JPS6057021B2 - Manufacturing method for neoplasm diagnostic agent - Google Patents

Description

[Detailed description of the invention] The present invention relates to a diagnostic agent for neoplasm.

More specifically, one-way plate immunodiffusion (S, R.1, D
, ) and a method for producing a gel plate containing an NEA antibody used in diagnosing neoplasms by immunoelectrodiffusion method (Laurel 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 some antigenic substances have been reported. Examples include α-fetoprotein for primary hepatocellular carcinoma and carcinoembronic 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 that occur in humans. The present inventor investigated the existence of a unique antigenic substance common to a wide variety of neoplasms, and as a result, discovered a novel antigenic substance, NEA.

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. (1) NEA is
Uktalony test using antibodies against NEA, which is present in fetal tissues (mainly small and large intestines), fetal serum, amniotic fluid, meconium, various neoplastic tissues (mainly malignant neoplasms), and the patient's serum and ascites. I can prove things. However, in the Uktalony method, NE is not present in non-neoplastic tissue from neoplastic patients, normal human serum, serum from non-neoplastic patients, and ascites.
It is not possible to prove the existence of A. Furthermore, antiserum against NEA was added to non-neoplastic patient tissue extract antigen, normal human serum,
Inactivation or attenuation of NEA antibody activity is observed even when neutralizing and absorbing serum from non-neoplastic patients is used. The Uktakani test proves that the NEA antibody activity is inactivated or attenuated by the absorption procedure.

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), agar gel, starch, etc. as a support, PH8, fl, ionic strength 0
．． The gamma globulin fraction was electrophoresed using a barbital buffer of 0.025-0.1, and the known immunoglobulins (immunoglobulins GlA, .M, .D and E), other serum gamma globulins and immunological This difference is proven by the Uktalony test method and immunoelectrophoresis method.

(3) The extinction coefficient of NEA is E? Out ¥280 = 0.93
It is 6.

The protein content was measured by the Lowry method using bovine serum albumin as a standard. (4) NEA is
As a result of molecular weight measurement by column chromatography using Sephadex G-200 (trademark: Pharmacia), it was a protein with a molecular weight of 100,000±20,000. (
5) NEA was determined by chromatography using a basic anion exchanger to have an ionic strength of 0.05 and a pH of 7. It has the property of being eluted together with immunoglobulin G without being adsorbed to the ion exchanger when it is incubated with O buffer.

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

(7) NEA has a pH of 7. It has the property of precipitating in a 2.6 molar ammonium sulfate solution. NEA is a specific antigen common to neoplasms with the above characteristics, but in the present invention, NEA obtained by immunizing animals with this NEA
Manufacture a diagnostic agent for neoplasms using antibodies.

The diagnostic agent of the present invention is used in diagnosis by one-dimensional plate immunodiffusion method and immunoelectrodiffusion method (Laurel method). The configuration of the present invention is as follows.

The NEA antibody is dissolved together with the support, and this is solidified into a flat plate. NEA antibody is NEA or NEA<5NE
It is obtained by immunizing a non-human animal with a complex of antibody A and collecting antiserum.

As the support, those commonly used in one-dimensional plate immunodiffusion methods can be used.

Examples include agar, agarose, starch, and polyacrylamide gel. Conventional methods are used to obtain a gel plate from this support and NEA antibody.

For example, the support is heated and dissolved in a buffer solution, and NEA is added to the solution.
It can be obtained by adding and mixing the antibody, pouring the resulting solution onto a glass plate or into a plastic container, and cooling and solidifying it. To perform diagnosis using the gel plate obtained according to the present invention, a hole is made in the gel plate and patient's serum is injected into the hole. In the one-way immunodiffusion method, the presence or absence of a sedimentation ring is observed after this is left for a certain period of time. In addition, in the immunoelectrodiffusion method, an electric current is passed through a gel plate into which serum is injected, and the presence or absence of a sedimentation zone is observed.
The appearance of a sedimentary ring or zone indicates the presence of NEA in the serum, and can be diagnosed as a neoplastic patient.
Next, the present invention will be explained in more detail by showing Examples and Experimental Examples. Experimental Example 1 Partial Purification of NEA from Cancer Tissue A frozen 100y breast cancer resected tissue was cut into dice.
A 5-fold volume of physiological saline was added to the mixture in a homogenizer, and the mixture was homogenized at 4°C.

