JPS5949544B2 - Method for producing NEA antibody for neoplasm diagnosis - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an antiserum for diagnosing neoplasms using a novel fetal antigenic substance NEA (hereinafter simply referred to as NEA) for neoplasms.

Studying the specific antigenic substances of neoplasms in humans is an important issue for the pathogenesis, prevention, treatment and diagnosis of neoplasms.

In recent years, attempts have been made to search for antigenic substances specific to neoplasms, and some antigenic substances have been reported. Examples include α-fetoprotein for primary hepatocellular carcinoma and carcinoenpronic antigen (CEA) for adenocarcinoma.

However, these antigenic substances are restricted to certain organs, system organs or histopathological forms of neoplasms.
For this reason, we have explored the existence of neoplasm-specific antigenic substances that are common to a wide variety of organs and histopathological forms of neoplasms, have produced specific antibodies against these antigenic substances, and have also developed antigen-specific antigens using these specific antibodies. Establishment of a detection method is expected to encourage vigorous progress in the diagnosis, prevention, and treatment of various neoplasms occurring in humans. Therefore, it is an object of the present invention to provide a method for producing NEA antibodies that can be used for diagnosing a wide variety of neoplasms.

As a result of exploring the existence of unique antigenic substances common to a wide variety of neoplasms, the present inventor discovered that NE, a novel antigenic substance.
I found A.

The NEA of the present invention is neoplasmembryoni.
It is an abbreviation for "cantigen" and was named by the present inventor.

NEA has the following properties.

C. NEA is an Uttalony test method that uses antibodies against NEA. It can be proven that it exists.

However, the Uktaroni test method cannot prove the presence of NEA in non-neoplastic tissues of neoplastic patients, normal human serum, serum and ascites of non-neoplastic patients. Furthermore, N.E.
Even if antiserum against NEA is neutralized and absorbed with non-neoplastic patient tissue extract antigen, normal human serum, and non-neoplastic patient serum, NEA
No inactivation or attenuation of antibody activity was observed, and NEA antibody activity may be inactivated or attenuated by absorption procedures using fetal tissue extracts, fetal serum, certain malignant neoplastic tissue extracts, or patient serum. Proved by the Uktaroni method.

NEA is therefore a specific antigen common to fetuses and neoplasms.

2) NEA is cellulose acetate membrane, Pevicon C-
870 (vinyl chloride/vinyl acetate complex) using agar gel, starch, etc. as a support, pH 8.6, ionic strength 0.02
γ by electrophoresis using barbital buffer of 5 to 0.1
Uktaroni 30 is electrophoresed in the globulin fraction and is immunologically different from known immunoglobulins (immunoglobulins G, A, M, D and E) and other serum gamma globulins.
−=′. 7;. '::Go≠Everyone-ゝ10m 0.936.

The protein content was measured by the Lowry method using bovine serum albumin as a standard. 4) NEA is the result of molecular weight measurement by column chromatography using Sephadex G-200 (trademark Pharmacia),
It is a protein with a molecular weight of 100,000 and a molecular weight of 20,000.

5) NEA was determined by chromatography using a basic anion exchanger at an ionic strength of 0.05 and a pH of 7. When developed with O buffer, it has the property of being eluted together with immunoglobulin G without being adsorbed to the ion exchanger.

6) The isoelectric point of NEA is PH9. l~9.4.

7) NEA has a pH of 7. O, has the property of precipitating in a 2.6 molar ammonium sulfate solution.

As mentioned above, NEA is a specific antigen for neoplasms, and neoplasms can be diagnosed by examining the presence of NEA in serum.

The present invention uses this NEA to produce NEA antibodies for neoplasm diagnosis.

The configuration of the present invention is as follows.

A non-human animal is immunized with NEA or a complex of NEA and NEA antibody, and antiserum is collected. In the present invention, NEA
may be used unpurified or purified.

For example, fetal serum, amniotic fluid, serum from neoplastic patients, ascites from neoplastic patients, extracts from neoplastic tissues, meconium extracts, and those purified by protein fractionation and purification can be used. As the protein fractionation and purification method, a method commonly used in the field of immunology is used.

