JPS6044620B2 - Method for separating antigens and antigen-antibody conjugates - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating an antigen and an antigen-antibody conjugate from a solution containing the antigen and the antigen-antibody conjugate.

More specifically, the present invention relates to a method for separating an antigen and an antigen-antibody conjugate, which comprises bringing activated carbon microcapsules having selective adsorption ability into contact with a solution containing the antigen and the antigen-antibody conjugate. Conventionally, various methods have been established and put to practical use for separating unreacted antigen and antigen-antibody conjugates in antigen-antibody reactions in radioimmunoassay methods, but each method has advantages and disadvantages.

Furthermore, at present, adsorption methods are considered to be superior for processing a large number of such samples in a short time in clinical diagnosis and the like. The adsorbents used in this adsorption method include ion exchange resins, talc, florisil, cellulose powder, activated carbon, and dextran charcoal powder. It has the non-specificity of adsorbing conjugates, and also non-specifically adsorbing serum proteins, so it strongly inhibits the adsorption of antigens that are not bound to antibodies, which is not necessarily satisfactory. It wasn't something. The present inventors have conducted various studies on methods for separating antigens and antigen-antibody conjugates from solutions containing unbound antigens and antigen-antibody conjugates generated by their binding in antigen-antibody reactions. As a result,
It has been found that activated carbon microcapsules having the selective adsorption ability as described below specifically adsorb only antigens from the solution, and do not adsorb any antigen-antibody conjugates at all.

Furthermore, it has been found that activated carbon microcapsules having selective adsorption ability are excellent in that even if serum proteins are present in the solution, they do not inhibit adsorption. The present invention was completed based on the above-mentioned various findings, and consists of an antigen and an antigen obtained by bringing activated carbon microcapsules having selective adsorption ability into contact with a solution containing an antigen and an antigen-antibody conjugate. An object of the present invention is to provide a method for separating an antigen-antibody conjugate with high purity and to easily obtain an antigen-antibody conjugate that is extremely useful in immunoassay.

The antigen used in the present invention is as generally defined, and is, for example, a substance that triggers the production of antibodies or that reacts specifically with antibodies.

Furthermore, an antigen-antibody conjugate is similarly defined immunologically, and is, for example, an antigen-antibody conjugate produced by an antigen-antibody reaction between the above-mentioned antigen and an antibody capable of binding to it. An example of the above-mentioned antigens and antigen-antibody conjugates is an antigen-antibody conjugate consisting of an antiserum (anti-pork insulin) that is an antibody obtained by administering the antigen porcine insulin to another species of animal, and porcine insulin. A certain porcine insulin-antibody conjugate, and an antigen-antibody conjugate consisting of porcine glucagon and an antiserum, which is an antibody obtained by administering the antigen porcine glucagon to another species of animal, are porcine glucagon-antibody conjugates. Furthermore, antigens that can be adsorbed to activated carbon and have a molecular weight of about 30,000 or less, such as AC
T.H. ．． TSHl parathyroid hormone, thyroid hormone,
Examples include renin, angiotensin, gastrin, calcitonin, oxytocin, vasopressin, testosterone, aldosterone, progesterone, cortisone, etc. Antigen-antibody conjugates thereof include conjugates with antibodies capable of binding to the above antigens. Further, the solution containing an antigen and an antigen-antibody conjugate in the present invention is a solution containing an antigen-antibody conjugate obtained by reacting a pair of antigens with an antibody as described above. . The activated carbon microcapsules having selective adsorption ability used in the present invention contain activated carbon as a core material and have selective adsorption ability, and are manufactured as follows.

