JPH0246284A - Production of human alpha-phetoprotein - Google Patents

Abstract

PURPOSE: To enable the easy production of human α-fetoprotein in high reproducibility at a low cost by using human hepatocarcinoma cell secreting mainly human α-fetoprotein and secreting only negligible amount of albumin.

CONSTITUTION: Human hepatocarcinoma cell subjected to expansion process and acclimatization process is inoculated in a culture flask holding a liquid medium containing a carbon source, a nitrogen source, inorganic salts and vitamins, added with penicillin G, streptomycin and Na₂SeO₃ and free from bovine fetus serum and the cell is cultured at 37°C. The supernatant of the cultured product (containing α-fetoprotein and albumin at a weight ratio of (10-20):1) is concentrated to a volume of 1/(10-20) and purified. The α-fetoprotein is purified by the electrophoresis on a prepared polyamide gel. The purified α-fetoprotein is concentrated to a volume of about 1/10 after removing the acrylamide and finally freeze-dried to obtain α-fetoprotein having a purity of ≥98% and free from albumin. The production is easily performed in high yield and reproducibility by this process.

COPYRIGHT: (C)1990,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods and means for detecting tumor-associated antigens, particularly suitable for the fields of diagnosis and therapy.

α-fetoprotein (hereinafter referred to as AFP) is
It is a fetal alpha-globulin that is structurally similar to albumin.

Most of AFP exists in fetal serum and rapidly decreases after birth, decreasing to 1-6μ9/Q in healthy adults, but in patients with teratoma, hepatopathy, primary malignant liver cancer, and fetal pathologies ( fossil pathologica
tus), AFP a levels in human body fluids are significantly increased.

Quantification of AFP in serum or other body fluids is a fundamental and practical clinical parameter for the diagnosis of the above-mentioned pathological conditions and evaluation of appropriate treatment.

Immunization methods commonly used for the quantification of AFP require large amounts of pure α-fetoprotein for the preparation of specific antibodies and for control standards.

Currently, α-fetoprotein is obtained by extraction and purification from tracheal blood, fetal blood, and ascites fluid of patients with primary malignant liver cancer.

However, such methods are not always satisfactory, especially in terms of the raw materials used.

In fact, there are limits to the use of such raw materials, and because they are easily variable, α-fetoprotein cannot be reproducibly prepared in large amounts. Furthermore, since serum proteins (particularly albumin) are present in these raw materials at much higher concentrations than APP,
It is necessary to use purification methods that are complicated and not always sufficient. For example, immunoaffinity (im
Purification method using munoaff inity (B.
NoorgardPedersen rScard.
J. lmmunol. Suppl. J4
(1976) M. M. Baig “Analytical Biochemistry (Anal.
m. ) J (1980), 101, 200) makes it possible to obtain α-fetoprotein with good purity, but with partially degraded or fragmented α-fetoprotein that can still react with anti-α-fetoprotein antibodies. Substances cannot be removed from purified materials. Furthermore, in the elution method from the immunoaffinity column, the AF present in the eluate
Denatures P.

Thus, there has been a need for a simple, reproducible and inexpensive method (which eliminates the above-mentioned problems) as well as a means for the preparation of alpha-fetoprotein.

According to the present invention, such requirements can be met by a method using human hepatoma cells that secrete α-fetoprotein as a main protein component, but secrete albumin only in an amount negligible compared to α-fetoprotein. .

Furthermore, the object of the present invention is to contain carbon sources, nitrogen sources, inorganic salts and vitamins, penicillin G1 streptomycin and Na. Human hepatoma cells, which secrete α-fetoprotein as the main protein component, but only in negligible amounts compared to α-fetoprotein, were cultured in a liquid medium containing no fetal bovine serum supplemented with SeOs. The present invention provides a method for preparing human α-fetoprotein, a tumor-associated antigen (useful in the diagnostic and therapeutic fields), which comprises separating and purifying α-fetoprotein under non-denaturing conditions.

Another object of the invention is to use the obtained α-fetoprotein for the production of specific monoclonal and polyclonal antibodies.

Yet another object of the invention is in the field of therapy for the preparation of pharmaceutical compositions against primary hepatic tumors, in the field of diagnostics against hepatopathy, primary hepatic tumors, teratocarcinomas and fetal physiopathologies. (physiopath
The objective is to use the above-mentioned antibodies for the determination of

Yet another object of the present invention is to provide a kit for quantifying α-fetoprotein in human clinical samples using AFP and the above-mentioned antibodies.

The present invention contains carbon sources, nitrogen sources, inorganic salts and vitamins, including penicillin G, streptomycin and NatSe.
Surprising finding that human hepatoma cells conditioned in O3-supplemented medium without fetal bovine serum secrete α-fetoprotein as the main protein component, but only in negligible amounts compared to AFP. It is based on The acquisition of such properties by such cells makes it possible to implement the method of the present invention.

