JPH07121875B2 - Method for producing immunoglobulin fraction - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing an immunoglobulin fraction that can be used as an antibody in an immunochemical assay.

2. Description of the Related Art Immunoglobulins consist of two H chains and L chains in each class. Its representative class is Ig
The structure of G is, for example, edited by Hirokichi Kikuchi, “Medical Immunology,” P85 (198
1), shown in Nankodo. H chain has a molecular weight of about 50,000, L
The chains are said to have a molecular weight of about 20,000 and are linked by a single disulfide bond. The N-terminal sides of the H chain and L chain jointly form an antigen-binding site. The C-terminal sides of the two H chains jointly form the Fc region, and are linked by two disulfide bonds. From the results of the chemical structure study of immunoglobulin, it was confirmed that Fa
It is known that b and Fc can be respectively obtained by pepsin decomposition to obtain F (ab ') ₂ and a fraction in which the Fc-corresponding portion is further finely decomposed.

Fab, Fab ′ or F (ab ′) ₂ has a region that binds to an antigen, and it is said that the amino acid sequence varies at this portion to give specificity to the antigen-antibody reaction. On the other hand, the Fc region has a constant amino acid sequence and does not show antigen-binding ability, but has complement-fixing ability. The structure of the Fc region varies depending on the immunoglobulin class and subclass, and also varies depending on the animal species.

Problems to be Solved by the Invention As described above, the Fc region of immunoglobulin does not have antigen-binding activity, but there is an effector function that is an action on the complement and cells of the antibody, but the immunoglobulin is either in vitro or in vivo. When used as an antibody in an antigen-antibody reaction in the body, it often gives an inconvenient aspect. Often used in vitro for immunological diagnosis, the Fc region can be one of the causes of nonspecific reaction. For example, one of the interfering factors in serum is rheumatoid factor, which binds to the insolubilized or denatured Fc portion of IgG.
Therefore, if the rheumatoid factor is present in the serum, the above reaction proceeds in addition to the desired antigen-antibody reaction, which causes erroneous determination. In addition, inactivation (for example, test serum at 56 ℃
The substance group removed by heat passivation)
Often reacts with the Fc portion, giving non-specific results.
In the reaction in vivo, immunoglobulin is used for imaging and immunological treatment, but when the administered animal is different from the above immunoglobulin-producing animal, the antibody against the Fc portion is likely to be generated, the effect is reduced, and side effects are generated. Appears.

In order to improve such a defect, Fab, Fab ′ or F (ab ′) _{2 obtained} by removing the Fc portion from immunoglobulin is often used. F (ab ′) ₂ and Fab ′ obtained by further reduction are commonly used as antibodies for in vitro or in vivo antigen-antibody reaction, and are expected to be used more frequently in the future.

F (ab ') ₂ is obtained by treating immunoglobulin with pepsin. However, the difficulty of limited degradation from pepsin varies depending on the type of animal producing immunoglobulin. Usually, add about 1/50 amount of crystalline pepsin to immunoglobulin in 0.05M or 0.1M acetate buffer at pH 4.5, digest at 37 ° C for 15 to 72 hours, and add sodium hydroxide. Harmonize The product is fractionated by, for example, column chromatography on Sephadex G-75 or G-150 (Falmatia, Sweden) to obtain F (ab ').
₂ is obtained. In addition, F (ab ′) ₂ is added to 2
− Obtained by treating with a reducing agent such as mercaptoethanol and purifying by column chromatography such as Sephadex G-25 [Utsumi Shou: Medical Chemistry Laboratory 4 (Shunsuke Ueda), Nakayama Shoten, Tokyo, P108 (1972) : Journal of Immunoassay Volume 4
See page 209 (1983)].

However, even if immunoglobulins obtained from mice and rats are treated under the above conditions, F (ab ′) ₂
It has been reported that it cannot be obtained (see the above Journal of Immunoassay, especially pages 230-231).

Means for Solving the Problems In view of the above-mentioned circumstances, the present inventors have made earnest studies, and as a result, when the pH in the pepsin treatment is made more acidic, F (ab ′) ₂ can be efficiently produced. As a result of the discovery and further research, the present invention was completed.

The present invention provides immunoglobulins obtained from rodents at pH
A method for producing immunoglobulin F (ab ′) ₂ which is characterized by treating with pepsin at 2 to less than 4.

Examples of rodent (Rodentia) animals in the method of the present invention include:
For example, rodents (rats, Myomorphia) are preferable,
Examples include mice, rats, guinea pigs,
Examples include hamsters.

As the immunoglobulin in the method of the present invention, IgG, IgM,
Examples thereof include IgE and IgA, and IgG is particularly preferable, and IgG ₁ and IgG _2a are preferable.

The amount of immunoglobulin when the treatment of the method of the present invention is performed is about 0.1 to about 3 W / V% in the reaction solution, more preferably about 0.3 to 1 W / V%.