That is, a high molecular weight hydrophilic copolymer obtained by reacting a polymer material having a hydroxyl group with a difunctional or more functional crosslinking agent capable of reacting with the hydroxyl group of the polymer material while forming an ether crosslinking group. Activated carbon microcapsules having selective adsorption ability, characterized by being microcapsules in which activated carbon is dispersed, and a basic solution in which activated carbon is dispersed and a polymer substance having hydroxyl groups are dissolved in the steric voids during coalescence. selective adsorption characterized in that the encapsulation medium is dispersed in an encapsulation medium liquid under stirring, and then reacted with a difunctional or higher-functional crosslinking agent capable of reacting with the hydroxyl group of the polymer material while forming an ether crosslinking group. Method for producing activated carbon microcapsules with functional properties (Patent Application No. 1983-61468)
It is. That is, in the above method, activated carbon is first dispersed to prepare a basic solution in which a polymer substance having hydroxyl groups is dissolved. It may be about 0.5 to 50μ, and the amount used is preferably 10 to 50μ.
It is about 60%. Examples of polymeric substances having hydroxyl groups include polysaccharides such as dextrin, dextran, starch, agar, agarose, cellulose, and polyglycols, methyldextran, ethyldextran, hydroxypropyldextran, methylcellulose, ethylcellulose, and ethylhydroxyethylcellulose. Derivatives of the above-mentioned polysaccharides, products obtained by partial depolymerization thereof, small fragments thereof, polyvinyl alcohol, etc. may be used. It may be about 30% or more of the weight, preferably about 40 to 90%.In addition, in dispersing the activated carbon and dissolving the polymer substance having hydroxyl groups, both are sequentially and simultaneously dissolved in a basic solution, for example, in a basic solution. Solutions of any substance exhibiting these properties can be used, with alkali metal hydroxides such as sodium hydroxide being most suitable, as well as substances such as quaternary ammonium compounds, alkali metal carbonates or It may be dispersed and/or dissolved in a solution of an alkaline earth metal carbonate or an alkaline earth metal hydroxide, or it may be dispersed or/and dissolved in a separate basic solution and then the two may be combined. It is preferable to swell the polymeric substance having a hydroxyl group in advance with water and dissolve it in a basic solution.Then, this solution is dispersed in an encapsulation medium liquid under stirring. A liquid that is immiscible with, non-reactive with, and capable of dissolving the cross-linking agent described below,
Any material that can form W/0 droplets may be used, such as aliphatic hydrocarbons such as hexane and heptane, alicyclic hydrocarbons such as cyclohexane and cyclopeptane, and aromatic hydrocarbons such as benzene, toluene, and xylene. Hydrogen, halogenated hydrocarbons such as chloroform, carbon tetrachloride, dichloromethylene, and cycloethylene, or aromatic hydrocarbons such as dichlorobenzene and chlorobenzene, polyvinyl acetate, acetyl cellulose, acetyl butyl cellulose, and ethylene-acetic acid. 0.1 to 15% dissolution of emulsion stabilizer such as vinyl copolymer, or liquid paraffin such as liquid paraffin, pharmacopoeia paraffin, halogenated paraffin, methyl silicone oil, phenyl silicone oil, methyl phenyl silicone oil and other silicone oils, and if necessary, lanolin derivatives with a low 1LB value, glycerin, ethylene glycol, propylene glycol alkyl esters, sorbitan alkyl esters, polyoxyethylene, etc., which are dissolved in the encapsulation medium liquid. Sorbitan alkyl esters, polyoxyethylene alkyl ethers, polyoxyethylene alkyl esters, polyoxyethylene-polyoxypropylene copolymers, polyoxyethylene alkylphenol derivatives, polyoxyethylene alkyl amides, polyoxyethylene glycerin alkyl esters Examples include those to which surfactants such as, animal oil, vegetable oil, mineral oil, etc. are added, and the encapsulation medium liquid used may be used at least three times the amount of the solution. Further, a bifunctional or higher crosslinking agent capable of reacting with the hydroxyl group of the polymer substance while forming an ether crosslinking group is added to this dispersion and allowed to react. However, it may be a difunctional or higher functional material that can react with the hydroxyl group of the above-mentioned hydroxyl group-containing polymer substance, such as epichlorohydrin, dichlorohydrin, 1,2-3,4-diepoxybutane,
Bisepoxy-propyl ether, ethylene glycol-bisepoxypropyl ether, 1,4-butane-diol-bisepoxypropyl ether, etc. are mentioned, and the amount used is 113% based on the polymer material.
It is preferably about 1 to 3 times to 1 to 2 times. By adding a crosslinking agent to the dispersion in this way,
The polymeric material and crosslinking agent react to yield microcapsules of a high molecular weight hydrophilic copolymer gel containing activated carbon. The structure of the activated carbon-containing microcapsule obtained in this way is such that the adsorbent particles are suitably dispersed in the steric voids in the high molecular weight hydrophilic copolymer gel without their adsorption sites being sealed. Activated carbon microcapsules having selective adsorption ability thus formed can be recovered, washed, and optionally dried according to known solid-liquid separation means. Another example is the following method.

