclonal antibody ALC-1 (10mug/ml) is applied to the plate as the second antibody at the rate of 100mul per hole, allowed to stand at the room temperature for 1hr and it is washed by PBS. Then, ABTS substrate liquid is applied thereto at the rate of 100mul per hole to be brought into a reaction at the room temperature for 30min. and a 5% SDS solution is applied thereto at the rate of 100mul per hole. Then, the relation is stopped. As a result, in the serum of lung cancer patients 14 out of 34 cases were positive cases with OD415 exceeding 0.4.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to the field of clinical diagnosis, and the present application provides a method for quantifying a lung cancer antigen that can be applied to the clinical diagnosis of human lung cancer, and a kit for use in the clinical diagnosis.

BACKGROUND OF THE INVENTION Various methods for diagnosing lung cancer have been developed and used, such as X-ray photography, bite examination, fiberscope, and cytology. All of these methods, with the exception of some indirect X-ray imaging methods, are only performed on patients suspected of having lung cancer, and have not been used for early detection of lung cancer through so-called mass screening.

Problems to be Solved by the Invention In mass medical examinations, various tests for adult diseases are performed using blood and serum. If lung cancer can be diagnosed using blood or serum, it would be very useful because it would allow early diagnosis of lung cancer.

Means for Solving the Problems The present inventors have developed a method for serodiagnosis of lung cancer using a sandwich enzyme immunoassay using anti-human lung cancer-specific monoclonal antibody ALC-1 as a first antibody and a second antibody. They discovered that this can be done quickly and completed the present invention.

The present invention will be explained in detail below.

According to the present invention, an anti-human lung cancer specific monoclonal antibody ALC-1 (hereinafter referred to as the first antibody) is immobilized on a solid phase, a human serum sample is reacted with this to bind a lung cancer antigen, and then an anti-human lung cancer specific monoclonal antibody ΔLC-1 (
By labeling a second antibody (hereinafter referred to as a second antibody) with an enzyme, binding it to the lung cancer antigen bound to the first antibody, and measuring the amount of bound second antibody based on the reaction between the labeled enzyme and the substrate. , lung cancer antigens in serum samples can be quantified.

Anti-human lung cancer monoclonal antibody ALC-1 is a monoclonal antibody previously disclosed by the present inventors in Japanese Patent Application No. 59-46683, and is a hybridoma cell line ALC-1 (IFO50045) that has the ability to produce the monoclonal antibody.
) can be manufactured as follows.

8- to 10-week-old BA treated with Bristane [5 ml of 2,6,10,14-tetramethylpentadeca7 (Pristane) Q, administered intraperitoneally and kept for 2 weeks]
LB/C female mouse l:, ALC-1 cells 2-4X10"
"Seven cells are injected intraperitoneally. The hybridoma turns into ascites cancer in 10 to 21 days. Ascites fluid is collected from these mice,
Salting out with 50% ammonium sulfate, phosphate buffer (disodium phosphate 1.83 g, potassium phosphate 0.21 g, salt 7.65 g, resin water 11
, pH 7.2 or below (abbreviated as PBS) and NaCl 0.5
The mixture is dialyzed against a solution containing M and passed through a column of Sephacryl 5300 (manufactured by Pharmacia Fine Chemicals) (bed volume 750 ml) at a flow rate of 15 ml/hr, and the IgM fraction is collected and used as a purified monoclonal antibody.

Determination of antibody intib is performed by the Quchterlony method (double immunodiffusion method) (Introduction to Immunology Experiments, Biochemistry Experiment Methods 15, Gakkai Publishing Center, P, 74, 1981).

The amount of protein was quantified using the Folin method and absorbance at 280 nm (1,4 (OD2eo)'', immunoglobulin 1
mg/ml).

The specificity of the obtained monoclonal antibody was determined by its reactivity with normal or tumor tissue or its membrane components derived from various human organs obtained from multiple specimens, with various human normal or tumor cell culture lines, or with the membrane components thereof. Reactivity with human fetal cell culture lines or their membrane components, reactivity with conventionally known carcinoembryonic antigens (e.g. CEA), normal reactivity, reactivity with patient human serum, etc. were determined using enzyme-linked immunosorbent assay, Fluorescent antibody method,
This is performed using an immunohistological determination method (PAP method), etc.

Enzyme labeling of the monoclonal antibody ALC-1 was carried out using peroxidase, urease, alkaline phosphatase, β-galactosidase, etc. as enzymes, and a periodic acid cross-linking method [Journal of Histochemistry and Cytochemistry (J, Histachem, 1993)]. C
ytochem,) 22.1084-1091, (1
974)).

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

Dissolve 5 mg of peroxidase in 1 ml of 0.3 M sodium bicarbonate buffer (pH 8,1) and add fluoro-2°4-dinitrobenzene (DNP) solution Q, 1%.
1 ml of 0.06 M periodic acid is added thereto, and the mixture is allowed to react at room temperature for 30 minutes. This is dialyzed against a 0.01 M carbonate buffer at pH 9.5 to obtain a dialysate containing peroxidase having a DNP-formed aldehyde group. 5 μ of the antibody was added to this dialyzed solution and allowed to react at room temperature for 2 hours at a pH of 7.2. OIM! Dialyze with J-acid Babfur, and the solution after dialysis is diluted with 5ephadex.
Gel filtration is performed using G-100 column chromatography. The active fraction is used as an enzyme-labeled antibody.

