JPS6049866B2 - Purification method for NEA and NEA antibodies - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying a novel neoplastic fetal antigen NEA and its antibodies (hereinafter referred to as NEA and NEA antibody, respectively).

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. For example, α-fetoprotein for primary hepatocellular carcinoma or carcinogenic antigen (CEA) for adenocarcinoma.
etc. 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, produced unique antibodies against these antigenic substances, and further developed antigen detection methods using these specific antibodies. It is expected that the establishment of such a law will encourage exciting advances in the diagnosis, prevention, and treatment of various neoplasms occurring 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.

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

NEA has the following properties.

1NEA is a Uktalony 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 Uktalony 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
Antiserum against EA non-neoplastic 2 patient tissue extracted antigen,
Even if the neutralization absorption operation is performed with normal human serum or non-neoplastic patient serum,
No inactivation or attenuation of NEA antibody activity was observed, and 3NEA antibody activity was inactivated or attenuated by absorption procedures using fetal tissue extracts, fetal serum, certain malignant neoplastic tissue extracts, or patient serum. is proven by the Uktalony test method.

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

2NEA is cellulose acetate membrane, Pevico 3C-
870 (vinyl chloride, vinyl acetate complex), agar gel,
PH8.6 using starch etc. as a support. Ionic strength 0.0
The gamma globulin fraction was electrophoresed using a barbital buffer of 25 to 0.1, and the known immunoglobulins (
Immunoglobulins G, 4, A, M, D and E) are immunologically different from other serum gamma globulins, as proven by the Uktalony test method and immunoelectrophoresis method.

The extinction coefficient of 3NEA is E? ? (280n1μ)=0.
It is 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).

When 5NEA is subjected to chromatography using a basic anion exchanger with a buffer having an ionic strength of 0.05 and a pH of 7.0, it has the property of being eluted together with immunoglobulin G without being adsorbed to the ion photoexchanger.

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.

Therefore, NEA and NEA antibodies are important as diagnostic reagents for neoplasms, and the present invention is a method for purifying NEA and NEA antibodies.

The present invention uses affinity chromatography methods to purify NEA and NEA antibodies. That is, when purifying NEA having the above properties or an NEA antibody that is an antibody against it, a NEA-containing solution is brought into contact with a combination of NEA antibody and a support, or a NEA
contacting the antibody-containing solution with the NEA-support combination;
Then, the antigen-antibody complex formed on the support is dissociated and N
There are purification methods for recovering EA or NEA antibodies. A more detailed explanation is as follows.

First, NEA or NEA antibody is bound to a support such as Sepharose (trade name, Pharmacia) or Sephadex (trade name, Pharmacia). The conjugate is NEA
Alternatively, it can be obtained by adding a support activated with cyanogen bromide or the like to a solution of NEA antibody and stirring at a low temperature. The resulting conjugate is used as a separating agent. To purify NEA, a conjugate of a support and an NEA antibody is used, and to purify a NEA antibody, a conjugate of a support and NEA is used. NEA
Or, fill a column with a conjugate of NEA antibody and support,
A solution containing NEA or NEA antibody for the purpose of purification is passed through this. Only NEA or the NEA antibody reacts with the NEA antibody bound to the support or with NEA to form a complex, and other contaminant components flow out. The complex is then dissociated to obtain the desired NEA or NEA antibody. Dissociation of the complex can be achieved by flowing an aqueous solution with a pH of about 10 to 12 through the column. According to the purification method of the present invention, highly purified raw materials of NEA or NEA antibodies can be obtained not only from raw materials with a high content but also from raw materials with a low content.

Furthermore, the column used in the present invention can be used repeatedly by regenerating it. Other purification methods for NEA and NEA antibodies include ammonium sulfate salting-out method, gel P filtration method, electrophoresis method, ion exchange resin, etc., but these purification methods alone cannot achieve high immunological purity. It's difficult to get things.