That is, activated carbon microcapsules have crosslinking polymerization selective adsorption ability, in which activated carbon is dispersed in the steric voids in a high molecular weight crosslinked polymer obtained by subjecting an unsaturated monomer having an ethylenic double bond to a crosslinking polymerization reaction with a crosslinking agent. And, a high molecular weight crosslinked polymer characterized in that activated carbon is dispersed in a solution of an unsaturated monomer having an ethylenic double bond and a crosslinking agent, and this is subjected to a crosslinking polymerization reaction in an encapsulation medium liquid. This is a method for producing activated carbon microcapsules having cross-linked polymerization selective adsorption ability in which adsorbent powder is dispersed in steric voids (Japanese Patent Application No. 1983-9855). That is, in the above method, first, an unsaturated monomer having an ethylenic double bond and its crosslinking agent are dissolved in a solvent, but the unsaturated monomer having an ethylenic double bond to be used is one that can be polymerized. Either hydrophilic monomers or lipophilic monomers may be used, such as vinyl chloride, vinyl acetate, vinyl ethers such as vinyl ethyl ether, allyl derivatives such as allyl alcohol and allyl amine, styrene, butadiene, acrylic acid, methacrylic acid, and croton. Examples include hydrophilic monomers and lipophilic monomers such as acid, 5N-dimethylacrylamide, dimethylaminostyrene, and vinylsulfonic acid, and two or more of these monomers may be used in combination. For example, esters of acrylic acid, methacrylic acid, or crotonic acid with polyhydric alcohols, divinylbenzene, acrylic anhydride, methacrylic anhydride, N, N″-dimethylenebisacrylamide, aldehydes such as glyoxal, ketones,
Examples include allyl dihalogenide, alkyl dihalogenide, disulfohalogenide, and polybasic acids, but preferably crosslinking agents include N,N-dimethylenebisacrylamide, divinylbenzene, and glyoxal. . In addition, the amount of crosslinking agent to be used with respect to the unsaturated monomer having an ethylenic double bond is preferably about 0.5 to 50%, and the amount of the unsaturated monomer having an ethylenic double bond dissolved in a solvent is preferably about 0.5 to 50%. The concentration is usually about 5% or more, preferably about 10 to 50%, and the concentration and amount of the unsaturated monomer having an ethylenic double bond and its crosslinking agent are determined according to the desired crosslinking polymerization selective adsorption capacity. The selection may be changed as appropriate to obtain activated carbon microcapsules having the following properties. Activated carbon is further dispersed in the obtained solution. Preferably, the activated carbon is in the form of as fine a powder as possible, and the ratio of activated carbon to be used is relative to the resulting activated carbon microcapsules having cross-linked polymerization selective adsorption ability. Usually less than about 60%, preferably about 10 to 30%, and if a large amount of activated carbon is used, the activated carbon microcapsules that have cross-linked polymerization selective adsorption ability will become brittle, and if a small amount of activated carbon is used, the activated carbon microcapsules will become brittle. Since the adsorption capacity per unit weight of activated carbon microcapsules having crosslinked polymerization selective adsorption capacity becomes low, the amount may be changed as appropriate to obtain a product with high adsorption capacity and strong hardness. Next, this dispersion liquid is dispersed in an encapsulation medium liquid and emulsified.
The encapsulation medium liquid used is an oil-based encapsulation medium. A liquid or aqueous encapsulation medium may be used. When using an oil-based encapsulation medium liquid, the monomers and crosslinking agent mentioned above are hydrophilic, and water or a mixed solution of a hydrophilic organic solvent and water, as well as activated carbon, can be well incorporated during the crosslinking polymerization reaction! Sodium carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, sorbitol, maltodextrin, gelatin, gum arabic as necessary.
In the case where a cross-linking polymerization reaction is carried out by dissolving any water-soluble substance that increases viscosity in an aqueous solution, the encapsulation medium liquid includes the above-mentioned hydrophilic monomer and cross-linking agent. Liquids that are non-reactive, non-reactive with water, and poorly miscible with water are sufficient, such as aliphatic hydrocarbons such as hexane and heptane, and alicyclic hydrocarbon groups such as cyclohexane and cycloheptane. , aromatic hydrocarbons such as benzene, toluene, xylene, halogenated hydrocarbons such as chloroform, carbon tetrachloride, dichloromethane, dichloroethane, perchloroethylene, or halofogenated aromatic hydrocarbons such as dichlorobenzene, chlorobenzene, polyvinyl A 0.1 to 15% solution of emulsion stabilizers such as acetate, acetyl cellulose, acetyl butyl cellulose, and ethylene-vinyl acetate copolymer, or liquid paraffin such as liquid paraffin, pharmacopoeial paraffin, and halogenated paraffin. Examples include silicone oils such as methyl silicone oil, phenyl silicone oil, and methyl phenyl silicone oil, and if necessary, lanolin derivatives with a low HLB value, glycerin, noethylene glycol, propylene glycol, etc., which are dissolved in the encapsulation medium liquid. alkyl esters, sorbitan alkyl esters, polyoxyethylene sorbitan alkyl esters, polyoxyethylene alkyl ethers,