Add to this an equal volume of physiological saline (soda nitride 0.0
After stirring at 4° C. for 4 hours to extract the protein, the mixture was centrifuged at 5000 gravity (G) for 3 times. The uppermost fat 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 fat layer on the top 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 measured by the Lowry-Folln method and was found to be 73m91ml. 3ml of this concentrated liquid,
Diameter 2.6C1 filled with Sefadex G-200
A column with an R1 length of 90 c1 (bed volume 460 m1) was used to achieve a pH of 8. Gel filtration was performed in a barbital buffer with an ionic strength of 0.05, and the eluate was fractionated. During elution, 2
Four peaks were seen on the spectrophotometer at 807 nμ. That is, the first peak is a fraction containing proteins of 19S or higher, including α2-macroglobulin, immunoglobulin M1β-riboprotein, etc., and the second peak is a fraction containing immunoglobulin. Globulin fraction, third peak is 4.5S containing albumin
The fourth peak was a fraction containing substances with even smaller molecular weights. NEA was eluted between the second and third fractions.

That is, NEA was added to the fraction with a molecular weight of 120,000 to 80,000.
Approximately 90% of the sample was eluted. 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,
This crude NEA fraction was filtered through an ultrafiltration membrane (passable molecular weight 10).
000 or less) to concentrate to 1 volume, which is approximately 1 volume of the original tissue volume. Next, the length is 33Cml, the width is 801cm, and the thickness is 2C! R.L.
An electrophoresis tank was filled with Pepicon C-870 as a support, barbital buffer with a pH and ionic strength of 0.05 was used, and crude NEA was placed in a sample groove located 10 crn from the cathode side of the electrophoresis layer. Inject 5 mt of fraction concentrate, 8
Electrophoresis was performed for 1 hour at a constant current of 0 mA. From the sample groove to the cathode side 3
~7C! Since the NEA exists at a distance of rl, the support in this area is separated and
After adding molar phosphoric acid saline and stirring,
The supernatant was collected by centrifugation at rpm1. To this supernatant, 100 ml of the same buffer was added, and after stirring,
The supernatant was collected by centrifugation under the same conditions. In order to remove Pepicon C-870 that is slightly mixed in this collected liquid,
The collected liquid was filtered through a glass filter. Approximately 200m1 (
7)? Pour the liquid into a protein bag [Carl Schleicher Ultra
ulsenNO. Molecular weight that can pass through lOOl: 25,000]
was used to concentrate to 5 mt. The same operation as above was performed 5 times, and the resulting concentrate was stored at 4°C. Obtained NEA
showed almost the same electrical mobility as immunoglobulin G. Immunoglobulin G and A were detected as contaminant proteins in this specimen by Uktalony test method and immunoelectrophoresis method. In addition, by the above method, approximately 50% of NEA was recovered, calculated from the NEA in the initial physiological saline extract, and the degree of purification was approximately 10 (@
It was hot. Experimental Example 2 Production of NEA antibody Partially purified NEA obtained in Experimental Example 1 was mixed with protein 1 in physiological saline.
A solution prepared by mixing and emulsifying 0.5 ml of this solution with Freund's complete adjuvant was injected intracutaneously, 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.
ml was boosted, and two weeks later, the same NEA emulsion (1 ml) was boosted again.

Blood was collected 10 days after the last immunization, and the serum was separated and inactivated by incubation at 56°C for 3 minutes, followed by adding sodium nitride to a concentration of 0.05% for preservative purposes. To 1 ml of this antiserum, 1/3 volume of normal human serum and 5 m9 of colostrum purified γ-globulin were added, heated at 3TC for 1 hour, and then incubated at 4°C for 4S. After incubating for 30 minutes, the mixture was centrifuged at 4°C and 300 [Pm], and the supernatant liquid was collected. The NEA antibody specificity of the antiserum after such treatment was determined by the Uktalony 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. The reason why colostrum-purified γ-globulin was used to neutralize the antiserum is because the antigen-extracted tissue is breast cancer tissue.
There is a possibility that the partially purified A specimen contains a secreted antigen of immunoglobulin A, and antibodies against it may have occurred in the prepared antiserum, so the objective is to absorb and remove this antibody. γ-globulin purified from colostrum containing secreted immunoglobulin A antigen was used. Experimental Example 3 Isolation and Purification of NEA Using an NEA-specific antibody, NEA is isolated and purified by affinity chromatography.

