JPS62297755A - Reagent for diagnosing interstitial pneumonia - Google Patents

Abstract

PURPOSE:To enable sensitive diagnosis of the activity of a patient's disease condition by incorporating the monoclonal antibody KL-6 belonging to the IgG1 class which reacts with the human II-type alveolar epithelium, line bronchial epithelium, and bronchial gland serous cells into a titled reagent. CONSTITUTION:The mouse monoclonal antibody of the globulin class IgG1 which has the affinity to the human tissue including the human lung tissue and detects the soluble antigen in blood by an immunological measurement method is prepd. The soluble antigen in the serum detected by the antibody is quantitatively determined, by which the index to evaluate the activity of the disease condition of the interstitial pneumonia is obtd. More specifically, this reagent contains the monoclonal antibody KL-6 belonging to the IgG1 class which reacts with the human II-type alveolar epithelium, fine bronchial epithelium, and bronchial glad serous cells. The activity of the patient's disease condition is thus sharply diagnosed.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention (Field of Industrial Application) The present invention uses a mouse monoclonal antibody that has an affinity for human lung tissue, and uses a mouse monoclonal antibody that has an affinity for human lung tissue to infect patients with various types of interstitial pneumonia in Baijiu. II! as a diagnostic agent that can be used to qualitatively or quantitatively quantify existing soluble antigens! It is something to do.

(Prior Art) Interstitial pneumonia is a disease characterized by cellular infiltration of alveolar septa. The etiology of this disease is most common, and is caused by various pathological conditions including idiopathic interstitial pneumonia, sarcoidosis, hypersensitivity pneumonitis, collagen pneumonitis, pneumoconiosis, radiation pneumonitis, and drug-induced pneumonitis. It is a generic term. Because the etiology of these diseases is often unknown, there are few effective treatments, and there are only a few cases in which the condition improves in response to the administration of adrenocortical hormones, and in many cases, the condition improves rapidly. Or it is a group of diseases that gradually worsen and lead to death due to respiratory failure.

Incidentally, the average prognosis for idiopathic interstitial pneumonia is reported to be around 5 years. In addition, when diagnosing interstitial V1 pneumonia, a pathological search is essential. It is difficult to extract the main site of the lesion using a biopsy, so open pupil biopsy may be performed under general anesthesia.In cases where histological support cannot be obtained, the chest line It must be treated as a clinical diagnosis case by referring to examinations, pulmonary function tests, and medical history.In addition, in cases where the progression of interstitial pneumonia is chronic and long-term, it is necessary to determine whether the disease is in an active stage or not. It has always been difficult to determine whether the patient is in the active stage.There are only three diagnostic methods that are currently accepted as indicators of activity. 1. Search for pathological images from open lung biopsy as described above, 2. Check for radioisotope accumulation on scintigrams using 67Ga-citric acid, and 3. This involves performing bronchoalveolar lavage using a mirror, calculating the number of cells in the lavage fluid, and identifying the type of cells.

(Problems to be solved by the invention) However, open lung biopsy [is an extremely invasive test method performed under general anesthesia, and it is difficult to use this test as an opportunity to detect acute exacerbation of interstitial pneumonia. It is also reported that there are quite a few. Furthermore, since the diagnostic method using 67Ga-citric acid uses radioactive substances, it cannot be carried out except at medical institutions with sufficient facilities, and it is medically extremely undesirable to administer large amounts of radioactive substances to the human body. Therefore, it is impossible to implement it frequently. The third bronchoalveolar lavage method is not the open lung biopsy culm mentioned in the first method, but because it involves inserting a bronchoscope into the airway, the pain of thinking is not unusual as it is accompanied by a decrease in ventilation volume. , frequent implementation is discouraged. Therefore, LDH in the facial serum is generally used as an indicator of the activity of interstitial pneumonia.
Hydrogenase) level measurements are frequently used, but changes in L D +1 values are extremely insensitive, and most cases of Lee with increased IDH levels develop very severe acute exacerbation, and this is limited to this case. Therefore, it is difficult to say that it is an excellent indicator for understanding the disease status of interstitial pneumonia.

Therefore, the development of an index that can accurately and easily evaluate the activity of interstitial pneumonia is considered to be of great value.