Surfactants such as polyoxyethylene alkyl esters, polyoxyethylene-polyoxypropylene copolymers, polyoxyethylene alkylphenol derivatives, polyoxyethylene alkyl amides, polyoxyethylene glycerin alkyl esters, animal oil, vegetable oil, or mineral oil. When using a low boiling point solvent as the encapsulation medium liquid, a closed system such as a reflux device may be used, and preferably a solvent that is difficult to evaporate such as liquid paraffin, silicone oil, toluene, etc. may be added. , benzene etc. When using an aqueous encapsulation medium, the monomers and crosslinking agents mentioned above are lipophilic, and water-poorly miscible solvents capable of dissolving them, such as perchloroethylene, carbon tetrachloride, benzene, i・
Encapsulation medium in which a lipophilic monomer and a crosslinking agent are dissolved in a single or mixed solvent such as toluene, chlorobenzene, dichlorobenzene, cyclohexane, amyl alcohol, isoamyl alcohol, or butyl acetate to perform a crosslinking polymerization reaction. The liquid does not dissolve the above-mentioned hydrophilic monomers and cross-linking agents, can stably hold their droplets, is non-reactive with them, and has an interface that is poorly miscible with the above-mentioned solvents. Any aqueous solution of an active agent or a hydrophilic protective colloid may be used, for example, a surfactant in water. Solutions include alkylbenzenesulfonic acids such as Nurex R1, Nurex C-1, and Nurex Paste H;
0.01-5%, preferably 0.25-2% aqueous solution of anionic surfactants such as alkyl ester sulfonates of organic dibasic acids such as Ravisol B, higher alcohol sulfate ester sodium salts such as Syntreckis The aqueous solution of the hydrophilic protective colloid is preferably 0.05 to 5% of sodium carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, gelatin, gum arabic, sodium alginate, sorbitol, maltodextrin, etc. 1-2
% aqueous solutions are used, and further examples include combinations of these surfactants and hydrophilic protective colloids.
The amount of the encapsulating medium used is sufficient to emulsify and disperse the dispersion, and may be about twice or more, preferably about 3 to 5 times, the amount of the normal dispersion.
Furthermore, in this emulsified and dispersed system, a cross-linking polymerization reaction is sufficiently caused, and this cross-linking polymerization reaction can be carried out using ordinary means such as ammonium persulfate, potassium persulfate, sodium persulfate, peroxide, etc. hydrogen,
Addition of catalysts such as benzoyl peroxide, hydrogen peroxide, trialkyl aluminum, sodium chlorate, etc., or metals that may be present at different valence levels such as copper, iron or tin, or by irradiation with light or radiation. It can be made to react. The above catalyst may be added directly to a solution containing an unsaturated monomer having an ethylenic double bond and its crosslinking agent, but some crosslinking polymerization reaction will occur until an emulsified and dispersed system is obtained. This does not particularly affect the ability to obtain microcapsules having a certain crosslinking polymerization selective adsorption ability. Furthermore, by sufficiently carrying out the crosslinking polymerization reaction, activated carbon microcapsules with activated carbon dispersed in the steric voids of the gel-like high molecular weight crosslinked polymer, which has crosslinking polymerization selective adsorption ability, are precipitated. It is obtained by separating and washing based on known solid-liquid separation means. Furthermore, in activated carbon microcapsules with selective adsorption ability obtained by various methods such as in-liquid curing coating method, phase separation coating method, and in-liquid drying method, the wall material polymer is a cellulose ester derivative, such as cellulose acetate, Cellulose propionate, cellulose butyrate, cellulose acetate propionate, cellulose acetate butyrate, cellulose acetate phthalate, hydroxypropyl methyl cellulose phthalate, cellulose acetate dibutylamino hydroxypropyl ether,
hydroxypropyl methylcellulose trimellitate,
Cellulose nitrate, cellulose sulfate, cellulose phosphate,
In the case of cellulose-based wall material polymers such as cellulose benzoate, methylcellulose, and ethylcellulose, the microcapsules can be saponified as needed without disintegrating or dissolving them in order to improve their acid resistance, alkali resistance, and solvent resistance. Activated carbon microcapsules with selective adsorption ability and cellulose wall material (
(Japanese Patent Application No. 49-32686).

