JPH0712319B2 - Monoclonal antibody screening method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] The present invention relates to a method for screening a monoclonal antibody. More specifically, the physiological activity of an enzyme antigen is used as an index to determine the physiological activity of the enzyme antigen. The present invention relates to a method for simultaneously screening a monoclonal antibody that exerts an influence (inhibits or activates a physiological activity) and an antibody that simply binds to an enzyme antigen and has no influence on the physiological activity at all.

According to this method, only the antibody-producing cell line of interest can be selected and screened extremely efficiently from among the fused cells, and in particular, a monoclonal antibody for analysis or purification of enzyme reaction can be efficiently obtained. Therefore, the productivity and production efficiency of the monoclonal antibody are greatly increased, and the monoclonal antibody is used not only in the immunochemistry but also in the enzyme industry.

[Conventional technology]

Monoclonal antibodies that have been put into practical use or are being studied for a wide range of uses such as purification, therapeutic treatment, diagnostic use, and research use include, for example, polyethylene glycol (PEG) obtained by splenocytes or peripheral blood cells immunized with an antigen and myeloma cells. In this case, it is necessary to select a fused cell clone producing and secreting an antibody against the antigen of interest from the obtained fused cells. is there. Therefore, in general, in order to detect a monoclonal antibody contained in a supernatant obtained by using a fused cell culture supernatant as a sample, a passive agglutination reaction in which an antigen is immobilized on sheep erythrocytes or latex particles in advance, or an antigen is detected in advance. Enzyme immunoassays and radioimmunoassays (EIA, ELISA, EMI) immobilized on microtiter plates and cubes
Methods such as T, RIA, etc. have been developed and widely used (Nobuhiro Hattori “From viral hepatitis to hepatocellular carcinoma” (1982)
-7-10) Cancer and Chemotherapy, p439-443) However, in these conventional methods, the principle of detection is that an antibody binds to an immobilized antigen, so that the antigen retains physiological activity like an enzyme. If the detected monoclonal antibody affects the physiological activity of the antigen, it cannot be determined whether or not the inhibitory or activated monoclonal antibody to the enzyme antigen and the bound monoclonal antibody are simultaneously and conveniently detected from the initial stage of screening. It was difficult.

Further, in the conventionally used screening methods, the antigen is often immobilized directly on the solid phase, and a highly pure antigen preparation is required. Therefore, it is not only necessary to use a large amount of very expensive enzyme, but also a great deal of labor is required for immobilization, and complete immobilization itself is a difficult task.

[Disclosure and Effect of Invention]

In order to solve these drawbacks, the present inventor has conducted various researches from various technical fields such as enzyme chemistry, immunochemistry and biochemistry, and as a result, uses an enzyme as a simple labeling substance as in the conventional method. Instead of focusing on the utilization of the physiological activity itself held by the enzyme antigen, on the basis of this, we considered this method using the physiological activity of the antigen as an index,
He has invented a method for screening a monoclonal antibody against an enzyme antigen.

The present invention is to first prepare a monoclonal antibody-producing cell against an enzyme antigen and apply it to it. The preparation can be carried out by a conventional method, and the animal is sensitized intraperitoneally or the like with the antigen in the presence or absence of an adjuvant, and if necessary, further immunization is performed to immunize the animal with the antigen, Antibody-producing cells such as splenocytes and lymph node cells are removed from animals. This was fused with myeloma cells using polyethylene glycol (PEG), etc.
Hybridomas are selected by culturing in AT medium (selection medium consisting of hypoxanthine / aminopterin / thymidine).

From the thus obtained hybridomas, only the desired antigen-producing cell line, in other words, only the desired monoclonal antibody is screened extremely accurately and efficiently by this method.

The principle of detecting a monoclonal antibody according to the present invention will be described with reference to FIG.