For example, salting out method, electrophoresis method, gel filtration method, ion exchange method, affinity chromatography method, centrifugation method,
Ultra deep filtration method, isoelectric point fractionation method, organic solvent fractionation method, cone (
fractionation method of COhn), modified method of Spiro (SpirO), N
Add EA antibody to collect NEA-NEA antibody complex,
Examples include a method of separating NEA from this, and a method of removing contaminants by adding antibodies against contaminants other than NEA. A complex of NEA and NEA antibody can be obtained, for example, by mixing the ridge and NEA antibody in a solution.

An animal other than a human is immunized with the above-mentioned NEA or a complex of NEA and NEA antibody, and blood is collected after a certain period of time to obtain an antiserum.

Examples of animals used include domestic rabbits, goats, horses, and cows.When immunizing, use Freund's complete adjuvant to immunize with NEA or NEA (5) emulsified complex with NEA antibody, and It is preferable to immunize not only once but several times in order to obtain antiserum with a high antibody titer.To remove antibodies other than NEA antibodies contained in the obtained antiserum: Normal human serum , immunoadsorption method using extracts obtained from non-neoplastic patient ascites and serum, and non-neoplastic tissue as antigens; affinity chromatography to which the above-mentioned antigen is fixed; affinity chromatography to which NEA is fixed; Add NEA to the antiserum and
The EA-NEA antibody conjugate was isolated and NE
A method for separating antibodies can be used.

Diagnosis of neoplasms (N
EA detection), the following method can be used.

1) Uttalony [wad immunodiffusion method and Uttalony [wad immunodiffusion method] by Cohn's modified method 2) Single plate immunodiffusion method (S.R.I.D.) 3) Electrophoresis method (immunoelectrophoresis method,
When using this method (immunoelectrophoresis, electroimmunodiffusion), since both the NEA antibody and the analyte migrate to the γ-globulin fraction, the NEA antibody or the analyte is carbamylated and transferred to the albumin side. Use it so that it can move easily.

Even when the NEA antibody and the subject are subjected to this carbamylation treatment, their immunological activity as antibodies or antigens is not inactivated. 4
) Radioimmunoassay (RIA) 5) Enzyme antibody assay (ELISA) 6) Method using bacteriophage 7) Complement fixation reaction (CF) 8) Hemagglutination reaction (PHA, RPHA) 9) Immune adhesion reaction (A ) Diagnosis of neoplastic patients using the NEA antibody and immunodiffusion method among the above methods revealed gastric cancer, colon cancer, rectal cancer, pancreatic cancer, primary liver cancer, uterine cancer, and ovarian cancer. 27 out of 84 patient sera of breast cancer, leukemia, and lymphosarcoma were positive.

In contrast, all 111 sera from normal subjects and non-neoplastic patients were negative. In addition, the results of diagnosis using the one-way plate immunodiffusion method showed that 3 out of 98 cases of neoplastic patient serum
Seven cases were positive, serum 1 of normal and non-neoplastic patients.
All but one of the 41 cases were negative. Therefore, the NEA antibody according to the present invention can be used for specific diagnosis of neoplasms in general, and malignant neoplasms in particular. It should be noted that the Uktalony immunodiffusion method and the one-way plate immunodiffusion method are relatively insensitive methods, so if a more sensitive method is used,
It is thought that the diagnosis rate will further increase.

Next, a method for purifying NEA, a method for producing NEA-NEA antibody complex, a method for producing NEA antibody, a method for purifying NEA antibody,
The present invention will be explained in more detail by showing the results of neoplasm diagnosis using antibodies as Examples and Experimental Examples.

Experimental Example 1 Partial Purification of NEA from Cancer Tissue Frozen 1009 breast cancer removed tissue was cut into dice.
Add 5 volumes of physiological saline in a homogenizer and heat at 4°C.
Homogenized for 10 minutes.