Immunologically highly purified or single products can be obtained by using the method of the present invention alone or in combination with other purification methods. As mentioned above, NEA is a neoplasm-related antigen, and establishing a method for detecting NEA from serum etc. is useful for diagnosing, preventing, and treating neoplasms. Immunological detection methods that utilize antigen-antibody reactions are often used to detect trace substances in serum, but the detection reagents used for this detection require immunologically high purity. Ru. Therefore, immunologically highly pure or single NEA and NEA antibodies obtained by the method of the present invention are useful as a detection reagent for NEA, particularly as an immunological detection reagent. Next, examples and experimental examples will be shown.

Experimental Example 1 Partial Purification of NEA from Cancer Tissue A frozen 100 y breast cancer resected tissue was cut into dice, and 5 times the volume of physiological saline was added in a homogenizer and homogenized for 1 minute at 4°C.

Add to this an equal volume of physiological saline (soda nitride 0.0
After stirring at 4°C for an hour to extract the protein, the mixture was centrifuged at 5,000 gravity (G) for 3 cycles. The uppermost fat layer was removed and the supernatant liquid was collected.
The resulting supernatant was centrifuged again at 200 g for 3 minutes, a small amount of the uppermost fat layer was removed, and the supernatant 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/ml by the LOly-FOlln method. 3ml of this concentrated liquid was filled with Cephadex G-200 and the diameter was 2.6C77!
, length 90C! Gel filtration was carried out in a barbital buffer with pH 8.6 and ionic strength 0.05 using a column of RL (bed volume 460 ml), and the eluate was fractionated. During elution, 2807T1. Four peaks were observed in the P spectrophotometer. That is, the first peak is a fraction containing proteins longer than μs, including α2-macroglobulin, immunoglobulin M1β-riboprotein, etc., the second peak is a globulin fraction containing immunoglobulin, and the third peak is albumin. including 4
．． The 5S fraction, the fourth peak, was a fraction containing substances with even smaller molecular weights. NEA was eluted between the second and third fractions. That is, the molecular weight is 120,000 to 800
Approximately 90% of NEA was eluted in the 00 fraction. 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 operation to obtain coarse N
The EA fraction was collected, and the crude NEA fraction was shrunken to about 1/10 the volume of the original tissue using an ultrafiltration membrane (molecular weight that can pass through it: 10,000 or less). Next, length 33cm, width 8
Pepicon C was used as a support in an electrophoresis tank with a thickness of 2G per cm1.
-870, using a barbital buffer with a pH of 8 and an ionic strength of 0.05.
Add 5 ml of crude NEA fraction concentrate to the sample groove provided at the cm site.
was injected and electrophoresed for 16 hours at a constant current of 80n1A. Since 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 mL (7) PH
7.5, O. After adding O5M phosphate buffered saline and stirring, the mixture was centrifuged at 3000 rpm to collect the supernatant. To this supernatant, 100 ml of the same buffer was added and stirred, followed by centrifugation under the same conditions to collect the supernatant.

Pepicon C7-870, which is slightly mixed in this collected liquid,
The collected liquid was filtered through a glass filter to remove it. Approximately 200 TrLt (7) Protein bag with deep fluid [Carl Schieicher]
) manufactured by Ultrahulsen NO. lOO, molecular weight that can pass through: 25,000] and concentrated to 5 ml.

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 Production of NEA antibody Partially purified NEA obtained in Experimental Example 1 was diluted with protein amount 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, which was injected in divided doses into the hind legs and abdomen of domestic rabbits weighing approximately 2k9, subcutaneously and intramuscularly. .