(Means for Solving the Problems) The present invention is a mouse monoclonal antibody of globulin class IgG1 that has affinity for human tissues including human lung tissue and detects soluble antigens in blood by immunoassay. The aim is to produce an antibody and quantify the soluble antigen in baijiu that is detected by the antibody, thereby using it as an index to evaluate the activity of the pathological condition of interstitial pneumonia.

That is, the present invention relates to a reagent for diagnosing human interstitial pneumonia characterized by containing a monoclonal antibody K[-6 belonging to the IClG1 class that reacts with human alveolar type II epithelium, bronchiolar epithelium, and bronchial gland serous cells. .

In the following description, unless otherwise specified, cells 1 tissues, etc. are human cells 1 tissues.

- Monoclonal antibodies directed against human lung tissue used in the present invention can be prepared by, for example, producing hybridomas by cell fusion of murine myeloma cell lines and mouse murine cells immunized with a lung adenocarcinoma cell line derived from human lung tissue, and culturing the monoclonal antibodies against human lung tissue. It is produced by selecting antibodies in the supernatant that react with lung tissue, and then selecting antibodies that detect soluble antigens in the supernatant using immunological techniques.

By using the monoclonal antibody according to the present invention and quantifying the soluble antigen in the surface serum to which this antibody specifically reacts, it is possible to acutely diagnose the activity of the pathological condition of interstitial pneumonia. The present inventors discovered this for the first time.

The monoclonal antibody of the present invention can be produced as follows. That is, mice, rats, etc. are immunized with human lung tissue or human lung cancer cell lines, etc., tile cells are obtained from the immunized animal, these are fused with myeloma cells, and the resulting fused cells are cloned to produce human lung cancer cells. cell type epithelium and bronchiolar epithelium,
Fused cells that produce antibodies that react with bronchial gland serous cells are selected, cultured, and the antibodies recovered. The obtained antibodies were purified, some of the purified antibodies were labeled with enzymes, radioactive t1, and isotopes, and the Sand-Deutsche method was performed using unlabeled and labeled antibodies to identify soluble antigens with more than 11 million molecules in serum. Select fused cells to detect. Immunization method.

The cell fusion method, the fused cell selection method, the +) Ndoidge method, etc. can be carried out by known conventional methods.

More specifically, the monoclonal antibody of the present invention can be produced as a diagnostic reagent, for example, as follows.

First, mice are immunized with lung cells. The animal to be immunized is not limited to mice, but murine animals such as rats or other animals may be used; however, it is usually preferable to use mice. Furthermore, normal lung tissues, homogenates thereof, or antigens collected therefrom may be used instead of lung cancer cells.

For example, BALB/C mice are inoculated with lung cancer cells, normal lung tissue, homogenates thereof, or antigens collected therefrom several times at intervals of several days to several weeks. per animal between vaccinations
Preferably, 105 to 108 cells are used at one time. Thereafter, the glenoid is removed from the mouse, and antibody-producing cells are obtained by centrifugation. Since these cells do not have the ability to proliferate, they are fused with cells that have the ability to self-propagate. Myeloma cells are particularly preferred as cells with self-propagation ability. It is preferable to use myeloma cells from the same species of animal. Furthermore, it is preferable to select myeloma cells that do not produce antibodies. Antibody-producing cells and myeloma cells are mixed with a cell fusion agent such as polyethylene glycol to perform cell fusion. The ratio of antibody-producing cells to myeloma cells used is preferably 1:1 to 10:1 in cell number ratio. The tq'd fused cells are separated by limiting dilution method, and after the separated fused cells are grown, the antibodies produced in each well are treated with various types of antibodies using known methods such as fluorescent antibody method or enzyme antibody method. React with cells, tissues, etc.
From the results, hybridomas producing the desired antibody are selected. The selected hybridomas are grown by in VitrO culture method or in vivo transplantation method to obtain antibodies.

The in VitrO culture method can be carried out, for example, as follows. That is, hybridomas are cultured in an appropriate culture medium (for example, complete RPMI medium) until the growth limit is reached, and the culture supernatant is collected. The monoclonal antibody secreted into Tsuchiura can be purified and used as an IqG fraction by the following method. That is, the culture supernatant is precipitated with 40% saturated ammonium sulfate, and then passed through a diebulamine ethyl cellulose ram equilibrated with 2011M phosphate buffer at pH 7.8 to remove impurities before use.

