JP2961986B2 - Method for separating and purifying polyclonal antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antibody which specifically reacts with a certain antigen (hereinafter referred to as "antigen-specific antibody").
And a method for easily separating and purifying an antigen-specific antibody under mild conditions from a polyclonal antibody containing an antibody that does not specifically react with the antigen (hereinafter referred to as an antigen non-specific antibody).

[0002]

2. Description of the Related Art Monoclonal antibodies are antigen-specific antibodies having a single antibody binding site,
It is useful for clinical diagnosis, treatment and purification of substances. As a means for obtaining a monoclonal antibody, there is a production method developed by Koehler and Milstein [Nature, 2
56, 495-497 (1975), European Journal of Immunology, 6, 51
1-519 (1976)], the monoclonal antibody obtained by this method has not only antigen specificity,
It has an excellent feature that it is uniform in class, subclass, binding ability to antigen, and the like. However, in order to produce a monoclonal antibody by this method, various complicated steps are required.In particular, it takes time to screen cells producing an antigen-specific antibody, and special equipment for cell culture is used. There is a problem that high cost is required to perform.

[0003] As a simple method for purifying an antigen-specific antibody to such an extent that it can be applied to clinical examination or immunological research means, there is a method of performing several kinds of adsorption operations to separate and purify a polyclonal antibody. As such a separation and purification method,
For example, there is a method using an affinity gel in which protein G, protein A, an antigen, or the like is bound to Sepharose. However, the method for separating and purifying the antigen-specific antibody using the affinity gel requires eluting an acid or alkaline liquid that is severe for the antigen-specific antibody when the adsorbed antigen-specific antibody is eluted from the gel. After separation and purification, there is a problem that the activity of the antigen-specific antibody may decrease and the cost is high.

Recently, as a method for easily separating antibodies under mild conditions, a method using column chromatography using hydroxyapatite as a carrier has been attempted. For example, JP-A-63-146896 discloses an example in which monoclonal antibodies classified into various classes or subclasses and serum immunoglobulins of mouse, human, rabbit and the like (both IgG and corn fraction 2) are separated. It is shown. In general, antigen specificity and type (class,
Subclass) is a property of antibodies that are completely independent of each other, and there is no correlation between the possibility that antibodies can be separated by antigen specificity and the possibility that antibodies can be separated by type. The antibody separation examples described in the above-mentioned patent publications have examined the possibility that antibodies can be separated by class or subclass, and were originally included in polyclonal antibodies by column chromatography using hydroxyapatite as a carrier. There is no example showing that the antigen-specific antibody contained therein can be separated from the antigen non-specific antibody contained as an impurity.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides a polyclonal antibody containing a non-antigen-specific antibody in addition to an antigen-specific antibody, easily and easily under a mild condition for the antigen-specific antibody.
It is intended to provide a method for separating and purifying an antigen-specific antibody.

[0006]

Means for Solving the Problems The present inventors have made intensive studies to solve the above problems, and as a result, column chromatography using a specific hydroxyapatite aggregate as a carrier separates an antigen-specific antibody in a polyclonal antibody . The present invention has been found to be an effective means for achieving this, and the present invention has been completed. That is, the present invention has a pore volume of 1 to 5 ml / g.
Hexagonal columnar or needle-like hydroxyapatite
Contact aggregates using column chromatography with a carrier, a polyclonal antibody containing an antigen nonspecific antibody
A method for separating and purifying a polyclonal antibody, comprising separating and purifying an antigen-specific antibody in the method.