To this, add an equal amount of physiological saline (sodium nitride) to 0.
After stirring at 4° C. for 48 hours to extract the protein, the mixture was centrifuged at 5,000 gravity (G) for 30 minutes. The uppermost fat layer was removed and the supernatant liquid was collected. The resulting supernatant was centrifuged again at 2,000 gravity (G) for 30 minutes, the topmost small fat layer was removed, and the supernatant was collected. This supernatant was placed in a cellulose tube for dialysis, and polyethylene glycol (average molecular weight approximately 15
,000) and concentrated to about 1/20 volume. The protein concentration after concentration is LOIYTOlin.
) method, the result was 73~/ml. 3ml of this concentrated liquid was filled with Cephadex G-200 and the diameter was 2.6CT! 11 Length 90CTfL (floor capacity 460m0
Using a column, gel filtration was performed in barbital buffer with pH 8.6 and ionic strength 0.05, and the eluate was fractionated. During elution, four peaks were seen on a 280 mμ spectrophotometer. That is, the first peak is the fraction containing 19S or higher proteins, including Φ-macroglobulin and immunoglobulin M1β-lipoprotein, the second peak is the 7S globulin fraction containing immunoglobulin, and the third peak is the fraction containing albumin. The fourth peak was a fraction containing a substance with an even smaller molecular weight. NEA was eluted between the second and third fractions. That is, molecular weight 120,000~
Approximately 90% of the NEA was eluted in the 80,000 fractions.
The eluate between the second and third peaks, excluding the second and third peaks, was collected and used as a crude NEA fraction. Repeat the above operations to collect the crude NEA fraction, and concentrate this crude NEA fraction to approximately one-tenth the volume of the original tissue using an ultrafiltration membrane (passable molecular weight 10,000 or less). did. Then the length is 33
CTn1 width 8Cr! An electrophoresis tank with L1 thickness of 2 CTn was filled with Pevicon C-870 as a support, pH 8.6,
Using a barbital buffer solution with an ionic strength of 0.05, 5 ml of the crude NEA fraction concentration solution was injected into a sample groove provided at a position 10 cIn from the cathode side of the electrophoresis layer, and electrophoresis was performed at a constant current of 80 mA for 16 hours. Since the NEA exists at a distance of 3 to 7 cm from the sample groove to the cathode side, the support in this area was separated and 100 m1 (7) PH7.5, O. After adding O5 molar phosphate buffered saline and stirring, 3,000 rpm15
The supernatant was collected by centrifugation for a minute. To this supernatant, 100 ml of the same buffer was added and stirred, followed by centrifugation under the same conditions to collect the supernatant. In order to remove a small amount of Pepicon C-870 mixed into the collected liquid, the collected liquid was passed through a glass filter. Approximately 200 m10) of the finished liquid was placed in a protein bag [Carl Schleicher,
Schleicher) UltrahisenN
5T
Concentrated to n1. The same operation as above was performed 5 times, and the obtained concentrate was stored at 4°C. The electrical mobility of the obtained NEA was almost the same as that of immunoglobulin G. Immunoglobulin G and A were detected as contaminant proteins in this preparation by Uktaroni test method and immunoelectrophoresis method. In addition, by the above method, NEA was calculated from the NEA in the initial physiological saline extract to approximately 50 (F6
was recovered, and the degree of purification was about 100 times higher. Experimental Example 2 Partial purification of NEA from ascites of cancer patients
The concentrated solution was centrifuged at 20,000 gravity (G) for 30 minutes to collect the supernatant.

