JPS6055779B2 - Manufacturing method for neoplasm diagnostic reagents - Google Patents

Description

[Detailed description of the invention] The present invention also relates to a method for producing a reagent for diagnosing neoplasms.

It is. More specifically, the present invention relates to a method for producing microparticles bound to NEA or NEA antibodies, which are used in diagnosing neoplasms by immunological detection methods based on agglutination reactions (PHA-HI method, RPHA method). 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 several antigenic substances have been reported. Examples include α-fetoprotein for primary hepatocellular carcinoma and carcinogenic antigen (CEA) for traumatic injuries. However, these antigenic substances are restricted to certain organs, system organs or histopathological forms of neoplasms. For this reason, we have investigated 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 used these specific antibodies. It is expected that the establishment of a method for detecting antigens in humans will lead to exciting advances in the diagnosis, prevention, and treatment of various neoplasms that occur in humans. As a result of our search for the presence of solids that are common to a wide variety of neoplasms, the present inventor discovered that this is a novel antigenic substance! I found BM.

The NEA of the present invention is neoplasmembryoni.
cnti is an abbreviation for thickness n, and was named by the present inventor.

NEA has the following properties.

iNHA is a uterine test method that uses antibodies against NEA, and is used to test fetal tissues (mainly small and large intestines), fetal serum, amniotic fluid, meconium, various neoplastic tissues (mainly malignant neoplasms), and patient serum and ascites. It can be proven that it exists.

However, the Ukronie 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.A.
No deactivation or attenuation of NEA antibody activity was observed when antiserum was neutralized and absorbed using non-neoplastic patient tissue extract, normal human serum, and non-neoplastic patient serum. The Ukuroni test method proves that NEA antibody activity is inactivated or attenuated by absorption using serum, certain malignant neoplastic tissue extracts, or patient serum.

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

2NEA is cellulose acetate membrane, Pevicon C-8
70 (vinyl chloride, vinyl acetate complex), agar gel, starch, etc. as a support, PH8, EL, ionic strength 0.0
The known immunoglobulin G
, A.M. D and E) are immunologically different from other serum gamma globulins, as proven by the Uktalony test method and immunoelectrophoresis method.

Is the absorption coefficient of 3NEA E? ? 280=0.936.

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

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

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

7NEA 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 reagent of the present invention can be used for agglutination reactions (PHA-HI method, RPHA
It is used when detecting NEA by the method (method).

The reagent of the present invention is obtained by binding NEA or NEA antibody to microparticles.

The microparticles used in the present invention include PHA-HI method, R
Materials commonly used in the PHA method can be used.

Mammalian and avian blood cells are particularly preferred, but other particles of about 1 to 1 micron such as polystyrene latex, polyester latex, vinyl chloride, bentonite, glass beads, etc. can also be used.

NEA can be obtained from the above-described NEA-containing composition using a conventional protein fractionation and purification method. NEA antibodies give animals NE
A or NEAI:. Obtained by immunization with a complex with NEA antibody. To bind microparticles to NEA or NEA antibodies, commonly used reagents are used.

For example, glutaraldehyde, formaldehyde, tannic acid, bisdiazodized benzidine, chromium chloride,
Examples include carbodiimide. The general method for producing the reagent of the present invention using sheep red blood cells will be explained as follows.

Sheep red blood cells are suspended in phosphate buffered saline to a concentration of approximately 5%, and 11 mol of a glutaraldehyde solution adjusted to 1 to 5% in phosphate buffered saline is added to this and stirred at room temperature for 3 to 7 hours. .

After washing with physiological saline, add phosphate buffered saline to 5%
Glunotaraldehyde produces sheep red blood cells. 0 for this
．． 5 by adding an equal amount of 0.001-0.02% tannic acid solution.
After stirring for ~2 minutes, wash with physiological saline.