To a solution prepared by adding 500 ml of the NEA-specific rabbit antiserum prepared in Experimental Example 2 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 60 minutes, the mixture was centrifuged at 5000 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 of a saturated ammonium sulfate solution with a pH of 7.8
After adding 6 volumes and centrifuging at 5000 r'Pm, to the obtained supernatant, add 1/4 volume of the same saturated ammonium sulfate solution to make a 33% ammonium sulfate solution,
After 6 mL, the precipitate was centrifuged at 500 Pm and the γ globulin fraction was collected. The precipitate was adjusted to pH 8. O, dissolved in approximately 15 ml of 0.01 molar phosphate buffer. 15ml of this solution
1, the diameter of Cephadex G-50 is 2.6CT! 11
Desalting and buffering were performed by fractionating by gel filtration using a 40 d long column and the above-mentioned phosphate buffer, and collecting the first eluted protein fraction. Furthermore, this solution was added to a protein concentration of 70m9 using a protein bag.
The concentration was adjusted to 1 ml. Then, the activated DEAE-
Cellulose was sized to a diameter of 2.6 cm using the phosphate buffer.
Length 40C77! The concentrated solution was added to the column, and the concentrate was sucked into the column bed. The phosphate buffer solution was then filled to the top of the bed and continued to flow. Most of the immunoglobulin G is not absorbed by DEAE-cellulose, but all other proteins are adsorbed. Therefore, the effluent was collected in portions and the absorption of 280r1rrL 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 10ml91ml using a protein bag. Next, 5 mg of the above-mentioned immunoglobulin G having NEA antibody activity was added per ml of gel to 50 ml of Bromsyan-activated Sepharose A gel, and the mixture was reacted for 6 hours at room temperature (20-25°C) to fix the immunoglobulin. Thereafter, unreacted immunoglobulin was thoroughly washed away with phosphate buffered saline. This Seph Arrows a gel is 2.5mm in diameter. Thickness, length 20C
A 7I column was filled with the phosphate buffered saline, and the partially purified NEA2Om9 (protein amount) obtained in Experimental Example 2 was dissolved in 10 ml of saline and added to it, and the NEA antibody fixed on the Sepharose-small gel was added. A NEA antigen-antibody complex was produced in the gel. After washing unreacted proteins with the phosphate buffered saline, a 0.2M sodium carbonate solution was added to the column to dissociate and elute NEA.

The collected NEA solution was diluted with 0.05M phosphate buffered saline (
After dialyzing to pH 7.5), it was concentrated and stored at 4°C.
The obtained NEA showed a single sedimentation line in the Uktarony method and immunoelectrophoresis using normal human serum prepared in Experimental Example 1 and anti-NEA serum before neutralization with colostrum-purified γ-globulin, and further showed a single precipitation line. 10% SDS (sodium dodecyl sulfate) polyacrylamide gel disc electrophoresis of isolated NEA showed a single band. As a result, the obtained NEA is a highly purified isolated specimen. Experimental Example 4 Production of NEA Antibody from Isolated and Purified NEA The purified NEAlOOμy obtained in Experimental Example 3 was added to 0.5 mL of raw. It was dissolved in saline and mixed with an equal amount of Freund's complete adjuvant to emulsify the solution.

1ml of this emulsion was injected intradermally, subcutaneously, and intramuscularly into the hind limbs and abdomen of a rabbit weighing approximately 2k9, and then boosted 12 times with an equal amount of antigen-adjuvant emulsion in the same manner every 2 weeks. did. Blood was drawn 10 days after the last injection and serum was separated. Next, the serum was kept at 56° C. for 3 times to make it inactive, and for preservation, sodium nitride was added to a concentration of 0.05%. That the obtained antiserum contained only NEA antibodies was demonstrated by the Uktalony method and immunoelectrophoresis using breast cancer tissue extract as an antigen. Example 1 Production of a gel plate containing NEA antibodies and detection of NEA in serum using the same NEA in serum of patients with malignant neoplasms
Considering that the amount can be spread over a relatively wide range, two types of plates with different antibody concentrations were prepared to prevent detectability due to excess antigen or inability to detect due to insufficient antigen, and both plates were injected with the original serum at the same time. We attempted to increase the NEA detection rate by measuring the NEA detection rate.

1) Production of gel plate containing NEA antibody PH8. O.
l mol, Tris-HCl buffered saline (0.1 for preservative)
(containing sodium nitride), agarose is heated to dissolve at 1.2% and gelled.

The temperature of this agarose gel was set to about 50°C, Experimental Example 4
The NEA rabbit antiserum obtained was heated to about 50℃ and mixed into the gel at an antiserum concentration of 2% for the high concentration plate and 0.4% for the low concentration plate, and then transferred to a glass plate (or (in a plastic container) to prepare a 1.2-inch thick agarose gel plate containing NEA antiserum. This flat plate has a diameter of 4m with a certain sufficient interval! An antigen hole of n was opened. )) Detection of NEA in test serum by one-way plate immunodiffusion method 10 μ' of test serum was simultaneously injected into the antigen holes of each of the above-prepared antibody high concentration plate and low antibody concentration plate, and the serum was added to the gel. After the sample was absorbed, the high concentration plate was allowed to stand for an hour in a humid box at room temperature, and the sedimentation ring was observed to determine whether or not NEA was detected.

Claims (1)

[Claims] 1. A method for producing a neoplasm diagnostic agent, which comprises dissolving an NEA antibody together with a support and solidifying it into a flat plate.