The supernatant liquid has a pH of 7.5 and an O. O5 molar phosphate buffered saline was added to make the total volume 11 (protein content approximately 3%). Add ammonium sulfate to this to make a 1.5M ammonium sulfate solution, and add sodium hydroxide to pH 7. Adjusted to O. After leaving this solution for 60 minutes,
Centrifuged on day 0 n. The supernatant was collected and ammonium sulfate was added to make a 2.6M ammonium sulfate solution.
After 60 minutes, centrifugation was performed at 10,000 rpm for 30 minutes. More than 60% of NEA was collected in the sediment. The precipitate was dissolved in barbital buffer with pH 8.6 and ionic strength 0.05, and the total volume was adjusted to 200 d. This solution was subjected to gel filtration using Cephadex G-50 (trademark, Pharmacia) using the same buffer, and the first eluted protein fraction was collected, followed by desalting of ammonium sulfate and buffering with barbital. . The protein concentration of this ammonium sulfate fraction was approximately 70,719/d.
This solution 5a was prepared and subjected to electrophoresis. That is, length 3
Pevicon C-870 was filled as a support into an electrophoresis tank with a width of 8CTfL and a thickness of 2C7rL, and the pH was 8.6.
Use a barbital buffer with an ionic strength of 0.05, and apply 10C from the cathode side of the electrophoresis tank! The solution 5d was injected into the sample groove provided at the position n, and electrophoresis was performed at a constant current of 80 mA for 16 hours. After that, 3~7C from the sample groove to the cathode side! ! The support at a distance of L was separated, and 100a of pH 7.5, O. O
After adding 5M phosphate buffered saline and stirring, 3,000r
pm, and centrifuged for 15 minutes to collect the supernatant. Furthermore, 100 μm of the same buffered saline was added and stirred, followed by centrifugation under the same conditions, and the supernatant was passed through a glass filter.

2.57 nI with protein bag! Concentrated into
2.5 ml of this concentrated liquid was filled with Sephadex G-200 and the diameter was 2.6C! ! L1 length 90cm (floor capacity 4
6011LI) column, pH 7.5, O. O5
Gel filtration was performed in molar phosphate buffer, and the eluate was aliquoted into each test tube. During elution, two absorption peaks were observed on a 280 mμ spectrophotometer. The first peak was the 19S fraction containing immunoglobulin M, and the second peak was the 7S fraction containing immunoglobulin. NEA elutes slightly later than the second peak. That is, molecular weight 120,000-80
About 90% or more of NEA was present in the fraction corresponding to ,000. This fraction was collected, concentrated in a protein bag, and stored at 4°C. Immunoglobulin G and A were detected as contaminants in the obtained NEA specimen by Uktaroni test method and immunoelectrophoresis method. The degree of purification of the obtained NEA was about 50 times higher. Example 1 Production of NEA antibody The partially purified NEA obtained in Experimental Example 1 was dissolved in physiological saline to reduce the protein amount to 1.
0.5ml of this solution.
An equal amount of Freund's complete adjuvant was mixed and emulsified and the solution was injected intradermally, subcutaneously, and intramuscularly into the hind legs and abdomen of a domestic rabbit weighing approximately 2 kg.

Two weeks later, NEA emulsion 1 was prepared in the same manner as above.
d, and two weeks later, the same NEA emulsion 17! Ll was boosted again.

Serum was collected 10 days after the last immunization, and after separation, the serum was incubated at 56°C for 30 minutes to inactivate it, and then sodium nitride was added to a concentration of 0.05% for preservative purposes. To 1 WLI of this antiserum, 1/3 volume of normal human serum and 5 volumes of colostrum purified γ-globulin were added, incubated at 37°C for 1 hour, then incubated at 4°C for 48 hours, and then incubated at 4°C for 30 minutes. ,00
It was centrifuged at 0 rpm and the supernatant was collected. The NEA antibody specificity of the antiserum after such treatment was determined by the Uktaroni test method and immunoelectrophoresis using original breast cancer tissue extract as an antigen, and it was found that it contained no antibodies other than NEA antibodies. Proven. The reason why colostrum-purified γ-globulin was used to neutralize the antiserum is that the antigen-extracted tissue was a breast cancer tissue, and there is a possibility that the NEA partially purified specimen may contain secreted immunoglobulin A antigen. There is a possibility that antibodies against this may have occurred in the prepared antiserum.
For the purpose of absorbing and removing this antibody, γ-globulin purified from colostrum containing a secreted immunoglobulin A antigen was used. Example 2 Production of NEA antibody Partially purified NEA obtained in Experimental Example 2 was mixed with protein 1 in physiological saline.
mg/ml, and mixed and emulsified 0.5 ml of this solution with an equal amount of Freund's complete adjuvant, and injected dividedly into the hind legs and abdomen of domestic rabbits weighing approximately 2 kg, subcutaneously, and intramuscularly. .