The method for producing human α-fetoprotein according to the present invention comprises: (a) containing a carbon source, a nitrogen source, an inorganic salt and a vitamin;
Penicillin G1 streptomycin and NatSeO+
α-fetoprotein is secreted as the main protein component in a liquid medium containing no fetal bovine serum, but
culturing human hepatoma cells that secrete albumin in negligible amounts compared to the α-fetoprotein at about 37°C;
(b) separating a supernatant containing crude alpha-fetoprotein from the cell disruption material; (c) separating the crude alpha-fetoprotein from the supernatant and purifying it by electrophoresis on a preparative polyacrylamide gel; Includes each step.

Hepatocytes suitable for the method according to the invention are human hepatoma cells.

Among these are human hepatoma cells that have a high degree of differentiation and can produce α-fetoprotein, albeit in minimal amounts.

According to the invention, such cells are expanded by manipulation according to known general methods and then conditioned by culturing in a medium containing no fetal bovine serum.

In practice, the culture was carried out at 37°C in minimal medium (MEM) supplemented with fetal bovine serum (10% by weight), Co, 5% and air 95%.
Expansion of the cells is performed by culturing the cells for at least 2 weeks under an atmosphere (90% humidity) containing %.

During this time, the medium was changed at least twice every week with the same volume of the same medium supplemented with fetal bovine serum, and the cells that had reached confluence were incubated with trypsin 0.25%, 0.536 mM EDTA and 5
Separate by trypsinization with Earle's salt solution containing mM NaOH at room temperature (20-25° C.) for approximately 20 minutes.

The cells thus isolated are inoculated into flasks containing the same minimal medium plus fetal bovine serum at a distribution of 5X to' cells per lCw''.

After the expansion step, the cells were collected and treated with penicillin G (50H/ff1) containing carbon source, nitrogen source, inorganic salts, and vitamins.
2), streptomycin (501N9/MQ), Na
Acclimation is achieved by inoculating flasks containing liquid medium without fetal bovine serum supplemented with 2SeO3 (3X 10-'M).

Such an acclimatization step may be carried out at a temperature of or near 37°C, C
o, 5% and an atmosphere containing 95% air (humidity 90%
%) for at least 3 weeks.

The medium is periodically replaced with the same volume of the same medium, and cells that have reached confluence are separated by trypsinization for about 3 minutes at room temperature with Earle's salt solution having the composition described above.

During this time, the amounts of AFP and albumin secreted into the medium are quantified by electrophoretic analysis on polyacrylamide gels to check the conditioning of the cells.

The results obtained (shown in Figures 1 and 2) show that AFP increased from 15% to 40% and albumin decreased from 40% to less than 3% of total protein.

In fact, it contains carbon sources, nitrogen sources, inorganic salts, vitamins, penicillin G1 streptomycin and NaxSe0
Cells are inoculated into culture flasks containing a liquid medium containing no fetal bovine serum supplemented with No. 3 and cultured at or near 37°C.

Periodically collect the medium from each flask, replace it with an equal volume of the same medium, and centrifuge to separate the supernatant from cell disruption material.

Combine the supernatant liquid (containing crude α-fetoprotein and albumin at an α-fetoprotein:albumin weight ratio of 10:1 to 20:1) and make a volume of approximately 1/LO-1/20 compared to the initial volume. Concentrate and purify.

Since the amount of albumin present in the supernatant is lower than that of AFP, simple purification methods can be used and mild operating conditions can be utilized.

In the method of the invention, α-fetoprotein is purified by electrophoresis on preparative polyamide gels. By operating in this manner, the albumin present in the supernatant (which exhibits a different migration than APP due to its charge) is completely separated and α-fetoprotein under negative conditions is obtained.

Purification by electrophoresis was performed by placing a polyacrylamide gel (composition shown in Example 2) in 0.024 M Lys buffer and 0.19 M glycine (pH 8.3), and applying a potential difference of 1 to 15 mA to 12 to 15 mA. This is done by applying it for 16 hours.

After this period of time, the portion of the gel containing alpha-fetoprotein is identified, isolated, and cut into 11-2x cubes. This gel cube was then dissolved in 50mM Tris buffer (
α-fetoprotein is eluted by adding the mixture to pH 8J) and stirring at a temperature of 4° C. at least twice.

The eluates are combined, centrifuged twice to separate residual acrylamide, concentrated to about 1/10 volume, and finally freeze-dried.

In this way, α-fetoprotein with a purity of 98% or more is obtained. Western blot analysis (using an anti-human albumin antibody) shows that the α-fetoprotein obtained does not actually contain albumin.