Further, as an in vivo transplantation method, for example, it can be carried out as follows. That is, a hybridoma is injected into the body, for example, into the peritoneal cavity of a nude mouse, allowed to grow as a tumor within the body of the nude mouse, and antibodies are collected from the serum or ascites of the nude mouse. More specifically, 2,6,10,14-tetramethylpentadecane was injected intraperitoneally for 0.5 d in advance (5 to 10 days before) into BALB/C mice to which hybridomas were to be transplanted, and then hybridomas of 5 x 106 ~ 107 cells are implanted intraperitoneally. When the transplanted mouse is kept for an appropriate period of time, a tumor of Vybrid-7 is formed in the abdominal cavity, and the abdomen becomes enlarged. As a result, high concentrations of monoclonal pooled antibodies are produced in the ascites and serum, and these are collected. In addition to the monoclonal antibody of interest, the ascites and serum contain polyclonal bamboo antibodies derived from the mouse itself, but since they do not react with human tissue, they are used without being isolated. Also, as in the case of the in VijrO culture method, in
It is also possible to use the IoG fraction in the in vivo transplantation method.

Using the monoclonal antibody KL-6 thus obtained, the antigen in serum can be quantified by an immunological method to diagnose human interstitial pneumonia.

Enzyme immunoassay (E
IA), radioimmunoassay (RIA), and fluorescein immunoassay (FIA).The monochrome antibody Kl-6 can be used in any of these methods, and can be made into a form suitable for these methods and used as a reagent. These reagents may be manufactured by known methods.In these immunoassays, monoclonal antibody KL It is measured by forming a reaction product between a reagent containing -6 and an antigen.

In the measurement, the L-clonal antibody KL-6 is labeled and used for the measurement. As labeling agents, enzymes commonly used in FIA such as horseradish peroxidase, β-D-galactosidase, and alkaline A-sulfatase are used.
I at RIA.

For FIA, commonly used labeling agents such as fluorescein isothiocyanate can be used, and any labeling agent can be used as long as the activity of the labeling agent can be measured.

When the labeling agent is an enzyme, a substrate is used to measure its activity. As a substrate, for example, 5-aminosalicylic acid-H2 is used as a substrate for horseradish peroxidase.
Substrates for β-D-galactosidase include O2,0-phenylenediamine-H0, 4-aminoantipyrine-H2O2, etc.; −
Examples include galactopyranoside.

Furthermore, monoclonal antibody KL-6 can be attached to insoluble carriers used in conventional immunoassays, such as beads and plates made of synthetic resins such as polystyrene, polyethylene, polyacrylic, Teflon, and plastic. I can do it.

In addition, it is also possible to adopt a measurement method that does not require the label,
Measurement can be performed by forming a reaction product between the monoclonal antibody KL-6 and the antigen in serum, and applying hdl such as turbidimetry, PEG precipitation method, and latex agglutination method.

To give a more specific example, the purified monoclonal antibody can be labeled with horseradish peroxidase, for example, by the periodic acid method as an enzyme labeling method for 1-6.

Furthermore, as a 125-labeling method with a radioactive isotope, for example, a lactoperoxidase method can be used.

The binding inhibition method and the Sand-Deutsch method are considered to be preferable methods for detecting soluble antigens in serum, but since the binding inhibition method requires purification of a large amount of antigen, the Sand-Deutsch method is generally used. is used. To carry out the sandwich method, first, purified monoclonal antibodies are adsorbed onto beads such as polystyrene to form antibody-labeled beads, which are then reacted with serum in a test tube. If soluble antigen is present in the serum, it will bind to the antibody. After washing, enzyme or
When a labeled antibody labeled with I is reacted and washed, the labeled antibody is indirectly bound to the beads in proportion to the amount of antigen in the serum, and then the bound enzyme is suspended or
M of soluble antigen in serum by measuring the amount of I
It is possible to quantify.

(Example) Example 1 1) Immunization 8-week-old mBALB/cv mice were 5×1
06 lung adenocarcinoma-derived cell line (hereinafter referred to as VHRC-LCR), and then 8 x 106 cells were intraperitoneally injected twice at 2-week intervals.

2) Cell fusion Three days after the final immunization, the spleen was taken out and passed through a stainless mesh to prepare a cell suspension. This 8.
4X 107 tile cells and 4.2X 107 8 azaguanine-resistant myeloma cells P3-NSI-Ao4/1 (N
After centrifugation, 1 d of 45% polyethylene glycol (average molecular weight 1 fi 6000) was added and gently stirred for 2 minutes. After washing, the cell mixture was suspended in RPMI medium containing 10% tallow serum (complete RPMI medium) and placed in a 96-well microculture plate at 106 cells per well.
The seeds were sown at a ratio of 0.1. After 24 hours, complete RPMI medium (+-IA T
Medium) 0.1#11! added.