Hereinafter, the present invention will be described in detail. The object of separation and purification in the present invention is a polyclonal antibody containing an antigen non-specific antibody. A polyclonal antibody is obtained as an immunoglobulin from an antiserum obtained by immunizing an animal with a certain antigen (A). Therefore, an antibody (A) that specifically reacts with the antigen (A) is always contained in the polyclonal antibody as an antigen-specific antibody. On the other hand, before immunizing the animal with the antigen (A), the antibody (X) is produced by an immune response in the animal body, and the animal is immunized with the antigen (A).
In many cases, other types of antigens (B) enter the animal as impurities together with the antigens (A), and thus react specifically with the antigens (B). An antibody (B) that does not specifically react is also produced. The polyclonal antibodies to be separated and purified in the present invention contain these antibodies (X) or antibodies (B) as non-antigen-specific antibodies.

[0008] In the present invention, the animal for obtaining the polyclonal antibody to be separated and purified, and the antigen (A) used for immunizing the animal are not particularly limited. And sheep, rabbits, rats, etc., and antigens (A) include, for example, protein antigens such as bovine serum albumin, bovine gamma globulin, egg albumin, chimney hemocyanin, egg white lysozyme and purified tuberculin protein; dextran, ficoll and lipopolysaccharide. Polysaccharide antigens of sugar; cellular antigens such as sheep red blood cells, lymphocytes and tumor cells; artificial antigens in which hapten groups such as DNP, TNP or BPO are bound to carriers such as proteins, polysaccharides or cells. When polyclonal antibodies are to be separated and purified with high precision according to the present invention, it is preferable to reduce the proportion of antigen (B) contaminated as an impurity when immunizing animals with antigen (A).

The carrier of the column used in the present invention is fine.
Hexagonal or needle-like hydroxy with a pore volume of 1-5 ml / g
It is an agglomerate made of sheapatite, more preferably an agglomerate made of hexagonal columnar or acicular hydroxyapatite having a pore volume of 1.5 to 3 ml / g. Pore volume is 1-5
The use of an aggregate made of ml / g hexagonal columnar or needle-like hydroxyapatite hardly causes column clogging and is suitable for separating a large amount of polyclonal antibody on an industrial scale. Even when a polymer substance such as a protein that is difficult to purify is contained, a sufficiently high flow rate can be obtained under normal pressure, and a long-time treatment can be performed. Here, the pore volume refers to the total volume of the pores contained in the hydroxyapatite aggregate, and the mercury contact angle is 130 °,
It is a value measured by the mercury intrusion method under the condition that the surface tension is 484 dyn / cm. If the pore volume is less than 1 ml / g, it is difficult to obtain a sufficient flow rate, it is easy to cause clogging, and the amount of adsorbed antibody may not be sufficient.
If the pore volume exceeds 5 ml / g, the strength of the aggregate may not be sufficient for long-term use.

The preferred average particle size of the hydroxyapatite aggregate in the present invention is 60 to 100 μm.
It is preferable that the proportion of microaggregates less than m is 1 vol% or less. 1 vol% of micro-aggregates with a particle size of less than 10 μm
When these are exceeded, these micro-aggregates enter the pores, easily cause clogging, and cause a decrease in separation performance,
Further, when the column is filled with the hydroxyapatite aggregate, it may be necessary to remove fine particles present in the supernatant by decantation.

Hexagonal columnar or acicular hydroxyapatite can be produced, for example, by synthesizing hexagonal columnar or acicular crystals of monetite and then hydroxylating by adding an alkali, with a preferred pore volume of 1 to 5 ml / g. In order to obtain a hydroxyapatite aggregate having a structure, when a hexagonal columnar or acicular monetite crystal is subjected to an alkali treatment under heating, KOH or LiO is used as an alkali to be used.
It is preferred to select H.

In the separation and purification method of the present invention, the packing ratio of the column is preferably 10% or less, more preferably 2 to 8%, even more preferably 3 to 5%.
%. If the filling ratio exceeds 10%, it may be difficult to obtain a sufficient flow rate under normal pressure. Here, the filling rate (P) is a value calculated by the following equation.