Add phosphate buffered saline to prepare 5% glutaraldehyde and tannic acid treated sheep red blood cells. NE to this blood cell
To bind antibody A, use purified NEA antibody (5 μy
Add an equal amount of ~1TL9/Mt) and stir at room temperature for 10-6 minutes. In addition, in order to bind NEA, purified NE
Add an equal amount of A (1μy~1m9/ml) and incubate at room temperature for 10~
6. Detection of NEA in serum using the microparticles bound to NEA or NEA antibodies of the present invention is carried out as follows.

When NEA antibody-microparticles and coated serum are mixed in a test tube, if NEA is present in the serum, NEA. l5NE
Antibody A reacts and an agglutination reaction occurs, and as a result, an image of the bottom of the tube due to agglutination, which is different from natural sedimentation, can be seen with the naked eye.

Furthermore, if the serum does not contain NEA or the concentration of NEA is below the sensitivity of this reagent, the NEA antibody microparticles will naturally settle and a ring will be observed in the bottom tube. In the case of NEA microparticles, the same agglutination reaction as described above occurs due to the presence of NEA antibodies, but the coexistence of free NEA in this reaction system inhibits the agglutination reaction. Therefore, after adding an appropriate amount of NEA antibody to the test serum in advance, NEA microparticles are added thereto, and NEA in the serum is detected by observing the presence or absence of agglutination. Next, the present invention will be explained in more detail by showing examples and examples. Experimental Example 1 Partial Purification of NEA from Cancer Tissue A frozen 100f breast cancer removed tissue was cut into dice.
Add 5 volumes of physiological saline in a homogenizer and incubate at 4°C.
The mixture was homogenized at one level.

To this, add an equal amount of physiological saline (sodium nitride) to 0.
The mixture was stirred at 4° C. for an hour to extract the protein, and then centrifuged at 500 heel force (G) for 3 cycles. The fat layer in the uppermost soil layer was removed, and the supernatant liquid was collected. The obtained supernatant liquid was centrifuged again three times at 200 force (G), a small amount of the uppermost fat layer was removed, and the supernatant liquid was collected. This supernatant was placed in a cellulose tube for dialysis, and concentrated to 1 volume of about 2 powders in polyethylene glycol (average molecular weight: about 15,000). The protein concentration of the concentrate was determined to be 73 m9/Mt by the Lowry-Follin (LOIy-FOlln) method. 3 ml of this concentrate was filled with Sephadex G-200 and packed with a diameter of 2.6 crn1.
PH with a column of length o (bed volume 460 m1)
8. f) Gel filtration was performed in a barbital buffer with an ionic strength of 0.05, and the eluate was fractionated. 280 (at the time of elution)
7) Four peaks were observed on the mμ spectrophotometer.

That is, the first peak is 1? containing α2-macroglobulin, immunoglobulin M1β-riboprotein, etc. Does the second peak in the fraction containing the above proteins contain immunoglobulin? The third peak of the globulin fraction was a 4.5S fraction containing albumin, and the fourth peak was a fraction containing substances with smaller molecular weights.

NEA was eluted between the second and third fractions. That is, approximately 9 of NEA was added to the molecular weight fraction of 120,000 to 80,000.
0% was eluted. 2nd peak, excluding 2nd and 3rd peaks
The eluate between the peak and the third peak was collected and used as a crude NEA fraction. The above sampling was repeated to collect the crude NEA fraction, and this crude NEA fraction was collected.
The EA fraction was concentrated using an ultrafiltration membrane (passable molecular weight of 10,000 or less) to approximately 1 volume of the original tissue. Next, an electrophoresis tank with a length of 33 cm, a width of 8 cm, and a thickness of 2α was filled with Pepicon C-870 as a support, and p[18.
Using a barbital buffer with an e1s ionic strength of 0.05, 5 ml of crude NEA fraction concentrate was injected into the sample groove provided 10 cm from the cathode side of the electrophoresis layer, and 80n1A
Electrophoresis was carried out at constant current for 16 hours. 3 to 7 from the sample groove to the cathode side
Since the NEA exists at a distance of an, the support in this area is cut off and 100 m1 of pH 7.5. O. After adding O5M phosphate buffered saline and stirring, the mixture was centrifuged at 3000 rpm and the supernatant was collected. 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 this sampled liquid, the sampled liquid was filtered through a glass filter. Approximately 200 m1 of Oki liquid was poured into a protein bag [Carl, Sch
leicher) UItrahilsen NO. l
The solution was concentrated to 5 mL using a molecular weight of 25,000 that can pass through O.