1. First, an anti-immunoglobulin antibody is immobilized on an immobilization carrier.

As the anti-immunoglobulin antibody, anti-human immunoglobulin antibody is used in the case of mouse-derived hybridoma cells, and immunized animal species include goat, rabbit, sheep and the like. As the immobilization carrier, sepharose gel, sephadex gel, agarose gel, latex particles, polyacrylamide particles, polystyrene particles, tubes and the like are used, and the immobilization method of the anti-immunoglobulin antibody includes physicochemical action and covalent bond. However, it is preferable to use a covalent bond method such as the CNBr method in order to prevent substitution reaction between the immobilized anti-immunoglobulin antibody and the serum protein and monoclonal antibody contained in the hybridoma cell culture supernatant. To immobilize the anti-immunoglobulin antibody on the insoluble carrier.

Then, the monoclonal antibody contained in the hybridoma cell culture supernatant obtained by the above cell fusion treatment is mixed and suspended in an anti-immunoglobulin antibody-immobilized carrier, and the monoclonal antibody is immunoadsorbed to the immobilized anti-immunoglobulin antibody by an immune reaction. Let

2. Add and mix a certain amount of enzyme antigen to the monoclonal antibody-adsorbed anti-immunoglobulin antibody-immobilized insoluble carrier prepared in step 2. At room temperature or 37 ° C, an immunoreaction is carried out between the enzyme antigen in the free state and the monoclonal antibody in the solid phase, and after a certain period of time, the supernatant and the insoluble carrier are separated by centrifugation to separate the enzyme antigen from the monoclonal antibody. Stop the immune response. Even if the serum protein in the culture supernatant contains an enzyme having a physiological activity similar to the target enzyme antigen, the monoclonal antibody-adsorbed anti-immunoglobulin antibody-immobilized carrier prepared in 1 is used as a physiological buffer solution (PBS) or the like. And preferably
Similar physiological activities contained in serum can be eliminated by washing with PBS containing 0.1% BSA (pH 7.2) several times.

3.2 Measure the physiological activity of the enzyme antigen remaining in the centrifugation supernatant separated in 3.2 and the physiological activity of the enzyme antigen adsorbed on the insoluble carrier by immunoreaction with the monoclonal antibody by spectroscopic or electrode method . The monoclonal antibody was detected by the marked decrease in the enzyme antigen activity contained in the centrifugation supernatant separated in step 2. At the same time, the enzyme antigen activity adsorbed on the insoluble carrier was measured, and the adsorbed enzyme antigen activity was confirmed. The antibody is determined to be a monoclonal antibody bound to the enzyme antigen.

b. If the adsorbed enzyme antigen activity is significantly lower than that in the case of a, it is judged as an inhibitory monoclonal antibody against the enzyme antigen.

c. If the adsorbed enzyme antigen activity is markedly higher than that in the case of a, it is determined to be an activating monoclonal antibody against the enzyme antigen.

This screening operation consists of the above three steps 1, 2, and 3, but for enzyme antigens that are not contained in serum proteins and do not interfere with the measurement of physiological activity by serum proteins, perform steps 1 and 2 at the same time, that is, It is also possible to simultaneously add and mix the culture supernatant containing the monoclonal antibody and a certain amount of enzyme antigen to the anti-immunoglobulin antibody-immobilized carrier. A protein A-immobilized carrier can also be used as a method for specifically adsorbing a monoclonal antibody to an insoluble carrier.

Examples of enzyme antigens that can detect monoclonal antibodies by this screening method include Malate dehydrogenas
e, β-D-galactosidase, Glucose oxidase and Horse r
There are adish peroxidase and the like, but not limited to these enzyme antigens. In addition, since the polyclonal antibody contained in the antiserum can be adsorbed to the anti-immunoglobulin antibody-immobilized carrier by this screening operation-1, this screening method is used as an evaluation method of antiserum against enzyme antigen. It is also possible.

This screening method enables simultaneous detection of the inhibitory or activated monoclonal antibody and the bound monoclonal antibody, which had been detected by the combination of multiple screening methods up to now, and makes the production of monoclonal antibody against the enzyme antigen much more efficient. It can be expected that it will be widely used in various fields of enzyme industry and immunochemistry.