The antibody when immunoglobulin acts as an antibody is not particularly limited, and examples thereof include anti-carcinoembryonic antigen antibody, anti-hepatitis virus antigen antibody, anti-human chorionic gonadotropin antibody, anti-alpha-fetoprotein antibody, Anti-tetanus toxin antibody, anti-pertussis toxin antibody, anti-immunoglobulin antibody, anti-drug antibody, etc. may be mentioned.

Any known pepsin may be used in the method of the present invention. Examples of the pepsin include reagent pepsin such as pepsin 1: 60,000 (Sigma, USA) prepared by freeze-drying crystalline pepsin obtained from pig stomach mucus.

The concentration of pepsin in the above treatment is about 1/40 to 2 times the amount (weight) of immunoglobulin, and more preferably about 1/10 to 1/2 the amount (weight).

The pepsin treatment of the present invention is performed by treating immunoglobulins at pH 2 to less than 4. The treatment is preferably carried out in a buffer solution, and examples of the buffer solution include 0.1 M acetate buffer solution, 0.1 M glycine dry solution, and 0.5 M citrate buffer solution.

The pH of the treatment is about 2 to less than 4, but
It is preferably carried out at 2.5 to 3.5.

The pepsin treatment of the present invention is carried out at 4 ° C to 40 ° C for about 0.5 to 96 hours, more preferably about 35 to 38 ° C for about 1 to 2 hours.

The pepsin-treated product thus obtained is
It can be purified by a conventional purification means.

For example, the pepsin-treated product is neutralized to pH about 8 with an alkaline solution (eg, 1N-sodium hydroxide) and subjected to known gel chromatography (eg, Sephadex G-150 or -75, Ultrogel AcA44 (LKB (Switzerland, Sweden)) and F (ab ') ₂
Purified and collected as.

Immunoglobulin F (ab ′) thus obtained
₂ is a method known per se, for example, a pH neutral buffer solution using a reducing agent such as 2-mercaptoethylamine,
It can be Fab '.

These Fab ′ and F (ab ′) ₂ are (1) an antibody retained on a carrier, (2) an antibody labeled with a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or (3) a drug Any of the antibodies bound with can be used as a reagent in an immunochemical assay method known per se. For example, using the conditions of the present invention, a monoclonal antibody (F (ab ') ₂ fragment obtained by treatment with pepsin or
Fab 'fragment) was retained on a carrier and treated with pepsin in the same manner to obtain a monoclonal antibody (F (ab')).
_The sandwich enzyme immunoassay in which an enzyme-labeled antibody obtained by binding a ₂ fragment or Fab 'fragment) to an enzyme is combined has good sensitivity and almost no nonspecific reaction is observed. Also, using the conditions of the present invention,
Monoclonal antibody F (a obtained by treatment with pepsin
b ′) ₂ or Fab ′ can be used for an in vitro drug delivery system (drug delivery system) by chemically binding to a drug such as an anticancer drug.

By using the method of the present invention, immunoglobulin F (ab ′) ₂ fraction and Fab ′ can be produced in good yield in a short time from immunoglobulin obtained from a rodent.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Reference Examples and Examples.

Reference Example 1 (1) Purification of antigen 200 g of colorectal cancer tissue was shredded and 600 ml of 1% Tween 20
A 0.15 M NaCl solution containing Sigma (US) was added and crushed with a homogenizer for 10 minutes under ice cooling to prepare a suspension, which was further treated with an ultrasonic generator for 1 hour under ice cooling,
It was centrifuged at 12,000 rpm for 20 minutes. The supernatant was dialyzed against distilled water and then freeze-dried. Next, it was dissolved in 0.2 M citrate buffer (pH 6.5) and applied to a column (2.2 cm × 26 cm) of Concanavalin A-bound Sepharose 4B [Pharmacia (Sweden)] prepared using the same buffer. The substance retained on the column was eluted with a buffer solution containing α-methyl-D-mannocide. It was dialyzed against distilled water and then freeze-dried. Then 0.2M citrate buffer (pH 6.
Using 5), gel chromatography was carried out on a column (2.3 cm x 100 cm) of Ultrogel AcA-34 [LKB (France)], and a fraction of 280 to 350 ml was dialyzed against distilled water and freeze-dried. A purified antigen of human carcinoembryonic antigen (CEA) was obtained (5 mg).

(2) Preparation of monoclonal anti-CEA antibody 70 μg of the purified antigen obtained in (1) above was added to 150 μl of physiological saline.
And Freund's complete adjuvant [Freu
nd's complete adjuvant, "Immune biochemistry", Tachibana et al., No. 1
P. 26, Kyoritsu Shuppan Co., Ltd. (1967)] 250 μm was added and mixed well to form an emulsion, which was subcutaneously administered to BALB / C mice. Further, immunization with Freund's incomplete adjuvant was performed twice every two weeks, and the purified antigen was used as the final immunization.
400 μg obtained by dissolving μg in physiological saline was intravenously administered, and 3 days later, the spleen was taken out. Next, Dulbecco ’s modi
After washing well with fied MEM medium, the splenocytes 1 × 10 ⁸
The cells were mixed with 2 × 10 ⁷ mouse myeloma cells (P ₃ U ₁ ) and centrifuged at 700 rpm for 15 minutes to form a pellet. Next, 0.4 ml of 45% polyethylene glycol 6000 dissolved in RPMI-1640 was added, and 15 ml of RPMI-1640 was gradually added to dilute it, and then centrifuged at 700 rpm for 15 minutes to remove the cells.
It was dispersed in 100 ml of RPMI-1640 medium containing 20% fetal bovine serum. Then, 2.0 ml of each of the above cell dispersions was poured into a 24-well culture plate [Flow (USA)].
On days 5 and 8, half of the culture supernatant was replaced with HAT medium. When the antibody titer of the culture supernatant after 14 days was measured, a positive result was observed in 9 of 72 wells.