On the 2nd, 3rd, 5th, 7th, and 10th day after the start of culture, 0.1 m of the culture supernatant was discarded and 0.1 m of l-IAT medium was added.
#+1! added. After 12 days, hybridomas were observed to grow in all the wells.

3) Selection of hybridomas Hybridomas producing antibodies against VHRC-LCR cells were selected by enzyme antibody method. The enzyme antibody method was performed as follows.

VHRC-LCR cells were cultured in a 96-well microculture plate until reaching the growth limit, fixed with 0.25% glutaraldehyde for 5-7 minutes, and washed 5 times. 0.1 d of hybridoma culture supernatant was added and reacted at room temperature for 1 hour. After washing 5 times, 50 ml of horseradish BelA oxider L-labeled goat anti-mouse immunoglobulin was added as a second antibody and allowed to react for 1 hour.

After washing 6-7 times, add 1.1% hydrogen peroxide and 150 II
! g/lll1! 100/Zi! In addition, 1
Color was developed for 0 minutes at room temperature. Fifty portions of 10% oxalic acid were added as a stop solution, and the absorbance of 405 nlll was measured using a microplate spectrophotometer, and hybridomas with an absorbance of 0.02 or more were selected.

The selected hybridomas were transferred to a 24-well culture plate seeded with BALB/c mouse thymocytes (feeder cells), and treated with 100 μM hypoxanthin and 16
The cells were cultured in complete RPMI medium (HT medium) containing μM thymidine. After reaching the growth limit, V
Hybridomas producing antibodies against HRC-1 and cR cells were selected.

Next, the selected hybridomas were cloned by the limiting dilution method. That is, 50 cells/d! Or 1
The cells were diluted to 0 cells/d, dispensed into 96-well microplates pre-seeded with feeder cells (0.1 d each per culture plate, and cultured in l-IT medium for 2 weeks). When one hybridoma colony was formed in one well, it was taken out as a clone. VHRC-LCR by enzyme antibody method
Hybridoma clones were selected that secreted antibodies that reacted with cells but did not react with normal human lung fibroblasts.

Furthermore, among these clones, clones that reacted with human lung adenocarcinoma tissues were selected by immunoperoxidase staining of frozen sections. That is, human lung cancer, other organ cancers, and normal tissues were obtained using surgical materials, and 4IIIR frozen sections thereof were prepared. After fixation with acetone, the culture supernatant of the hybridoma clone was added and allowed to react at room temperature for 30 minutes. After thorough washing, the cells were reacted with a biotin-labeled anti-mouse TOG antibody (reacts with γ and λ chains) for 30 minutes at room temperature. After further washing, avidin and biodin-labeled horseradish 1 novipero sidase were added and allowed to react at room temperature for 1 hour. After thorough washing, 0.5 IPj/Id of diaminobenzidine and 0.5 IPj/Id of diaminobenzidine were added as substrates.
A 50mM t-Lis-HCl buffer (pH 7.0) containing 01% H2O2 was added to develop color.

In this way, the alveolar type epithelium, the bronchiolar epithelium.

Bronchial gland serous cells, thyroid follicular cells 1 Esophageal epithelium.

Cardiac gland cells, pancreatic duct epithelium, renal tubular epithelium, bladder transitional epithelium, endometrium, lung adenocarcinoma, lung squamous cell carcinoma, lung small cell carcinoma, stomach, duodenal papilla, bile duct, pancreas, colon, rectum, thyroid・Adenocarcinoma of the mammary gland 9 Although it responds to esophageal squamous cell carcinoma, it responds to bronchial epithelium, gastric surface mucinous Hp cells, and pyloric gland.

Duodenal epithelium, colon epithelium, rectal decathelium, hepatocytes, pancreatic exocrine cells, pancreatic endocrine cells, renal glomerular cells, cervical squamous cells, skin epithelium, uterine squamous cell carcinoma.

A hybridoma producing a monoclonal antibody that does not react with hepatocyte muscle was obtained and was named KL-6 cells, and the monoclonal antibody produced thereby was named KL-6 antibody.