Example 1. Preparation of anti-mouse immunoglobulin rabbit IgG-immobilized Sepharose 4B 100 μg of mouse immunoglobulin was subcutaneously injected together with Freund's complete adjuvant into Japanese domestic rabbits to prepare an antiserum against mouse immunoglobulin. After blood collection,
The serum was separated, and anti-mouse immunoglobulin rabbit IgG was purified by ammonium sulfate salting out and DEAE-cellulose.

Purified anti-mouse immunoglobulin rabbit IgG 0.1M NaHCO
Dissolve in ₃ (pH8.0) and mix with CNBr-activated Sepharose 4B.
After suspending, 4 mg of anti-mouse immunoglobulin rabbit IgG was covalently bound to 1 ml of Sepharose 4B. After the reaction, the remaining active groups are 0.
Blocking was performed with 1 M ethanolamine (pH 8.0) (Fig. 2).

2. Screening of mouse monoclonal antibody against yeast malate dehydrogenase BALB / c mouse spleen cells that were intraperitoneally sensitized with 50 μg of yeast-derived malate dehydrogenase (MDH) together with Freund's complete adjuvant and further boosted Cell fusion was performed with mouse myeloma cells (X Ag 8/6/5/3) using PEG (MW1500), and syngeneic mouse splenocytes were seeded in 24-well multiwells as feeder cells (2 × 10 ⁶ / well). ), HAT
The cells were cultured in a medium to prepare hybridomas with an efficiency of about 10 clones / well.

Using the anti-mouse immunoglobulin rabbit IgG-immobilized Sepharose 4B 50 μm prepared in the above 1, the hybridoma cell culture supernatant (1.0 ml) prepared above was added to this Sepharose 4B gel and suspended at room temperature for 1 hour. After the reaction, the gel was washed twice with PBS (pH 7.2) containing 0.01% BSA, and 0.1 units of yeast malate dehydrogenase dissolved in PBS (pH 7.2) containing 0.01% BSA was added. While suspending, react for 1 hour at room temperature, then centrifuge (5,000 rpm x 5 min)
Was separated into a supernatant and a gel. 200 μM NaDH and 500 μM
0.1 M potassium phosphate (pH 7.5) containing oxaloacetate was used as a substrate reaction solution of malate dehydrogenase, and 0.2 ml of the centrifugation supernatant was added to 2.0 ml to start oxygen reaction at 25 ° C. After 4 minutes, the decrease in absorbance at 340 nm was measured to determine the enzyme activity. At the same time, the gel after centrifugation was further added with 0.01% BS.
After washing twice with PBS containing A (pH 7.2), 1.0 ml of the enzyme reaction substrate solution was added to the gel and the enzyme reaction was carried out at 25 ° C for 10 minutes. After the reaction, the reaction was stopped by centrifugation, and the change in absorbance in the supernatant was measured. The results are summarized in FIG.

Of the hybridoma cells used for screening, M1
8A2, A3, A4, A7, A11 and B28 It was determined that the culture supernatant of any of the clones contained a binding monoclonal antibody that did not inhibit the enzyme activity, and only the M18A10 clone contained a monoclonal antibody that inhibits the enzyme activity. It was decided.

[Brief description of drawings]

FIG. 1 schematically shows the principle of the monoclonal antibody screening method according to the present invention, and FIG. 2 shows anti-mouse immunoglobulin rabbit IgG sepharose 4B.
FIG. 3 is a graph showing the results of screening mouse monoclonal antibodies against yeast MDH in FIG.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References S. Tomiyama, “Handbook of Monoclonal Antibodies” (1985-7-10) Kodansha P. 349 J. Immunol. Methods, 64 (1983) P.M. 31-37

Claims (1)

[Claims] 1. An anti-immunoglobulin antibody is immobilized on a carrier,
After reacting this with a monoclonal antibody against the enzyme antigen, the enzyme was allowed to bind, and after solid-liquid separation, the enzyme activity of the liquid part was measured, and at the same time, the enzyme activity of the fixed part was measured directly without separating the enzyme. Therefore, using the physiological activity of the enzyme antigen as an index, each of the inhibitory monoclonal antibody against the enzyme antigen, the activating monoclonal antibody, or the mere binding monoclonal antibody that does not affect the physiological activity is detected. Parallel screening method for monoclonal antibodies.