Next, cloning of these positive hybridomas was carried out by diluting with 20% fetal bovine serum and RPMI-1640 medium supplemented with BALB / C mouse thymus cells as a feeder, and repeating the limiting dilution method. Specifically, 5 kinds of hybridomas producing a monoclonal anti-CEA antibody were obtained. Among the hybridomas thus obtained, the hybridoma Mo272-11 (monoclonal antibody Mo-T2) was produced by the Fermentation Research Institute in Showa 60
Deposited under accession number IFO50033 on March 4, 1985.

The 5 types of hybridomas obtained above were intraperitoneally injected into BALB / C mice treated with mineral oil, and ascites were collected 2 to 3 weeks later.
A antibody was obtained. These monoclonal antibodies were salted out by the ammonium sulfate method to obtain globulin fractions (Mo-T1 to Mo-T6).

Example 1 Monoclonal anti-CEA antibody γ obtained in Reference Example 1 (2)
-Globulin fraction (Mo-T4) 5 mg at pH 1.0, 2.
0.1M acetate buffer adjusted to 0, 3.0, 4.0 or 5.0 respectively
It was dissolved in 1 ml, 1 mg of pepsin was added, and the mixture was reacted at 37 ° C for 2 hours. After neutralization with 1N-NaOH, the gel was passed through a Ultrogel AcA44 column (diameter 2 cm, length 75 cm) and eluted with 0.1 M borate buffer (pH 8.0). Elution patterns (measured by absorbance at 280 nm) obtained when treated at each pH
It is shown in FIG. That is, adjust the pH to 1,0,2.0,3.0,4.0 or
The case of adjusting to 5.0 is shown in FIG. 1, FIG. 2, FIG. 3, FIG. 4 or FIG. 5, respectively.

F (ab ') ₂ was eluted between about 50 fractions and about 54 fractions (volume of 1 fraction: 2.5 ml), but pH
At 3.0, the best elution pattern was obtained compared to other pH values. These F (ab ') ₂ fractions were pooled and serially diluted to prepare a test solution. Anti-mouse IgG antibody (Miles,
USA) was reacted with the above test solution using a 96-well microplate (Nunc, Denmark) in which 10 μg of each was adsorbed in each well, and then CEA and rabbit anti-CEA antibody horseradish peroxidase complex (DAKO) The antibody titer was determined by performing an enzyme-linked immunosorbent assay in which a solution was added. The usual o-phenylenediamine method was performed for color development, and the absorbance at 492 nm was measured.

The results are shown in Table 1, and the treatment at pH 3 gave the highest yield, and the antibody titer of the obtained F (ab ') ₂ was also the highest.

Example 2 According to the method of Example 1, pH 3.0, 37 ℃ 2 hours, or pH 3.0, 4 ℃,
Under the condition of overnight pepsin treatment, the amount of pepsin was changed to 5, 10 or 20 W / W% with respect to the amount of immunoglobulin. Second
The results are shown in the table, but the best conditions were pH 3.0 and 37 ° C for 2 hours.

Example 3 According to the method of Example 1, pH 3.0 and pepsin were changed at 4 ° C. for 2 days, 3 days or 4 days under the condition of 20 W / W% with respect to the amount of immunoglobulin. The results are shown in Table 3, but the conditions of pH 3.0, 4 ° C, and 3 days were good.

Example 4 According to the method of Example 1, pH 3.0 and pepsin at 20 W / W% with respect to the amount of immunoglobulin at 37 ° C. for 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours or 5 hours. I changed it. The results are shown in Table 4, but the conditions of pH 3.0, 37 ° C, 1 hour or 2 hours were favorable.

EFFECTS OF THE INVENTION According to the method of the present invention, immunoglobulin F (ab ′) ₂ obtained from a rodent can be efficiently produced. Therefore, the immunoglobulin fraction is industrially used as a reagent for immunochemical assay. Can be advantageously manufactured.

[Brief description of drawings]

1 to 5 show immunoglobulin F (a) obtained in Example 1.
The elution patterns of b ′) ₂ are shown respectively.

Claims (1)

[Claims] 1. An immunoglobulin obtained from a rodent
A method for producing immunoglobulin F (ab ') ₂ , which comprises treating with pepsin at pH 2 to less than 4.