Two weeks later, NEA emulsion 1 was prepared using the same procedure 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 stored at 56°C for 3 minutes to inactivate it, and then sodium nitride was added to a concentration of 0.05% for preservative purposes. . To 1 ml of this antiserum, 1/3 volume of normal human serum and 5 mg of colostrum purified γ-globulin were added and incubated at 3TC for 1 hour, then allowed to stand at 4°C for 3 minutes at 3000 r'Pm at 4°C.
The supernatant was collected by centrifugation. The NEA antibody specificity of the antiserum after such treatment was identified using the Uttalony test method and immunoelectrophoresis method using original breast cancer tissue extract as an antigen, and it was proven that it contained no antibodies other than NEA antibodies. . 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 EA partially purified specimen contains a secreted antigen of immunoglobulin A, and there is a possibility that antibodies against it may have occurred in the prepared antiserum, so this antibody should be removed by absorption. For this purpose, γ-globulin purified from colostrum containing secreted antigen of immunoglobulin A was used. Example 1 Isolation and Purification of NEA NEA is isolated and purified by affinity chromatography using a NEA-specific antibody.

NEA-specific rabbit antiserum 50r1L1 prepared in Experimental Example 2
An equivalent amount of PH7.5, O. To a solution of O5 molar phosphate saline buffer was added an equal amount of saturated ammonium sulfate with a pH of 7.8.
The solution was added. After 60 minutes, centrifugation was performed at 5000 r''Pm, and the resulting precipitate was dissolved in the phosphate saline buffer,
The lysate was prepared in an amount equal to that of the original antiserum.

Then, 1/4 volume of saturated ammonium sulfate solution with pH 7.8 was added, and after 6 times the mixture was centrifuged at 5000 rpm.
A quarter volume of the same saturated ammonium sulfate solution was further added to the obtained supernatant to obtain a 33% ammonium sulfate solution, and after 6 times the solution was centrifuged at 500 [Pm] to collect the γ globulin fraction of the precipitate.

The pH of the precipitate was 8. O, O. Ol mol phosphate buffer approx. 15
Dissolved in mt. Transfer 15ml of this solution to a Sephadex G-50 with a diameter of 2.6C! Fractionation was performed by a gel filtration method using a Rll length 40 cm column and the above-mentioned phosphate buffer solution, and the first eluted protein fraction was collected for desalting and buffering.

Furthermore, this solution was added to a protein concentration of 7 using a protein bag.
The concentration was adjusted to 0 mg/ml. Then the activated DEA
E-cellulose was cut to a diameter of 2.6 mm using the phosphate buffer described above.
After filling a CWi1 column with a length of 40C7n and adding the concentrated solution to this column and inhaling it into the column bed,
The above phosphate buffer solution was filled to the top of the pet and kept flowing.
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 of 280T1m 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 to 50 mL of Bromcyan-activated Sepharose heart gel per ml of gel, and the mixture was incubated at room temperature (20° to 25°C).
℃) for 6 hours to fix the immunoglobulin.

Thereafter, unreacted immunoglobulin was thoroughly washed away with phosphate buffered saline.

This Seph Arrow Heart Gel has a diameter of 2.6 cm and a length of 20 cm.
m column using the phosphate buffered saline,
Partially purified NEA2Om9 (protein amount) obtained in Experimental Example 1 was dissolved in 10 ml of saline and added thereto, and reacted with the NEA antibody fixed on the Sepharose-heart 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 0.05M phosphate buffered saline (PH7.5), concentrated, and incubated at 4°C.
Saved to.

The obtained NEA showed a single sedimentation line in Uktalony method and immunoelectrophoresis using normal human serum prepared in Example 1 and anti-NEA serum before neutralization with colostrum-purified γ-globulin. Isolated NEA in 10% SDS (
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 3 Partial purification of NEA from ascites of cancer patients
000) to 500 ml, and this concentrated liquid was
The supernatant was collected by centrifugation at 20,000 gravity (G).

