JPS6140067B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method of changing the charge of an antibody, and more particularly, to a method of changing the charge of an antibody, and more particularly to a method of changing the charge of an antibody.
The present invention relates to a method for changing the charge of an antibody, which is characterized by reacting a salt thereof, an acidic amino compound, or a basic amino compound with the antibody.

Conventionally, so-called immunoelectrophoresis, which utilizes antigen-antibody reactions, has been widely practiced for the purpose of qualitatively and quantitatively detecting antigens or antibodies, and its usefulness has been recognized in the fields of medicine and biology. However, in conventional immunoelectrophoresis methods, many antibodies or antigens require a long time to migrate, and in cross-immunoelectrophoresis in particular, there are many cases in which cross-phoresis is impossible or requires a long time for cross-phoresis. It was.
In addition, in the rocket immunoelectrophoresis method for analyzing antigens, quantitative analysis is not possible because the antibodies in the agar base migrate together with the antigen.
Quantitative analysis was possible only in the presence of a buffer that gave a constant pH value.

The inventors of the present application have conducted intensive research in order to eliminate the above-mentioned drawbacks, and as a result, we have found that antibodies can be treated with periodic acid and/or
The present inventors have discovered a method of changing the charge of an antibody by reacting it with a compound or a salt thereof, and an acidic or basic amino compound, and have completed the present invention.

Antibodies in the present invention can be obtained by salting out antiserum obtained from antibody-producing animals such as humans, cows, sheep, rabbits, and guinea pigs with a 50% ammonium sulfate solution, and dividing the fraction obtained by gel filtration, ion exchange chromatography, or Those purified by affinity chromatography etc., such as IgG,
Examples include IgM, IgD, IgE, IgA, etc. All of these antibodies are composed of an antigen-binding Fab region and an Fc region unrelated to antigen binding, and are distinguished from the Fab region in that the Fc region contains a small amount of oligosaccharide in addition to protein.

Examples of periodate salts used in the present invention include sodium salts, potassium salts, and calcium salts of periodic acid, which are used alone, in combination, or with periodic acid.

The acidic amino compound of the present invention means 2 in one molecule.
It refers to an amino compound having one or more carboxyl groups; examples of the former include acidic amino acids such as aspartic acid and glutamic acid, and examples of the latter include aminophosphoric acid compounds and aminosulfuric acid compounds. Aminophosphoric acid compounds include nucleotides with cytosine residues, adenine residues, guanine residues, etc., phosphates of vitamin _B1 , phosphoric acid esters of amino sugars such as 5-phosphoribosylamine, and creatine phosphate. Examples of aminosulfuric acid compounds include 2-aminoethanesulfonic acid (taurine), 2-aminopropanesulfonic acid, 3-aminopropanesulfonic acid, 1-aminopropane-
2-sulfonic acid, 4-aminobutanesulfonic acid,
5-aminopentasulfonic acid, o-aminobenzenesulfonic acid, p-aminobenzenesulfonic acid,
Examples include aminosulfonic acid compounds such as p-methylaminobenzenesulfonic acid and 1-anthracenesulfonic acid, and sulfuric acid ester compounds such as amino sugars such as glucosamine and galactosamine.

Furthermore, the basic amino compound of the present invention refers to a compound containing two or more amino groups in one molecule, such as diamines such as ethylenediamine and tetramethylenediamine, and polyamines such as spermine, spermidine, putrescine, and other polyamines. can be mentioned.

In the method of the present invention, when an antibody is brought into contact with periodic acid and/or its salt and an acidic or basic amino compound, the antibody and periodic acid and/or its salt are first reacted, and then the reaction product is Although the antibody may be reacted with an acidic or basic amino compound, preferably the antibody is reacted with periodic acid and/or its salt and the acidic or basic amino compound at the same time.

The amount of periodic acid and/or its salt used is 2 to 500 times the molar amount of the antibody, and the concentration is about 0.01M to 0.1M. The amount of acidic or basic amino compound used is 4 to 100,000 times the amount of the antibody.

As mentioned above, using periodic acid and/or its salt and an acidic or basic amino compound,
When treating antibodies in the step, use mild conditions that do not affect proteins at pH 4-9 and temperature 0-30℃.
Do this for 0.5 to 10 hours. In addition, when treating antibodies in two steps, the antibody and periodic acid and/or its salt are reacted at pH 3 to 7 and a temperature of 0 to 25°C for 0.5 to 10 hours, and then an acidic or basic amino compound and a pH 7
~10, at a temperature of 0 to 40°C for 0.5 to 10 hours.

After the reaction is completed, the reaction product is dialyzed to obtain an antibody with a changed charge. If it is desired to obtain a safer antibody with a changed charge, the antibody may be reduced using a reducing agent such as sodium borohydride, and then dialyzed again.

Since the antibodies obtained in this way have a positive or negative charge, they are difficult to detect using conventional qualitative or quantitative electrophoresis methods, such as cross-immunoelectrophoresis or rocket immunoelectrophoresis. In addition, the electrophoresis time required in the past can be significantly shortened, allowing for rapid analysis.