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 specimen by the Uktalony test method and the immunoelectrophoresis method. By the above method, about 50% of NEA was recovered, calculated from the NEA in the initial physiological saline extract, and the degree of purification was about 10%. Experimental Example 2 Partial purification of NEA from ascites of a cancer patient Ascites 2e of a gastric cancer patient was purified using an ultrap membrane (permeable molecular weight 15
000) to 500 ml, and this concentrated liquid was
The supernatant was collected by centrifugation at 20,000 gravity (G).

0.05M phosphate buffered saline having a pH of 7.5 was added to the supernatant to make the total volume 1e (protein content approximately 3%). Ammonium sulfate was added to this to make a 1.5M ammonium sulfate solution, and sodium hydroxide was added to adjust the pH to 7. Adjusted to O. After leaving this solution for 6 minutes, apply 10,000 r'P for 30 minutes.
Centrifuged at m. The supernatant was collected and ammonium sulfate was added to make a 2.6M ammonium sulfate solution.
After 30 minutes, the mixture was centrifuged at 1000 Pm for 30 minutes. More than 60% of NEA was collected in the sediment. Precipitate to pH8
．． It was dissolved in barbital buffer with an ionic strength of 0.05, and the total volume was made up to 200 ml. This solution was mixed with Cephadex G-50 (trademark, Pharmacia) using the same buffer solution.
The first eluted protein fraction was collected, desalted with ammonium sulfate, and buffered with barbital.
The protein concentration of this ammonium sulfate fraction was adjusted to about 70 mg/ml, and 5 mt of this solution was subjected to electrophoresis. That is, length 3301
Width 8Cr! An electrophoresis tank with a thickness of 11 and 20 mm was filled with Pepicon C-87 as a support, and the pH was 8.6 and the ionic strength was 0.
Using 05 barbital buffer, 10Cr! from the cathode side of the electrophoresis tank. 5 mt of the above solution is placed in the sample groove provided at the location 1.
was injected, and electrophoresis was performed for 2 hours at a constant current of 80n1A. After that, the support was separated from the sample groove at a distance of 3 to 7 cm toward the cathode, and 100 ml of pH 7.5, 0.05 M phosphate buffered saline was added and stirred, followed by centrifugation at 3,000 r'Pml. The supernatant liquid was collected. Furthermore, 100m1
After adding the same buffered saline and stirring, centrifugation was performed under the same conditions, and the supernatant was filtered through a glass filter. The Oki liquid was concentrated to 2.5 ml using protein bag. This concentrated liquid 2.
5m1 filled with Cephadex G-200 diameter 2
．． With a column of 9 cm long (bed capacity 460 m1),
Gel filtration was performed in a 0.05 molar phosphate buffer at pH 7.5, and the eluate was fractionated into each test tube. 280m at elution
Two absorption peaks were observed in the μ spectrophotometer. The first peak is the 19S fraction containing immunoglobulin M, and the second peak contains immunoglobulin G? It was a fraction. N.E.
A elutes slightly later than the second peak. That is, about 90% or more of NEA was present in the fraction corresponding to a molecular weight of 120,000 to 80,000. This fraction was collected, concentrated with protein bag, and then stored at 4°C. Obtained NE
In the A specimen, immunoglobulin G and A were detected as contaminants by the Uktalony test method and the immunoelectrophoresis method. Note that the degree of purification of the obtained NEA was about 5. Experimental Example 3 Production of NEA Antibody Partially purified NEA obtained in Experimental Example 1 was diluted with protein amount 1 in physiological saline.
A solution prepared by mixing and emulsifying an equal amount of Freund's Complete Aziband in 0.5 ml of this solution was injected intradermally, subcutaneously, and intramuscularly into the hind legs and abdomen of a domestic rabbit weighing approximately 2k9. .