When using peroxidase as the enzyme, add 550 mg of ABTS substrate solution [2,2'-azinopis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium to 0.
, 1M citrate buffer (a solution prepared by adding hydrogen peroxide IJJf!/ml immediately before use to a solution dissolved at pH 4, 2>11), and the color development is measured by absorbance at OD415 nm.As a substrate for urease. Teha 8 mg Bromocresol Purple, 100mg Urea and 0.2mM E
A solution containing DTA in 100 ml at pH 4.8 was used, and the measurement was carried out in accordance with the Journal of Immunological Methods (J, of Immunological Methods).
ds) 53.187-194. (1982). As a substrate for alkaline phosphatase, 1 μ/ml p-nitrophenyl phosphate was used as a substrate for alkaline phosphatase.
．． Use a solution dissolved in 1M jetanolamine buffer (pH 9,6).

Enzyme labeling can also be achieved via biotin-avidin binding. Biotin labeling was performed by J, HiStchem.

Cytochem, 27.1131-1139. (
1979).

Serological diagnosis is performed as follows.

Add the first antibody ALC-1 1 to a 96-well EIA plate.
Dispense 0 to 10011 g/ml into 50 to 200 g/well and leave at 4° C. for 1 to 2 nights or at room temperature for 2 to 4 hours. After washing with PBS, 1% bovine serum albumin (BSA)
- Add 200 m/hole of PBS and let stand at 4°C overnight or at room temperature for 2 hours.

After thoroughly washing the plate with PBS, add 50 to 100 tdl of serum specimen diluted 1 to 100 times to each well. 4
After leaving at ℃ overnight or for 2 hours at room temperature, wash thoroughly with PBS. Next, biotinylated second antibody or peroxidase-labeled second antibody (10-100x/A1
) was added at 50 to 100 JJIt/hole, and the mixture was further left at 4°C overnight or at room temperature for 2 to 4 hours. As a second antibody,
If biotinylated antibodies were used, the plate was washed with PBS.
After washing well with PBS, add avidin-biotin-peroxidase (10J1g/ml) at 50 to 10 ld/well, leave it for 30 minutes at room temperature, and wash well with PBS.Next, as a substrate solution, add ABTS substrate solution at 50 to 10 (bcl!/Add a hole, leave it for 10-30 minutes at room temperature, 5% SO3 solution 5
Add 0 to 100 m/hole to stop the reaction. OD of each hole
The 41 s value is measured, and the amount of antigen in the serum sample is calculated from the degree of color development. A normal value was determined by comparing the antigen amount in the serum of a healthy person and that in the serum of a lung cancer patient obtained in this way, and those exceeding the normal value were considered to be positive for lung cancer.

Examples of the present invention are shown below.

Example 1 96-hole EIA plate [Flow Laboratories (F
ALC manufactured by Low Laboratories)
-1 (10g/ml) was added 100n/well, left overnight at 4°C, washed with PBS, and added 1% BSA-PBS 200n/well.
100 n/well of healthy human serum (25 samples) and lung cancer patient serum (34 samples) were added to the plate, which was left overnight and thoroughly washed with PBS. After being left at 4°C overnight, the plate was thoroughly washed with PBS. . Next, as a second antibody, pyotinated anti-lung cancer monoclonal antibody ALC-1 (10,q/ml) was added for 100 s/well, left at 4°C overnight, washed well with PBS, and avidin-biotin-peroxidase. (V
(manufactured by IECTOR) (10g/ml) 100td
After adding t/hole and leaving it for 1 hour at room temperature, it was washed with PBS. Next, add ABTS substrate solution at 100μ/well and store at room temperature for 3
After 0 minutes of reaction, 100J11/well of 5% SDS solution was added to stop the reaction. The color development of each hole was measured using an absorbance meter (OD41S).
) was measured. As a result, as shown in Figure 1, there was only one positive case out of 25 with ODA15 exceeding 0.4 in serum from healthy individuals, but in serum from lung cancer patients, 0D41S
There were 14 out of 34 positive cases with a value greater than 0.4. This shows that lung cancer patients can be detected with a probability of 41% by the serodiagnostic method using this antibody.

Reference Example ALC-1 cells were added to 8-week-old BALB/c female mice treated with nibristane.
X10' cells/mouse were injected intraperitoneally. After 10-21 days,
The hybridoma turned into ascites cancer. Ascites was collected from mice with ascites (5 to 101'Ill/mouse) and centrifuged (3,00 rpm).

5 minutes) to remove solids.

The ascites from which the solid content had been removed was salted out with 50% ammonium sulfate, dialyzed with a solution containing 10.5M NaC in PBS, and transferred to a column with Sephacryl 300 (manufactured by Pharmacia Fine Chemicals) (bed volume 750m1) at a flow rate of 15m1. The IgM fraction was collected and used as a purified antibody.

[Brief explanation of drawings]

FIG. 1 shows the results of serum lung cancer diagnosis using the method of the present invention. 11 days

Claims (2)

[Claims] (1) Anti-human lung cancer-specific monoclonal antibody ALC-
1 (hereinafter referred to as the first antibody) is immobilized and reacted with a human serum sample to bind the lung cancer antigen, and then anti-human lung cancer specific monoclonal antibody ALC-1 (hereinafter referred to as the second antibody) is immobilized with an enzyme. 1. A method for quantifying a lung cancer antigen in a serum specimen, the method comprising: labeling with a second antibody, binding it to the lung cancer antigen, and quantifying the amount of the bound second antibody by measuring the activity of the labeled enzyme. (2) Human lung cancer antigen quantification consisting of a solid-phase anti-human lung cancer-specific monoclonal antibody ALC-1, an enzyme-labeled anti-human lung cancer-specific monoclonal antibody ALC-1, and a coloring system based on the labeled enzyme activity. kit for.