That is, the microcapsules are dispersed and saponified in an acid or alkali aqueous solution with an appropriately adjusted concentration without disrupting or dissolving the shape of the microcapsules, which is sufficient to remove the ester residue of the cellulose ester derivative from the microcapsules. do. Examples of acids used in this saponification include sulfuric acid, salt acids, lyso acids, and examples of alkalis such as sodium hydroxide, potassium hydroxide,
Examples include ammonia and triethylamine. Furthermore, during this saponification, the temperature, time, and catalyst may be selected as appropriate. Activated carbon microcapsules having selective adsorption ability and having cellulose as a wall membrane thus obtained can be obtained by known solid-liquid separation means. Furthermore, the adsorption ability of the activated carbon microcapsules with selective adsorption ability used in the present invention for methylene blue and the molecular sieving effect for various organic compounds will be described.

(1) Adsorption capacity for methylene blue Activated carbon microcapsules having various selective adsorption capacities obtained in Reference Examples 1 to 5 described later were used.

The amount used is equivalent to 1 g of activated carbon.

The methylene blue aqueous solution used is a 0.01% solution (
0D595mp:5.420) is used. As a measurement method, activated carbon microcapsules having each selective adsorption ability were added to a methylene blue aqueous solution, left to stand for 2 days, and then overnight, and the Oki liquid was measured at 0D595 mμ to measure the adsorption ability for methylene blue. As a control, untreated activated carbon (trade name: Strong Shirasagi, manufactured by Takeda Pharmaceutical Co., Ltd.: Control 1) and Adstar B1 (trade name: manufactured by Ados Kasei Co., Ltd.: Control 2) were used in the same manner.

As a result, as shown in Table 1, the activated carbon microcapsules used in the present invention were compared to the untreated control 1. It can be seen that its adsorption capacity hardly deteriorates compared to activated carbon, and it has an extremely superior adsorption capacity compared to Adstar B1, a commercial product of Control 2.

Also, selection of Reference Examples 1 to 5 used in the present invention! Instead of activated carbon microcapsules having adsorption ability, reference example 6 described below was used.
Using the various activated carbon microcapsules obtained in steps 9 to 9 (equivalent to 1 g of activated carbon), this was added to a 0.01% methylene blue aqueous solution (0D 5 parts mμ; 5.480), left for 24 hours (and then washed). 0D5 of the liquid
The adsorption capacity for methylene blue was measured by measuring at 95 mp, and the adsorption rate was determined.