Whether the charge of the antibody obtained by the method of the present invention has changed to negative or positive can be easily confirmed by comparing it with an untreated antibody using, for example, isoelectric focusing.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Human albumin was injected intramuscularly into several places on the back of the thighs of the front and back legs of rabbits along with a complete Freund's adjuvant band in a conventional manner. After 14 days, the same amount was injected again in the same manner, and after 14 to 21 days. Blood was collected and anti-human albumin serum was prepared. This anti-human albumin serum was salted out with a 50% aqueous ammonium sulfate solution, and the resulting precipitate was added to a 0.1M phosphate buffer (PH
6.8), purified by DEAE/cellulose column chromatography, and then passed through a Sephadex G-200 column to collect the 7S antibody fraction.

The anti-human albumin antibody thus obtained was diluted with 0.1M acetate buffer (PH) to a concentration of 10mg/ml.
6.0) Dissolved to 10ml. After adding 7 mg of ethylenediamine to this anti-human albumin antibody solution and adjusting the pH to 6.0 with acetic acid, 1 ml of 0.1M sodium periodate was added, and the mixture was allowed to react at room temperature for 2 hours. Next, 1 ml of 1M glycerol was added, and after being left at room temperature for 2 hours, it was dialyzed against phosphate buffered saline (hereinafter abbreviated as PBS) overnight, and then
10 mg of NaBH ₄ was added, treated for 2 hours, and dialyzed again.

The resulting antibody whose charge had changed to positive was subjected to electrophoresis for 1 hour in veronal buffer (PH8.6, ionic strength 0.05) using a cellulose acetate membrane in a conventional manner. At the same time, an untreated antibody was also electrophoresed as a control, and after the electrophoresis was completed, it was stained with Ponceau 3R (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in a 3% trichloroacetic acid aqueous solution to a concentration of 0.2% for 10 minutes. The electrophoresis position was confirmed by washing with an acetic acid aqueous solution. While the untreated anti-human albumin antibody moved slightly to the anode side from the sample addition position, the treated antibody moved to the cathode side, clearly changing its charge to positive.

Example 2 Antibody with positive charge obtained in Example 1
10mg of veronal buffer (PH8.6, ionic strength
0.05) was mixed at 60°C to prepare an agar plate with a gel thickness of 1.5 mm. An agar plate was prepared using untreated anti-human albumin antibody in the same manner.

Rocket immunoelectrophoresis was performed using these agar plates as supports. The electrophoresis conditions were as follows: sample holes with a diameter of 3 mm were made in a straight line slightly on the cathode side of the agar plate, 0.5 to 2.5 μg of human albumin was dispensed, and electrophoresis was performed at 10 Vol/cm. As a result, on agar plates containing untreated anti-human albumin antibodies, it took 2.5 hours to complete the formation of a precipitate with 2.5 μg of human albumin, whereas on agar plates containing pretreated antibodies, it took 2.5 hours to complete the formation of a precipitate.
It was completed in 1.5 hours. As a result, it was confirmed that antibodies whose charge changed to positive by the method of the present invention had rapidity in rocket immunoelectrophoresis.

Example 3 IgG fraction (manufactured by Miles, USA) of anti-human IgG-γ chain specific antibody (goat) was adjusted to 20 mg/ml.
It was dissolved in 5 ml of 0.2M acetate buffer (PH5.5). Dissolve 20mg of aspartic acid in this anti-human IgG-γ chain specific antibody and add 0.2M sodium periodate solution.
After adding 0.5 ml and standing at room temperature for 1 hour, the mixture was dialyzed against PBS overnight. Next, 10 mg of NaBH ₄ was added, treated for 4 hours, and then dialyzed against PBS again.

The resulting antibody whose charge has changed to negative is transferred to an Ampholine/PAG plate kit and an LKB multiphase electrophoresis device (manufactured by LKB, Sweden).
Isoelectric focusing was carried out for 90 minutes using the following instructions. After treatment with a protein fixative according to the recipe, the samples were stained with Coomassie, followed by decolorization, and the isoelectric position of the resulting antibody was confirmed.

Similarly, isoelectric focusing was also performed using an untreated anti-human IgG-γ chain specific antibody as a control. Untreated anti-human IgG-γ chain specific antibody has a strong PH of around PH 9.5 and PH 6.5.
In contrast, the treated antibody showed an isoelectric focusing image strongly around PH5.5, and showed an isoelectric focusing image around PH4.5 to 7, and the isoelectric point was clearly on the acidic side. It has migrated. As a result, it was confirmed that the charge of the antibody treated by the method of the present invention changed to negative.

Example 4 Rocket immunoelectrophoresis was carried out in the same manner as in Example 2, except that human albumin was replaced with human IgG, using the antibody obtained in Example 3 whose charge had changed to negative. On agar plates containing untreated human IgG-γ chain-specific antibodies, it took approximately 4 hours for precipitate formation to complete at a concentration of 2.5 μg of human IgG, whereas on agar plates containing treated antibodies, On the plate, it took about 1 hour. As a result, it was confirmed that antibodies whose charge was changed to negative by the method of the present invention had rapidity in rocket immunoelectrophoresis.

As is clear from the above examples, antibodies whose charge has been changed by the method of the present invention have superior properties in immunoelectrophoresis compared to untreated antibodies.

Claims (1)

[Claims] 1. A method for changing the charge of an antibody, which comprises reacting periodic acid or a salt thereof, and an acidic amino compound or a basic amino compound with the antibody.