The supernatant liquid has a pH of 7.5 and an O. O5 molar phosphate buffered saline was added to make the total volume 1e (approximately 3% protein). 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, 1000 [Pm
and centrifuged. The supernatant was collected, ammonium sulfate was added to make a 2.6M ammonium sulfate solution, and the mixture was centrifuged at 10,000 r'Pm for 6 times and 3 times. More than 60% of NEA was collected in the sediment. Precipitate to pH8
．． 6 was dissolved in barbital buffer with an ionic strength of 0.05, and the total volume was adjusted to 200 mL. This solution was mixed with Sephadex 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 m9/ml, and 5 ml of this solution was subjected to electrophoresis. That is, the length is 33 cT.
An electrophoresis tank with a width of 8 (:7n1 and a thickness of 2α) was filled with Pepicon C-870 as a support, and using a barbital buffer with a pH of 8.? The above solution 5TfLL was injected into the sample groove provided at the site, and electrophoresis was performed at a constant current of 80rr1A.Then, the support at a distance of 3 to 7 cm from the sample groove to the cathode side was cut off, and a 100 mt pH7. .05
Add molar phosphate buffered saline and stir at 3000 rpm1
The supernatant was collected by centrifugation. Another 100m1
After adding the same buffer solution and stirring, centrifugation was performed under the same conditions, and the supernatant liquid was filtered through a glass filter.

The p solution was concentrated to 2.5 ml using a protein bag. 2.5ml of this concentrated liquid was filled with Cephadex G-200, diameter 2.6cff1, length 90Cr! i (floor capacity 46
Gel filtration was performed in a 0.05 molar phosphate buffer at pH 7.5 using a 0 ml column, and the eluate was fractionated into each test tube. During elution, two absorption peaks were observed on a 280 mμ spectrophotometer. The first peak is the 1Cf5 fraction containing immunoglobulin M, and the second peak contains immunoglobulin? It was a fraction. NEA elutes slightly later than the second peak. That is, molecular weight 120,000 to 80,000
About 90% or more of NEA was present in the fraction corresponding to . This fraction was collected, concentrated in a protein bag, and then heated at 4°C.
Saved with. Immunoglobulin G and A were detected as contaminants in the obtained NEA specimen by the Uktalony test method and the immunoelectrophoresis method. In addition, the obtained NEA
The degree of purification was about 50 times higher.

Example 2 Purification of NEA-specific antibody Partially purified NEA prepared in Experimental Example 3 was purified at pH 7.5, 0.
Dissolved in 0.05 molar phosphate buffered saline to reduce the amount of protein to 10 m
It was adjusted to 9/ml.

Claims (1)

[Scope of Claims] 1. When purifying NEA having the following properties (i) to (vi), an NEA-containing solution is brought into contact with a bond of NEA antibody and a support, and then the antigen-antibody produced on the support is purified. It is characterized by dissociating the complex and recovering NEA. Purification method of NEA: (i) Electrophoresis using barbital buffer with pH 8.6 and ionic strength 0.025 to 0.1 is carried out into the γ globulin fraction. (ii) E■% (280 mμ) = 0.936 (measured by the Lowry method using serum albumin as the standard)
. (iii) Molecular weight 100,000±20,000 (measured by column chromatography using Sephadex G-200 (trademark: Pharmacia)). (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) PH7.0. , precipitated in a 2.5 molar ammonium sulfate solution. 2. When purifying the NEA antibody, which is an antibody against NEA having the following properties (i) to (vi), a solution containing the NEA antibody is brought into contact with a combination of NEA and the support, and then the antigen-antibody produced on the support is purified. It is characterized by dissociating the complex and recovering the NEA antibody. Purification method of NEA antibody: (i) PH8.6 ionic strength 0.
The gamma globulin fraction is electrophoresed using a barbital buffer of 0.025 to 0.1. (ii)E■%(2
80 mμ) = 0.936 (measured by the Lowry method using serum albumin as a standard). (iii) Molecular weight 100,000±20,000 (measured by column chromatography using Sephadex G-200 (trademark: Pharmacia)). (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 2.5 molar ammonium sulfate solution at pH 7.0.