4) Preparation of monoclonal antibodies (a) In VitrO culture method 105 antibodies/ml! Hybridomas were grown in culture medium (complete RP
MI medium) until the growth limit (2 x 106 cells/d) was reached, and the culture supernatant was collected. (However, it can also be used as an IOG fraction by the following method. Namely,
After precipitating the culture supernatant with 40% saturated ammonium sulfate, pl+7
．． It is used after removing impurities by passing it through a dietylaminopurcellulose column equilibrated with 20111M phosphate buffer (No. 8). ) (b) In vivo transfer IIi! Transplant the I-paid hybridoma into BΔL B/C mice in advance (5
~10 days ago) 2,6,10.14-tetramedylpentadecane was administered intraperitoneally. 5ml! Give it an injection. Next, 5 x 106 hybridomas are implanted intraperitoneally. When the transplanted mouse is kept for three weeks, a hybridoma tumor is formed in the abdominal cavity and the abdomen becomes enlarged. As a result, high I! ! Several monoclonal antibodies were generated and collected. In addition to the monoclonal antibody of interest, the ascites and serum contained polyclonal antibodies derived from the mouse itself, but since they did not react with human tissue, they were used without being isolated. (Also, it can be used as an ICJG fraction by the method shown in 4) (a). ) 5) Characteristics of t-clonal antibody KL-6 f1a) Reactivity with normal lung tissue The reactivity between KL-6 antibody and normal lung tissue was investigated by immunoperoxidase staining 3. −6 antibodies=
21- reacted with type ■ alveolar decathelium, bronchiolar epithelium, and bronchial gland serous cells, but did not react with type ■ IMi cell epithelium, bronchial epithelium, bronchial gland mucus cells, and interstitium.

b) Reactivity with lung tissue of interstitial pneumonia The KL-6 antibody reacted with regenerated alveolar ■-type epithelium in interstitial pneumonia and fibrotic sites in lung tissue of idiopathic interstitial pneumonia. Interstitial collagen fibers.

It did not react with lineal miscellaneous blast cells or inflammatory cells.

C) Reactivity with normal tissues other than lungs KL-6 antibody is thyroid I & 1 cell epithelium, esophageal epithelium.

Cardiac gland cells, pancreatic ductal epithelium, renal tubule skin, bladder transitional epithelium,
Reacted with endometrial cells, but gastric superficial mucosal cells.

Pyloric gland cells. Duodenal skin. Colonic epithelium, rectal epithelium.

Hepatocytes, pancreatic exocrine/endocrine cells, renal glomerular cells.

Cervical squamous epithelium, skin epithelium. It did not react with the blush method or white blood cells.

d) Reactivity of K L-6 antibody with cancer tissue is lung adenocarcinoma, lung squamous cell carcinoma, lung small cell carcinoma, gastric cancer, papillary carcinoma, cholangiocarcinoma, pancreatic cancer, and colon cancer.

It reacted with rectal cancer, papillary thyroid adenocarcinoma, breast cancer, renal cancer, and esophageal squamous cell carcinoma, but did not react with cervical squamous cell carcinoma or hepatocellular carcinoma.

e) Cell antigen that reacts with monoclonal antibody KL-6 Cells that react with KL-6 antibody are treated with periodic acid, neuraminidase, and protease, and then reacted with KL-6 antibody to determine the biochemical properties of the cell antigen. I looked into it. 9
After culturing VHRC-LCIt cells in a 6-well microculture plate until the proliferation limit was reached, they were fixed with 0.25% geltaraldehyde, washed thoroughly, and then placed at 1, 10.100 m
M periodic acid, 0.01,0.1. lu (unit)/
- Neuraminidase was added and reacted for 30 minutes, washed, and reacted with KL-6 antibody for 1 hour. After washing, i, Q
After adding I-labeled goat anti-mouse immunoglobulin of X 10 CpHl and reacting for 1 hour, after washing,
Cells were solubilized with 0.2N sodium hydroxide, transferred to a polystyrene tube, and radioactivity was measured using a gamma counter. As a result, the K for cells treated with periodic acid and neuraminidase was higher than that of untreated cells.
The binding of the L-6 antibody was reduced, and it was found that the cell antigen with which the KL-6 antibody reacts is a sugar chain antigen having sialic acid at the end. In addition, 5 x 106 VHRC-
LCR cells were reacted with 0.25% pronase for 30 minutes, washed, reacted with 05-KL-6 antibody for 30 minutes, washed, and then treated with 10/u FITC (fluorescein
Isodiocyanate) labeled goat anti-mouse I OG F
(ab') 27 Reacted with lagmen 1- for 30 minutes. After washing, the cells were fixed with 1% paraformaldehyde and the fluorescence was observed using a fluorescence microscope. As a result, the fluorescence of the pronase-treated cells was attenuated compared to the untreated cells. Therefore,
It was found that the KL-6 antibody-reactive antigen on cells also has a protein portion. From the above, it was revealed that the cell antigen with which the KL-6 antibody reacts is a glycoprotein, and the antigenic determinant is a sugar chain having a sialic acid at the end.