The filling rate can be easily controlled by the particle size distribution and pore volume of the hydroxyapatite aggregate. In order to prepare a column having such a low packing ratio, the above hydroxyapatite aggregate is dispersed in a solvent, and a slurry having an appropriate concentration may be packed in the column. A preferred solvent for dispersing hydroxyapatite is a solution used for column chromatography, for example, a 1 to 10 mM phosphate buffer.

The separation method of the present invention separates and purifies a polyclonal antibody by column chromatography, and can be specifically carried out, for example, by the following operation. That is, the carrier is packed in a column, and the polyclonal antibody is injected into the column, whereby the polyclonal antibody is adsorbed on the carrier. Thereafter, by supplying an appropriate mobile phase solution, the polyclonal antibody adsorbed on the carrier is eluted in ascending order of distribution to the carrier.
When a polyclonal antibody having a large distribution is separated, it is preferable to elute the polyclonal antibody by changing the composition of the mobile phase solution so as to reduce the distribution to the stationary phase. The separation method of the present invention can be sufficiently carried out under normal pressure, but can also be carried out under pressure if necessary.

Other separation operations may be performed according to a general column chromatography operation, but preferable conditions include, for example, the following. Flow rate: generally varies depending on the size of the column, but it is appropriate to use 0.5 to 1 ml / min for a thin column for analysis and 3 to 5 ml / min for a relatively thick column for preparative analysis.
For a 5 mm, 40 cm long glass open column,
3 ml / min is appropriate. Amount of mobile phase liquid passed: about 10 times the volume of hydroxyapatite aggregate packed in the column. Composition of mobile phase solution: phosphate buffer, more preferably potassium phosphate buffer. Elution conditions: It is preferable to elute a phosphate buffer from 1 to 10 mM to 300 mM by gradually changing the concentration from a low concentration to a high concentration. Specifically, from the time when the elution of the protein starts to increase sharply After the protein elution concentration reaches a maximum, until it is sufficiently lower than the maximum,
It is preferred to keep the concentration of the phosphate buffer constant. When the concentration of the phosphate buffer is changed stepwise and eluted, the antigen-specific antibody can be separated and purified from the polyclonal antibody with higher precision. Column temperature: room temperature. More preferably at 4 ° C. to prevent denaturation of the polyclonal antibody. Polyclonal antibody loading: about 1/10 of the maximum adsorption.

[0016]

EXAMPLES The present invention will be described more specifically with reference to the following examples. [Hydroxyapatite] First, an acicular hydroxyapatite aggregate was synthesized as follows. That is, 1 L of pure water was charged into a 3 L flask equipped with a stirrer and a condenser, heated to 95 ° C., and then 1 L of 0.5 mol / L aqueous solution of calcium chloride and 1 L of 0.5 mol / L aqueous solution of disodium phosphate were added. Was added dropwise over 5 hours to obtain an aggregate composed of monetite of acicular crystals. Subsequently, at the same temperature, 1 mol /
L of potassium hydroxide aqueous solution (300 ml) was added dropwise over 1 hour to obtain an acicular hydroxyapatite aggregate.

The acicular hydroxyapatite aggregate obtained as described above was evaluated for pore volume, particle size distribution and liquid permeability as follows. (Pore volume) The mercury contact angle is 130 ° and the surface tension is 4
Under the condition of 84 dyn / cm, the pore volume of the hydroxyapatite aggregate was measured by a mercury intrusion method. (Particle size distribution) The particle size distribution was measured using a laser diffraction type particle size distribution analyzer LA-500 manufactured by Horiba Seisakusho, and the average particle size was determined. (Liquid permeability test) A hydroxyapatite aggregate was packed into a glass open column having an inner diameter of 12.5 mm and a length of 40 cm so as to have a bed volume of 15 ml, and further added with 10 mM phosphate buffer (pH 6.8) to reduce the total volume. Adjusted to 30 ml. The buffer was passed under normal pressure, and the outflow rate (ml / Hr) per unit time was measured. The results of the pore volume, the average particle size, the liquid permeability test and the packing ratio are described below. Pore volume: 1.8 ml / g Average particle size: 89 μm (Ratio of 10 μm or less: 0.3%) Filling rate: 4.8% Liquid permeability: 240 ml / Hr