Two weeks later, NEA emulsion 1 was prepared in the same manner as above.
ml was boosted, and two weeks later, the same NEA milk.m1 was boosted.
Booster immunization was carried out again with 1 ml of the solution.

Blood was collected 10 days after the last immunization, and the serum was separated and inactivated by standing at 56° C. for 3 hours. Then, sodium nitride was added to a concentration of 0.05% for preservative purposes. 1 ml of this antiserum contains 1/3 of the customer's normal human serum, purified colostrum, and γ-globulin 57F! 9 was added and heated to 3TC for 1 hour, then placed in a 4°C leakage chamber for 3 minutes at 4°C and 3000 rpm.
The supernatant was collected by centrifugation. The NEA antibody specificity of the antiserum after such treatment was determined by the Ukuroni test method and immunoelectrophoresis using original breast cancer tissue extract as an antigen, and it was proven that it contained no antibodies other than NEA antibodies. It was done. However, the reason why colostrum-purified γ-globulin was used to neutralize the antiserum is that the antigen-extracted tissue was a breast cancer excised tissue, so there is a possibility that the NEA partially purified product may contain the secreted antigen of immunoglobulin A. γ-globulin purified from colostrum containing the secreted antigen of immunoglobulin A was used to absorb and remove this antibody. . Experimental Example 4 Isolation and Purification of NEA Using an NEA-specific antibody, NEA is isolated and purified by affinity chromatography.

To a solution prepared by adding 50 ml of the NEA-specific rabbit antiserum prepared in Experimental Example 3 and an equal volume of 0.05M phosphate buffered saline with a pH of 7.5, the same volume of a saturated ammonium sulfate solution with a pH of 7.8 was added. After 6 minutes centrifugation at 5000 rpm,
The obtained precipitate was dissolved in the above-mentioned phosphate buffered saline to prepare a solution having an amount equal to that of the original antiserum.

Next, 1/4 volume of saturated ammonium sulfate solution with pH 7.8 was added, and after 6 times the solution was centrifuged at 5000 r'Pm. To the obtained supernatant, 1/4 volume of the same saturated ammonium sulfate solution was added to 33. % ammonium sulfate solution, centrifuged at 5000 r''Pm after 6 times to remove the γ of the precipitate.
Globulin fraction was collected. Precipitate PH&0s0.0
Dissolved in approximately 15 ml of 1 molar phosphate buffer. This solution 1
5m, diameter 2.6CrI of Cephadex G-50
Desalting and buffering were carried out by collecting the first eluted protein fraction after separation by a gel p-filtration method using a column with an L1 length of 40 CWL and the above-mentioned phosphate buffer.
Furthermore, this solution was added to a protein concentration of 7 using a protein bag.
The concentration was adjusted to 0m9/ml. Then, the activated DE
AE-cellulose was cut to a diameter of 2.5 mm using the phosphate buffer described above.
6C7! The concentrated solution was added to the column and absorbed into the column bed, and then the phosphate buffer solution was continued to be filled to the top of the bed. 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 r1m was measured to obtain a protein fraction. In this way, immunoglobulin G was isolated and collected. Furthermore, the protein concentration of this fraction was concentrated and adjusted to 10 m9/ml using a protein bag. Next, the above-mentioned immunoglobulin G5m9 having NEA antibody activity was added per ml of gel to 50 ml of bromcyan-activated Sepharose a gel, and the mixture was 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 Sepharose a gel was packed into a column with a diameter of 2.60 mm and a length of 20 cm using the phosphate buffered saline, and the partially purified NEA2Om9 (protein amount) obtained in Experimental Example 1 was added to 10 mt of physiological saline. It was dissolved and added, and reacted with the NEA antibody fixed on Sepharose bile gel to produce a NEA antigen-antibody complex in the gel. 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 0.05 phosphate buffered saline (PH7.5), concentrated, and stored at 4°C. The obtained NEA was the anti-NE before being neutralized with the normal human serum and colostrum purified γ-globulin prepared in Experimental Example 3.
The Uktalony method and immunoelectrophoresis using A serum showed a single precipitation line, and furthermore, 10% S of isolated NEA
DS (sodium dodecyl sulfide) polyacrylamide gel disk electrophoresis showed it as a single band. As a result, the obtained NEA is a highly purified isolated specimen.