f) Molecular weight of soluble antigen that reacts with monoclonal antibody KL-6 To measure the molecular weight of soluble antigen that reacts with K1-6 antibody, cancerous pleural effusion was gel-filtered with Sepha[1-S4B].
The soluble antigen content in each fraction was quantified and determined using the sandwich enzyme antibody method described below. As a result, it was found that the molecular weight of the soluble antigen with which the K L-6 antibody reacts is at least 1 million or more.

6) Serum soluble antigen (K
Quantification of KL-6 antigen) a) Sandwich oxygen antibody method for quantification of KL-6 antigen A sandwich enzyme antibody method was performed for the quantification of KL-6 anti-ryo in serum. Polystyrene beads were used as the same phase. First, the beads were immersed in a purified KL-6 antibody solution (0.25 M phosphate buffer pH 7.5) at 50 m/d.
After incubating at 37°C for 1 hour, the mixture was cooled in ice water for 10 minutes and the IC mixture was used as antibody-adsorbing beads. The periodic acid method was used for enzyme labeling of the purified K i-6 antibody. That is, 5 IPJ of horseradish par No. 4] sidase to 1 #Il! Add 0.1-IX 1-fluoro-2,4-dinitrobenzene ethanol solution to the solution dissolved in 0.3M carbonate buffer pH 8.1, and after incubating for 20 minutes, dissolve in 0.01M carbonate buffer pH 9.5. was dialyzed. After dialysis, 1 d of 0.06M periodic acid was added and reacted for 30 minutes, then 1 d of 0,16M x brene glycol was added and reacted for 60 minutes, and dialyzed again against 0.01M charcoal [buffer 1] 89.5. and then equilibrated with the same buffer.
The essence of mg! 1IKL-6 antibody solution and reacted at room temperature. After 3 hours, 5 μ of sodium borohydride was added, and the mixture was left standing at 4° C. overnight. Thereafter, it was dialyzed against PBS, and the first peak obtained by gel filtration with a Sephadex G-200 column was used as a peroxidase-labeled KL-6 antibody.

The measurement procedure is as follows: First, add protein buffer (10% normal rabbit serum, 0.1% bovine serum albumin) to a glass tube containing KL-6 antibody absorbent.

40 at 0.15M PBS Ill 6.4)
A sample diluted twice was added for 0.3 d, and incubated at 37° C. for 3 hours.

Afterwards, it was washed 3 times with saline, and then washed with protein buffer for 10 minutes.
Veroxider 1-labeled K L-6 antibody diluted 0 times was added for 0.3 d, and the ink was incubated for 16 to 20 hours. After washing, transfer the beads to a polystyrene tube and incubate at 0.
3.0 PDA solution (0.3% o-phenyene
diamine dihydrochloride,
0.02% @2Q2゜0.05M C1trate
buffer (pH 4,0) and reacted for 30 minutes, then 1 d of 2NPA acid was added to stop the reaction, and the absorbance was 0 D.
492 was measured.

As a standard sample, a 160-fold dilution of cancerous pleural effusion caused by lung cancer was used as 640/d, as shown in Figure 1.
A standard curve was drawn using seven points of 0.2, 4, 8, 16, 32, and 64 u/d, and the sample was determined.

b) Healthy people, various malignancies! Distribution of serum KL-6 antigen levels in patients with LFR ulcers, benign lung disease patients, and patients with other inflammatory bamboo diseases As shown in Figure 2, the KL-6 antigen level in 80 healthy subjects was 218 ± 152 u/me. (Average (m1 standard deviation (
SD)) and cut m+2SD value 5820/d
It was set as the off value. The positive rate for cancer patients is 32% (32%) for lung cancer.
2/99).