Two weeks later, NEA emulsion 1 was prepared in the same manner as above.
m1 was boosted, and two weeks later, the same NEA emulsion 1a was boosted again.

Blood was collected 10 days after the last immunization, and the serum was separated and inactivated by standing at 56° C. for 30 minutes, followed by adding sodium nitride to a concentration of 0.05% for preservative purposes. To 1 ml of this antiserum, add 1/3 volume of normal human serum and 1/3 volume of cirrhosis patient ascites concentrate (protein content: 80~/ml) at 37°C for 1 hour, then at 4°C for 48 hours. After placing
The supernatant was collected by centrifugation at 3,000 rpm for 30 minutes at 4°C. The antiserum after this treatment was identified by the Uktaroni method and immunoelectrophoresis using raw ascites concentrate as an antigen, and it was proven that it did not contain any antibodies other than the NEA antibody. Experimental Example 3 Isolation and Purification of NEA Using an NEA-specific antibody, NEA is isolated and purified by affinity chromatography.

An equivalent amount of PH7.5, O. To a solution of O5 molar phosphate buffered saline was added an equal volume of saturated ammonium sulfate solution, pH 7.8. After 60 minutes, the mixture was centrifuged at 5,000 rpm, and the resulting precipitate was dissolved in the phosphate buffered saline to give a solution equal in volume to the original antiserum.

Then, 1/4 volume of saturated ammonium sulfate solution with pH 7.8 was added, and after 60 minutes, centrifugation was performed at 5,000 rpm. To the obtained supernatant, 1/4 volume of the same saturated ammonium sulfate solution was added. A 33% ammonium sulfate solution was prepared, and after 60 minutes, the γ globulin fraction of the precipitate was collected by centrifugation at 5,000 rpm. The precipitate was adjusted to pH 8. O
,O. Dissolved in approximately 15 ml of OlM phosphate buffer. Transfer 10 d of this solution to a Sephadex G-50 with a diameter of 2.6
I fractionated by gel filtration using a 40 ClrL column and the phosphate buffer described above, and collected the first eluted protein fraction for desalting and buffering. Further, this solution was concentrated and adjusted to a protein concentration of 70~/ml using a protein bag. Then, the activated D
EAE-cellulose was sized to a diameter of 2 using the phosphate buffer described above.
, 6 (7111) was packed into a column with a length of 40 cm, the concentrated solution was added to the column, and the concentrated solution was sucked into the column bed, and then the phosphate buffer solution was filled in the upper part of the bed and continued to flow.
Most of the immunoglobulin G is not adsorbed to DEAE-cellulose, but all other proteins are. Therefore, the effluent was collected in portions and the absorption at 280 nm was measured to obtain a protein fraction. In this way, immunoglobulin G was isolated and collected. Furthermore, the protein concentration of this fraction was adjusted to 10 W/ml using a protein bag. Next, immunoglobulin G5~ having the above-mentioned anti-A antibody activity was added per 1 d of gel to 50 ml of bromcyan-activated Sepharose 4B gel and allowed to react at room temperature (20-25°C) for 6 hours to fix the immunoglobulin. Thereafter, unreacted immunoglobulin was thoroughly washed away with phosphate buffered saline. This Seph Arrows 4B gel has a diameter of 2.6C! n1 length 20
A crrL column was filled with the phosphate buffered saline, and the partially purified NEA2O obtained in Experimental Example 1 (protein amount) dissolved in 10 ml of saline was added to the column, and the Sepharose-4B gel was fixed. A NEA antigen-antibody complex was produced in the gel by reacting with the NEA antibody. After washing unreacted proteins with the phosphate buffered saline, a 0.2M sodium carbonate solution (PHll.5) was added to the column to dissociate and elute NEA. The collected NEA solution was dialyzed against 0.05M phosphate buffered saline (PH7.5) and concentrated.
Stored at 4°C.