In the prior art, it could not be foreseen that the problem of preparing α-fetoprotein would be solved by a simple method such as the present invention.

In fact, it could not have been predicted that hepatoma cells produce α-fetoprotein as the main protein component and at the same time secrete negligible amounts of albumin.

The use of cells with these hitherto unknown properties made the method of the invention possible for the first time.

Such a method exhibits significant advantages over the current state of the art, among which ease of implementation, high yields of α-fetoprotein and reproducibility are achieved.

According to the invention, the latter advantage is due to the fact that the operation is performed using only one constant AFP source.

The α-fetoprotein obtained according to the method of the invention is therefore particularly suitable for the international standardization of diagnostic kits and the preparation of monoclonal and polyclonal antibodies (useful in the diagnostic and therapeutic fields) obtained by known general methods. Are suitable.

The drawings will be explained in detail.

FIG. 1 shows the results obtained on analytical polyacrylamide gel (PAGE).

L: Supernatant obtained from hepatoma cells at the beginning of acclimation shows the presence of two bands corresponding to AFP and albumin.

2: depending on the width of the supernatant obtained from the conditioned cells).

10 and 11: Supernatants obtained from unacclimated hepatoma cells from cultures on microcarriers show bands corresponding to albumin.

12,13 The supernatant obtained from hepatoma cells during the conditioning step shows that the albumin band gradually disappears.

Example 1 Acclimation of hepatoma cells Hepatoma cells 1ep G2 (Knowles et al. [Science (1980), 209.
p497-499) in minimal medium MEM (Eacle H) containing 10% fetal bovine serum.

“Science J (1959), 130. p 43
2-437) 1011 (2-437) inoculated into four culture flasks containing 2-437) at 37°C, CO.

2 in an atmosphere consisting of 5% and 95% air (90% humidity)
Incubated for a week.

The medium was changed twice a week, and cells that reached confluence were treated with trypsin 0.25%, EDTAO, 536
Earle salt solution containing mM and NaOH5+nM (J, R, Earle rJ, Natl, Canc
er In5tl (1943).

p 165.4) Separated by trypsinization using a 5-wing Q at 37°C for 20 minutes.

The cells thus isolated were re-inoculated into 4 flasks (175cx'') containing 25 cells of the same medium at a distribution of 5.0000 cells per cjI''.

By operating as described above, after another two weeks,
4 flasks containing liver cancer cells (175cJI”)
I got it.

Cells were then released by trypsinization under the same conditions as described above and placed in RPMI 1640 medium (GIBCO).
(Na2SeO33x10-”M, penicillin G5o1
19/11Q and streptomycin 50x9h (added with l and without fetal bovine serum) 25tnQ
8 flasks (175cR″) containing 2, lcR
The cells were inoculated so that 50,000 cells per cell were distributed.

maintained at 37° C. for at least 3 weeks under the same conditions as described above;
During that time, the medium was replaced with the same volume of the same medium twice per week.

Cells that reached confluence were detached by trypsinization for 3 minutes at room temperature and re-inoculated into another flask containing the same RPM11640 medium.

After another two weeks, the hepatoma cells were completely acclimatized.

That is, a large amount of α-fetoprotein was secreted into the medium, and albumin was secreted only in an amount negligible compared to AFP.

The changes in the yield of α-fetoprotein and albumin produced during various acclimatization steps were measured using Laemli (rN
atureJ 227. p 6g0-685. (1
Sodium sulfate (SDS)
) was quantitated by electrophoresis under non-denaturing conditions, and the results are shown in FIGS. 1 and 2.

APP and albumin in the supernatant varied from 15% to 40% and from 40% to less than 3% of total protein, respectively.

Example 2 a) Preparation of human α-fetoprotein from conditioned hepatoma cells Hep G2 cells conditioned as described in Example 1 were cultured in RPM1 1640 medium (GIBCO) (NazSe
Os 3x10-8M.

Penicillin G 50m9hQ and streptomycin 5
0mg/mQ added) 1 containing 30mQ
00 flasks (175cx'') were inoculated.

The flasks were incubated for 45 days at 37°C under an atmosphere consisting of 0.0025% and 95% air (90% humidity), during which time the medium was periodically collected, centrifuged to remove material from cell debris, and the same Replaced with medium. The supernatants were combined and stored at -30°C.

Operating in this manner, 2012 supernatants containing 43 mg/Q of crude alpha flask and approximately 219/Q of albumin were collected during the first 15 days.

b) Purification of α-fetoprotein Supernatant 5Q obtained in step a) above (crude AFP
215xy), the Minitan system (M.
A slab of less than 10,000 daltons was used to concentrate to a final volumetric volume of 400πQ. The concentrate was then loaded in a volume of 12 mQ' onto a preparative polyacrylamide gel (PAGE) in the absence of denaturing agents.