[Polyclonal Antibody] The following two kinds of anti-human serum albumin (hereinafter referred to as HSA) antibodies (IgG fraction of rabbit serum) were used. Sample: manufactured by Funakoshi Co., Ltd. (catalog number: NE-0
621-40) Sample: manufactured by the Institute of Medical Biology (catalog number: 127) As a result of separating the above two types of samples by affinity chromatography, in the case of a sample, the chromatogram of FIG. 1 is obtained. A chromatogram of 2 was obtained. The gel preparation method and elution conditions at this time are as follows. ○ Preparation of gel (1) After washing 20 ml of Sepharose 4B gel (trade name, manufactured by Pharmacia) with water,
Add 0 ml of water. 2 g of this gel in 20 ml of water in advance
Of bromine cyanide is added, followed by 2N
NaOH aqueous solution is added in an appropriate amount to adjust the pH value to 10.5 to
While keeping at 11, the sample was left for 8 minutes. (2) The gel treated in (1) above was washed with 1 L of cold 0.1 M carbonate buffer (pH 9.0), and then washed with HSA solution (HSA2).
(0 mg / carbonate buffer 10 ml) was added and left at 4 ° C. overnight. (3) Affinity chromatography was performed by adding 10 g of Tris base to the gel treated in the above (2), leaving it at 37 ° C. for 1 hour, and thoroughly washing it with a phosphate buffer until the pH value reached 7.4. A gel to be used for was prepared. ○ Elution conditions After loading the sample on the column filled with 10 ml of the gel obtained as described above (200 μL for the sample and 50 μL for the sample), first, unadsorbed substances are eluted with a phosphate buffer, Thereafter, the adsorbed substance was eluted with a 0.17 M glycine-hydrochloric acid solution (pH 2.5). 1 and 2
In the above, before the anti-human serum albumin antibody is eluted by passing the glycine-hydrochloric acid solution, an antibody that does not specifically react with albumin (antigen non-specific antibody) is eluted as an unadsorbed substance, so that Indicates that an antigen non-specific antibody is contained. It is known that rabbit IgG has no subclass, and according to the report of Attasii et al., HSA has seven antigenic determinants [European Journal of Biochemistry, Vol. 20 pages (1984)].

[Preparation of column] Inner diameter 10 mm, length 15 cm
The glass column was packed with hydroxyapatite aggregates dispersed in 1 mM phosphate buffer, and the bed volume was reduced to 1
0 cc. After pre-equilibration with 1 mM phosphate buffer (pH 7.5), 2 mg of sample was loaded. [Elution conditions] 1 mM phosphate buffer (referred to as solution A) and 300 mM
Elution was performed by passing a mixed solution adjusted to an appropriate concentration by premixing a phosphate buffer solution (hereinafter referred to as solution B) through a column. The concentration of the mixed solution was adjusted so that the mixing ratio of the solution B was 8% to 10%.
It was changed to 0% and increased gradually. In addition, the pH value of the column at the time of liquid passage is 7.5. [Measurement of Antibody Activity] The eluate was collected at 5 ml / tube by a fraction collector. The antibody activity of each fraction was determined by enzyme immunoassay (EIA method) [Ingvar et al.
Immunochemistry, Vol. 8, p. 871 (1971)
Year)].