The obtained NEA showed a single sedimentation line in the Uktaroni method and immunoelectrophoresis using the normal human serum prepared in Example 1 and the anti-NEA serum before neutralization with colostrum purified γ-globulin. Furthermore, 10% SDS of isolated NEA (
Sodium dodecyl sulfate) was shown as a single band by polyacrylamide gel disc electrophoresis. As a result, the obtained NEA is a highly purified isolated sample. Experimental Example 4 Purification of NEA-specific antibody Partially purified NEA prepared in Experimental Example 2 was purified at pH 7.5 and O.
Dissolve in O5 molar phosphate buffered saline and reduce the protein amount to 10~
/d.

This was added in twice the volume to bromcyan-activated Sepharose 4B gel and reacted at 4°C for 24 hours to fix the protein in partially purified NEA to the gel. Next, unreacted proteins were thoroughly washed away with the phosphate buffered saline solution, and then the unreacted protein was washed into a tube with a diameter of 1.5 mm. , length 20c! Packed into a column of n. To this column, purified rabbit immunoglobulin G solution (protein concentration 10~/TILI) containing the NEA antibody obtained by ammonium sulfate salting out and purification by ion exchange column chromatography in Experimental Example 3 was added.
was added and reacted with NEA fixed on Sepharose 4B gel to produce a NEA antigen-antibody complex in a gel. Next, after thoroughly washing and removing unreacted immunoglobulin G protein using the phosphate buffered saline, a 0.2M sodium carbonate solution (PHll.5) was added to the column, and NEA
Antibodies were dissociated and eluted. 1/5 volume of 1 molar glycine hydrochloride for the collected NEA antibody fraction? Liquid solution (PH2.5
) was added to neutralize it. This was then applied to Sephadex G25 column chromatography and eluted with 0.05M phosphate buffered saline to remove glycine.
The obtained NEA antibody showed a single precipitation line in the Uktaroni method and immunoelectrophoresis using anti-rabbit serum protein goat serum, and also showed a single precipitation line with anti-rabbit γ-globulin goat serum. Ta. Furthermore, no sedimentation line appeared in the Uktaroni method and immunoelectrophoresis using anti-human serum protein antiserum and anti-colostrum protein antiserum.

From these results, the obtained NEA antibody is a highly purified NE
This is rabbit immunoglobulin G consisting only of the A antibody. This NEA antibody showed an antibody titer approximately 100 times higher (relative to protein amount ratio) than the original serum. Example 3 Method for producing NEA antibody from isolated and purified NEA Purified NEAlOOμ9 obtained in Experimental Example 3 was 0.57!11
was dissolved in physiological saline, and this solution was mixed with an equal amount of Freund's complete adjuvant to emulsify.

1 d of this emulsion was injected intradermally, subcutaneously, and intramuscularly into the hind limbs and abdomen of a domestic rabbit weighing approximately 2 kg; further, the same amount of antigen-adjuvant emulsion was boosted twice every 2 weeks in the same manner. . Blood was drawn 10 days after the last injection and serum was separated. The serum was then kept at 56° C. for 30 minutes to inactivate it, and sodium nitride was added to a concentration of 0.05% for preservation. That the obtained antiserum contained only NEA antibodies was demonstrated by the Uktaroni method and immunoelectrophoresis using breast cancer tissue extract as an antigen. Example 4 Production of NEA antibody using NEA-NEA antibody complex Ascites of a primary hepatocellular carcinoma patient containing NEA (approximately 3,007 ml of ascites 11) was purified using a filtered membrane (permeable molecular weight: 10,000).
It was concentrated to Ll, and then centrifuged at 40,000G at 4°C for 30 minutes, and the supernatant was used as a NEA antigen solution.