This prepared gel (170x 135x 4.5mm) is
Acrylamide 10%, bisacrylamide 0.266
%, Tris (pHL3) OJ74M, ammonium persulfate 0.05%, TEMED (N, N, N', N'-
Tetramethyl-ethylenediamine) 0.083%.

On these gels, acrylamide gel 3.75%, bisacrylamide 0.1%, Tris (pH 6,8) 0.1
25M, ammonium persulfate 0.05%, TEMEDO
,083% gel (25x 130x 4.5mm
) were laminated.

0.0247M Tris buffer, 0.19M glycine (
The gel was run for 16 hours at 15 mA/gel in pH 8.3).

After the electrophoresis was completed, the gel was removed from the buffer, a strip of approximately 1 ci from both sides and a central strip of approximately 0.3 cM was cut out, and the remaining gel was stored at -20°C. After staining the α-fetoprotein in the strips with Coomassie blue, the strips were aligned with the rest of the gel to identify the AFP-containing gel portion.

These gel parts were separated, cut into 1-2H cubes, and added to 50mM) Lys buffer (p
H8,3) Eluted twice in 6'mQ/gel (total 2
0 hours).

The eluate (containing AFP as the main protein component) was
Centrifuge twice to remove residual acrylamide and use a vacuum filter Millipore CX 30 to remove ca.
710 and lyophilized.

In this way, 19 mg of α-fetoprotein with a purity of 9899% was obtained from the supernatant 1e (yield 45%).

Western blot analysis using an anti-human albumin antibody showed that the resulting α-fetoprotein did not contain albumin.

Figure 2 shows the results obtained by preparative analytical gel (PAGE), comparing various amounts of α-fetoprotein (2, 3, 4 and 5) purified as described above with commercially available α-fetoprotein (6, 7). , 8).

This allows slight non-uniformity to be observed.

[Brief explanation of the drawing]

FIGS. 1 and 2 are photographs showing the results of polyacrylamide gel electrophoresis showing changes in the amounts of AFP and albumin due to acclimatization and the effects of purification when operating according to the method of the present invention. (1 other person)

Claims (1)

[Scope of Claims] 1. A human hepatoma cell that secretes α-fetoprotein as a main protein component, but secretes albumin only in an amount negligible compared to α-fetoprotein. 2. The human hepatoma cell according to claim 1, wherein the weight ratio of secreted α-fetoprotein and albumin is 10:1 to 20:1. 3. In the product according to claim 1 or 2, in a liquid medium containing a carbon source, a nitrogen source, an inorganic salt, and a vitamin, and containing no fetal bovine serum and supplemented with penicillin G, streptomycin, and Na_2SeO_3, at or around 37°C. human hepatoma cells, which are obtained by acclimatizing human hepatoma cells at a temperature of 10:2 and for a time necessary to obtain α-fetoprotein and albumin in a mutual ratio of 10:1 to 20:1. 4. A method for producing human α-fetoprotein, wherein (a) the human liver cancer cells according to claims 1 to 3 are mixed with a carbon source, a nitrogen source, an inorganic salt, and a vitamin, and penicillin G
, cultured at about 37°C in a liquid medium containing no fetal bovine serum supplemented with streptomycin and Na_2SeO_3, (b) cell-disrupting substances are separated from the medium, and a supernatant containing crude α-fetoprotein as the main protein component is obtained. (c) separating the crude α-fetoprotein from the supernatant and purifying it by electrophoresis on a preparative polyacrylamide gel. 5. The method for producing human α-fetoprotein according to claim 4, wherein the medium used in step (a) is RPMI1640. 6. In the manufacturing method according to claim 4, the supernatant liquid in step (b) is separated at 3000 rpm. A method for producing human α-fetoprotein by centrifugation for 10 minutes. 7. In the manufacturing method according to claim 4, the electrophoresis in step (c) is performed in Tris buffer (pH 8.3) at 20%
A method for producing human α-fetoprotein, carried out at −25° C., current 10 to 15 mA/gel, 12 to 16 hours. 8. A method for using human α-fetoprotein, which comprises using α-fetoprotein obtained by the production method according to claims 4-7 for the preparation of special monoclonal and polyclonal antibodies. 9. A method of using monoclonal and polyclonal antibodies, wherein the antibody according to claim 8 is used for preparing a composition for treating primary human liver cancer. 10. The antibody according to claim 8 is α-
Methods of using monoclonal and polyclonal antibodies for the diagnosis of diseases causing increased fetoprotein. 11. A diagnostic kit for the qualitative and quantitative determination of α-fetoprotein in biological human samples, using α-fetoprotein obtained by the method according to claims 4-8 and specific monoclonal and polyclonal antibodies. An α-fetoprotein diagnostic kit characterized by the following.