Gastric cancer 0% (0/19), Otama cancer 0% (0/8), hepatocellular disease 0% (0/8), pancreatic cancer 44% (4/9), breast cancer 7
It was 5% (6/8).

The K L-6 antigen values in benign lung diseases are shown in Figure 3, and the positive rate was 10% (2/21) for alveolar pneumonia, 0% (0/15) for chronic bronchitis, and 11% for bronchial asthma. (1/9)
, emphysema 40% (4/10), bronchiectasis 40% (2/10)
15), pulmonary tuberculosis 43% (9/21), diffuse panbronchiolitis 40% (4/10).

Interstitial pneumonia was 58% (29,150).

1--6 antigen values in inflammatory diseases other than the lungs are the fourth
As shown in the figure, the positive rate for chronic hepatitis was 11% (1/9).
, liver cirrhosis 18% (2/11), pancreatitis 0% (0/14),
Cholecystitis θ% (0/7).

C) Serum KL-6 antigen value in patients with interstitial pneumonia As mentioned above, in the study of 50 cases of interstitial pneumonia, the serum KL-6 antigen value was
The positive rate for 6 antigens was high at 58%, but when classified by etiology, the positive rate was 67% (18/27) for idiopathic interstitial pneumonia, collagen disease lung Disease 44
% (4/9), sarcoidosis 40% (215),
Hypersensitivity pneumonitis 100% (2/2), radiation pneumonitis and drug-induced pneumonitis 50% (3/6), pneumoconiosis 0% (0/1)
)Met.

Serum KL-6 antigen levels and serum 1[))l (lactic dehydro
As shown in Figure 6, no significant correlation was found between the two activities, and the positive rate was
While the DH value was 11% (3/27), KL-6
The antigen value was 57% (16/28), and KL-6 antigen showed clearly superior sensitivity of 5.

67Ga, which is said to be used to evaluate the activity of interstitial pneumonia
- When we examined the KL-6 antigen levels in 15 cases of interstitial pneumonia for which citrate scintigraphy had been performed, we observed an accumulation of 67Ga in the lungs, as shown in Figure 7.
The KL-6 antigen positive rate in 0 cases was 80% (8/10), whereas in the 5 cases in which 67Ga accumulation images were not found, no KL-6 antigen positives were found, and the A strong correlation was observed.

KL-6 of 12 cases of interstitial pneumonia with follow-up observation
Figure 8 shows the changes in antigen values. KL was used in one case where subjective and objective findings improved and interstitial pneumonia showed a tendency to remit.
-6 antigen level decreased. Clinically, in the six cases in which little change in activity was observed, the fluctuations in Kl-6 antigen values were similarly small.

The five cases that showed clinical signs of worsening were all lung cancer cases, and radiation pneumonitis was caused by radiation therapy, but the K1-6 antigen levels after pneumonitis were the same for all types. There was an increase in

From the above, it is clear that serum K L-6 antigen is useful as an indicator of interstitial pneumonia activity.

(Effects of the Invention) When the reagent for diagnosing interstitial pneumonia of the present invention is used, the clinical condition of patients with interstitial pneumonia can be improved in 1. IJ t/I can be acutely diagnosed.

[Brief explanation of the drawing]

Figure 1 is a graph showing the standard curve for Kl-6 antigen;
The figure shows the distribution of 1-6 antigen values in serum, Figure 3 shows the distribution of KL-6 antigen values in serum of patients with benign lung disease, and Figure 4 shows the distribution of KL-6 antigen values in serum of patients with benign diseases other than lung disease. A diagram showing the distribution of serum KL-6 antigen values. Figure 5 shows serum KL in patients with interstitial pneumonia classified by etiology.
-6 antigen value distribution; Figure 6 is a diagram showing the correlation between serum KL-6 antigen value and serum LD l-1 value in patients with interstitial pneumonia;
Figure 8 shows the distribution of serum KL-6 antigen levels in patients with interstitial pneumonia as determined by 7G a-scintigram. be. Figure 1 KL-6 promotion t+Uchi news KL-6 1r
Attack -tl Shun'i X'IL Throat example

Claims (1)

[Claims] A reagent for diagnosing human interstitial pneumonia, characterized in that it contains a monoclonal antibody KL-6 belonging to the IgG_1 class that reacts with human alveolar type II epithelium, bronchiolar epithelium, and bronchial gland serous cells.