On the other hand, NEA-specific rabbit antiserum 50d produced in Example 1
was centrifuged at 4°C and 40,000G for 30 minutes, and the supernatant was used as a NEA antibody solution. The total amount of the above NEA antigen solution and NEA antibody solution was mixed and left at 37°C for 1 hour, and then at 4°C for 24 hours. Thereafter, this mixture was centrifuged at 10,000 G for 20 minutes at 4°C, the soil clear liquid was discarded, and the NEA antigen-antibody complex was collected as a precipitate. After adding 50 d of ice-cold saline to this precipitate and suspending the precipitate, 4°C.
The mixture was centrifuged at 10,000 G for 20 minutes, and the supernatant was discarded to obtain a precipitate again. Add the same amount of ice-cold saline again as above,
The same operation was repeated twice to obtain a precipitate. This NE
A antigen-antibody complex was added with PHll, O. Add 20 ml of l morglycine hydrochloride buffer and incubate with N for 30 minutes at 4°C.
The EA antigen-antibody complex was dissociated. Then add this solution to 4
The mixture was centrifuged at 30,000 G for 20 minutes at °C, and the supernatant was collected. To this supernatant, 0.4 mol of glycine-hydrochloric acid buffer was added, and the pH was adjusted to 7. It was adjusted to O and neutralized. By this neutralization, a NEA antigen-antibody complex was produced again, but the NEA
The operation from mixing the EA antigen solution and antibody solution to neutralization was repeated twice. After that, the neutralized solution was left at 37°C for 1 hour, then at 4°C for 24 hours, and then at 4°C for 20 minutes at 10.0°C.
It was centrifuged at 00G and the supernatant was discarded to obtain a NEA antigen-antibody complex. This complex was suspended in saline and the protein amount was 2~//
Adjusted to ml. 0.5 ml of this liquid and 0.5 ml of Freund's complete adjuvant were mixed to prepare an emulsion liquid. This emulsion was injected intradermally, subcutaneously, and intramuscularly into the hind legs and abdomen of a domestic rabbit weighing approximately 2 kg. After 2 weeks, 1 ml of NEA antigen-antibody complex emulsion prepared in the same manner as above.
1 was boosted.

Two weeks later, the mice were boosted again with 1 ml of the same emulsion. Blood was collected 10 days after the last immunization, and the serum was separated and inactivated at 56°C for 30 minutes.
Sodium nitride was added to give a concentration of 0.05%. This antiserum was identified by the Uktaroni test method and immunoelectrophoresis using the original ascitic fluid concentrate used to prepare the NEA antigen-antibody complex as an antigen, and it was proven that it did not contain antibodies other than NEA antibodies. Ta. Experimental Example 5 Detection of NEA in patient serum NEA in serum from various patients was detected using the Uktaroni method.

A 1.2% agarose plate was heated to pH 7.5, O. It was prepared using O5 molar phosphate buffered saline (containing 0.05% concentration of sodium nitride).

The thickness of the plate was 1.2 mm, the size of the antigen hole (test serum hole) and antibody hole was both 4 mm in diameter, and the distance from the center of both holes was 7 mm. NEA was detected by injecting 10 t1 of the anti-NEA rabbit antiserum prepared in Example 3 into the antibody hole and 40 t1 of the test serum into the antigen hole. In addition, using the high purity NEA obtained in Experimental Example 3 as an index, the sedimentation line that appears between this and the anti-NEA rabbit antiserum and the sedimentation line that appears when the test serum is used are completely different. It was confirmed that the sedimentation line that appeared in the test serum was due to NEA. The results are shown in the table below. As shown in the table above, NEA was detected in 27 (32.1%) of 84 neoplastic patient sera, but NEA was not detected in all 111 non-neoplastic patient sera, including normal human serum. Nakatsuta. Therefore, NEA has high specificity for neoplasms, and a serum NEA detection test using an NEA antibody can be used for the diagnosis of neoplasms. Experimental Example 6 Detection of NEA in patient serum Detection of NEA in patient serum by one-way plate immunodiffusion method Considering that the amount of NEA in malignant neoplasm patient serum covers a relatively wide range, detection due to antigen excess was performed. In order to prevent detection failure due to failure or lack of antigen, two types of plates with different antibody concentrations were prepared, and raw serum was measured simultaneously on both plates in an attempt to increase the NEA detection rate.