Example 5 Purification of NEA-specific antibody Partially purified NEA prepared in Experimental Example 2 was purified at pH 7.5, 0.
Dissolved in 0.05 molar phosphate buffered saline to reduce the amount of protein to 10 m
Adjusted to 9/Mt.

This was added in twice the volume to a small gel of bromocyan-activated Sepharose, and reacted at 4°C for 24 hours to fix the protein in the 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 with a diameter of 1.5 cm and a length of 20C77! was packed into a column.
To this column, purified rabbit immunoglobulin G solution (protein concentration 10 m9/ml) containing the obtained NEA antibody purified by ammonium sulfate salting out and ion exchange column chromatography in the experimental example was added, and cellulose? React with gel-fixed NEA, N
The EA antigen-antibody complex was made into 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 to remove NEA.
The antibody was eluted. Add 1/5 volume of 1 molar glycine hydrochloride buffer (PH2.5) to the collected NEA antibody fraction.
) was added to neutralize it. Next, add this to Cefdex G.
25 column chromatography plus elution with 0.05M phosphate buffered saline to remove glycine. The obtained NEA antibody showed a single precipitation line in the Uktalony method and immunoelectrophoresis using anti-rabbit serum protein goat serum, and also showed a single precipitation line with anti-rabbit γ-globulin goat serum. Furthermore, no sedimentation line appeared in the Uktalony method and immunoelectrophoresis using anti-human serum protein antiserum and anti-colostrum protein antiserum. From these results, the obtained NEA antibody is rabbit immunoglobulin G consisting only of highly purified NEA antibody. This NEA antibody showed a high antibody titer of about 10C@(relative to the protein amount ratio) of the original serum. Example 1 NEA antibody sensitized sheep red blood cells Sheep blood stored in Albaza solution was incubated at 4°C for 2000 r.
．． p. m. After removing plasma by centrifugation, the tube was washed 5 times with physiological saline at 2000 rpm. p. m. The ship sank in a centrifuge for a while.

Phosphate-buffered saline was added to the obtained sheep red blood cells to give a concentration of 5%, and 2.5% glutaraldehyde diluted with ice-cold phosphate-buffered saline was added to this suspension using a dropwise load for 11 mol. It dripped slowly. After stirring at room temperature for 5 hours,
Washed 5 times with physiological saline and heated at 2000 r'. P. m. So 5
Sheep red blood cells were centrifuged for minutes and treated with 5% glutaraldehyde in ice-cold phosphate buffered saline containing thimerosal. Next, an equal amount of 0.0025% tannic acid solution was added thereto, stirred once, and then centrifuged. Remove the supernatant and
After washing five times with physiological saline, it was centrifuged and phosphate buffered saline was added to obtain sheep red blood cells treated with 5% glutaraldehyde and tannic acid.

To this, the NEA antibody obtained in Experimental Example 5 (10 μy/ml)
After stirring for 3 minutes at room temperature, the solution was washed twice with saline, diluted with phosphate buffered saline, and treated with 10% glutaraldehyde and tannic acid.
Antibody sheep red blood cells were obtained. The detection sensitivity of NEA by the RPHA method using the obtained NEA antibody-seduced sheep red blood cells was 150.
It heated up at r1y/ml. Therefore, even the smallest amount of NEA in the test serum can be detected. Example 2 NEA-sensitized sheep red blood cells NEA (10
py/ml) was added thereto, and the mixture was stirred at room temperature for three times.

Then, 200[. P. m. After centrifuging at
% glutaraldehyde, tannic acid treated NEA sensitized sheep red blood cells were obtained. Using the obtained NEA-stimulated sheep red blood cells,
When NEA was detected by the PHA-HI method, the detection sensitivity was 2000 rly/ml.

Claims (1)

[Claims] 1. A method for producing a neoplasm diagnostic reagent, which is characterized by binding NEA or NEA antibody to microparticles.