[Results on Sample] FIG. 3 shows a chromatogram of the sample. The solid line in FIG.
The absorbance of the eluate at 0 nm, which is an index indicating the protein concentration, and the dotted line indicates the mixing ratio (%) of the solution B.
Represents the concentration of the phosphate buffer passed through the column,
This shows that the concentration of the phosphate buffer was increased stepwise. FIG.
Is HSA and bovine serum albumin (hereinafter BS) as antigens
Called A. ) Is performed, and the wavelength is 490 nm.
FIG. 3 shows the magnitude of antibody activity as a result of measuring the absorbance of the eluate in FIG. From this figure, an antibody specifically reacting with HSA is recognized as four peaks indicated by solid lines, and it can be seen that an antigen-specific antibody can be separated. In particular, fraction N of these four peaks
o. The peaks of 20 or more show a considerably high antibody activity in spite of the low protein absorbance in FIG. It can be seen that a specific antibody is contained. As described above, according to the present invention, it can be seen that antigen-specific antibodies can be easily separated under a mild condition of pH value 7.5.

Furthermore, in this separation example, it can be seen that the antigen-specific antibody which can be obtained as only one fraction when separated and purified by affinity chromatography is divided into four fractions. The present inventors presume that the reason why the antigen-specific antibody can be divided into four fractions may be due to the difference in the antigenic determinants of the antibody. Despite the fact that seven antigenic determinants are present in HSA, only four fractions were separated in this separation example, presumably because the peaks approaching each other overlapped. I have. In addition, HSA and BSA, which are antigens similar to each other,
The activity of the antibody specifically reacting with is shown by the solid line and the dotted line, respectively, but the peak of the solid line and the dotted line appear in almost the same fraction, and the ratio of the antibody activity of the peak of the solid line to the peak of the dotted line is It is considered that the difference between the peaks reflects the similarity and slight differences in the structure of the antigenic determinant.

[Results on the Sample] FIG. 5 shows a chromatogram of the sample. The contents indicated by the solid and dotted lines in this figure are the same as those in FIG. FIG. 6 shows the results of the antibody activity as in FIG. From FIGS. 5 and 6, the fraction No.
The eluate collected at 25 or more contains an antigen-specific antibody of considerably high purity. According to the present invention, the antigen-specific antibody can be easily separated under a mild condition of a pH value of 7.5. We can see that we can do it.

According to the method of separation and purification of the present invention, an antigen-specific antibody can be obtained more easily and at lower cost under mild conditions than the conventional affinity chromatography method. In the present invention, an antigen-specific antibody which can be obtained as only one fraction when separated and purified by affinity chromatography can be divided into a plurality of fractions by selecting appropriate elution conditions.

[Brief description of the drawings]

FIG. 1 HSA antibody manufactured by Funakoshi Co., Ltd. by affinity chromatography (catalog number: NE-0621-40)
9 is a chromatogram obtained when is separated.

FIG. 2 is a chromatogram obtained by separating HSA antibody (catalog number: 127) manufactured by Institute of Medical Biology by affinity chromatography.

[FIG. 3] HSA manufactured by Funakoshi Co., Ltd. by the method of the present invention.
It is a chromatogram when an antibody is separated.

[FIG. 4] HSA manufactured by Funakoshi Co., Ltd. by the method of the present invention.
It is an antibody activity chart at the time of separating an antibody.

FIG. 5 shows an HSA manufactured by the Institute of Medical Biology according to the method of the present invention.
It is a chromatogram when an antibody is separated.

FIG. 6 shows an HSA manufactured by the Institute of Medical Biology according to the method of the present invention.
It is an antibody activity chart at the time of separating an antibody.

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Hideki Kato 1 examiner, Nagoya Research Institute, 1-A, Funami-cho, Minato-ku, Nagoya-shi, Aichi Eiji Takahori (56) References JP 63-146896 (JP, A) JP-A-3-291300 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C07K 16/18 C07K 1/22 BIOSIS (DIALOG) JICST file ( JOIS) MEDLINE (STN) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A hexagonal column having a pore volume of 1 to 5 ml / g.
Is a method for separating and purifying an antigen-specific antibody in a polyclonal antibody containing a non-antigen-specific antibody, using column chromatography using a needle-like aggregate of hydroxyapatite as a carrier.