Low antibody concentration plates are difficult to observe with the naked eye because the sedimentation ring formed by the antigen-antibody complex is extremely thin. For this reason, the following method was used. After the antigen has sufficiently reacted with the antibody, the plate is immersed in a buffer solution, and unreacted antibodies are released from the agar into the buffer solution. After that, antibodies against the serum immunoglobulin of an animal of the same species as the used antibody are tested in an animal of a different species. The prepared antiserum is injected into each antigen hole and allowed to stand for a certain period of time. If an NEA antigen-antibody complex is present, the subsequently added antibody reacts with it, forming a sedimentation ring that can be observed with the naked eye. That is, it is a one-way immunodiffusion method using a two-antibody method. 1) Manufacture of one-way immunodiffusion plate PH8. O, O. Agarose is heated to dissolve and gel at 1.2% in 1 mol Tris-HCl buffered saline (contains 0.1% sodium nitride for preservation).

The temperature of this agarose gel was set to about 500C, and the NEA rabbit antiserum obtained in Example 3 was heated to about 50t, so that the antiserum concentration was 2 on the high concentration plate and 0.4 on the low concentration plate. The mixture was mixed into a gel at a ratio of 1,000 ml, and poured onto a glass plate (or in a plastic container) to prepare an agarose gel plate containing the NEA antiserum with a thickness of 1.2110 mm. Antigen holes with a diameter of 411 were drilled in this flat plate at regular and sufficient intervals. 2) Detection of NEA antigen in test serum by one-way plate immunodiffusion method Inject 10,111 samples of test serum into the antigen holes of each of the above-prepared antibody high concentration plate and low antibody concentration plate at the same time,
After the serum has been absorbed into the gel, the plate for high concentration is left undisturbed for 48 hours in a humid box at room temperature, and the sedimentation ring is observed for NEA.
The presence or absence of detection was determined.

On the other hand, the low concentration plate was immersed for 3 days in the same buffer solution used to prepare the gel plate to remove unreacted NEA antibodies in the gel. However, the buffer solution was replaced with fresh solution twice every day in the morning and evening. After that, the gel plate was taken out from the buffer solution, the buffer solution in the antigen hole was removed, and 10It11 of anti-rabbit immunoglobulin goat antiserum was injected into each antigen hole, and after the antiserum was absorbed into the gel. , and left to stand in a humid box at room temperature for 24 hours. Thereafter, the presence or absence of a sedimentation ring was observed, and NEA was detected. As shown in the table above, NEA was detected in 37 (37.796) out of 98 neoplastic patient sera, but NEA was detected in all 141 non-neoplastic patient sera, including normal human serum, except for one case. It wasn't done.

In addition, this case was serum from a patient who was clinically diagnosed as having liver cirrhosis, but who was suspected of having concurrent primary hepatocellular carcinoma. The above results demonstrate that detecting NEA in serum using this method is extremely useful for diagnosing malignant neoplasms.

Claims (1)

[Scope of Claims] 1. A method characterized by immunizing a non-human animal with a neoplastic fetal antigen substance NEA specified by the following properties (i) to (vi) and collecting antiserum. Method for producing NEA antibody for neoplasm diagnosis. (i) It is electrophoresed into the γ globulin fraction by electrophoresis using a barbital buffer solution with a pH of 8.6 and an ionic strength of 0.025 to 0.1. (ii) E^0^. ^1^%_1_c_m(280mμ
) = 0.936 (measured by the Lowry method using bovine serum albumin as the standard). (iii) Molecular weight 100,000±20,000 (However, Sephadex G-200 (trade name Pharmacia)
(measured by column chromatography using (iv) When developed in chromatography using a basic anion exchanger with a buffer having an ionic strength of 0.05 and a pH of 7.0, it is eluted together with immunoglobulin G. (v) Isoelectric point pH=9.1-9.4. (vi) Precipitation in a 2.6 molar ammonium sulfate solution at pH 7.0. 2. Immunizing an animal other than a human with a complex of neoplastic fetal antigen NEA specified by the properties (i) to (vi) in claim 1 and an antibody thereto;
NEA for neoplasm diagnosis, characterized by collecting antiserum
Method for producing antibodies.