The results are as shown in Table 2. Reference Examples 6 and 7 are activated carbon microcapsules obtained using the in-liquid curing coating method (orifice method) using a capillary, and the same amount of activated carbon was used. There was a considerable difference in the adsorption rate for each production example, and the adsorption rate was about 90%. Reference Examples 8 and 9 were activated carbon microcapsules obtained using the submerged drying method, and the adsorption rate was approximately 90%. The adsorption rate is approximately 90%, and these activated carbon microcapsules should be used to accurately quantify trace components in the body on the order of several μg to several Ng, but such an adsorption rate may cause measurement errors. However, it is still unsatisfactory as an adsorbent.

In comparison, the activated carbon microcapsules used in the present invention exhibit extremely good adsorption properties and are excellent as an adsorbent for immune reactions in quantifying trace components in living organisms. 1) Molecular sieve effect on organic compounds Activated carbon microcapsules having various selective adsorption capacities obtained in Reference Examples 1 to 5 described later were used.

The amount used is equivalent to 1 g of activated carbon content. The organic compounds to be subjected to molecular sieving used in the measurement are as follows, and all of these are 100% adsorbed on untreated activated carbon. A: Methylene blue
374B: Tuberactinomycin 7
98C: Insulin 5700D:
Egg white lysozyme 14000E: α-chymotrypsin 24500F: Semi-alkaline protease 30000G: Pepsin
35000H: Ovalbumin
450001: Serum albumin 67000J: γ-globulin 156000 Also, as a measurement method, activated carbon microcapsules with selective adsorption ability were
cm column was filled with the above organic compound (A-J
) was charged at a rate of 0.5 ml/min, then thoroughly washed with water, the effluent and the washing liquid were collected and combined, and for organic compound A, 0D5. mμ, organic compound B
-J was measured at 0D280 mμ, multiplied by the liquid volume, totaled, and compared with the absorbance value of the sample to calculate the adsorption rate for each organic compound.

As a result, as shown in Table 3, the various activated carbon microcapsules used in the present invention have a selective adsorption ability to sieve organic compounds of various molecular weights depending on their molecular weights. I can see that it is something.

In the above molecular sieve effect, organic compounds with a high adsorption rate, such as organic compounds showing an adsorption rate of about 50% or more, are referred to as adsorbent organic compounds, and organic compounds with a low adsorption rate or which are not adsorbed, such as organic compounds with a low adsorption rate, such as about 50% or more, for convenience. An organic compound exhibiting an adsorption rate of about 10% or less is called a non-adsorptive organic compound, and has a selective adsorption ability between adsorbable organic compounds and non-adsorptive organic compounds, that is, an adsorption rate of about 10 to 50%. If the molecular weight range of the organic compound is taken as the boundary of the molecular sieve, the results are obtained in Reference Examples 1 to 5 from Table 3 above. The boundaries of the molecular sieve of activated carbon microcapsules with selective adsorption ability are as follows.
That is, the molecular sieve obtained in Reference Example 1 has a molecular sieve of about 34,000~
The boundary of the molecular sieve obtained in Reference Example 2 is approximately 32,000 to 350 (A), and the boundary of the molecular sieve of the substance obtained in Reference Example 3 is approximately 27,000 to 3.
500 degrees, and the boundary of the molecular sieve obtained in Reference Example 4 is about 29,000 to 350 (4), and that of Reference Example 5.
The boundary of the molecular sieve obtained in the above is thought to be about 32,000 to 480 molecular weights. As mentioned above, the activated carbon microcapsules with selective adsorption ability used in the present invention have excellent adsorption ability in themselves, regardless of the manufacturing method, and the molecular weight of the antigen and the antigen-antibody conjugate is There is no limitation in any way as long as there is a difference between the two.

Next, in carrying out the present invention, first, a suitable antigen is selected, and antibodies thereof are raised in a foreign animal by immunological means, and blood is collected to obtain an antiserum.

By reacting the obtained antiserum with the antigen, an antigen-antibody conjugate is produced. In the present invention, activated carbon microcapsules having the selective adsorption ability as described above are brought into contact with a solution containing the antigen and the antigen-antibody conjugate, and this may be done by a batch method or by a column method. They may be brought into contact with each other. In addition, the amount of activated carbon microcapsules with selective adsorption ability to be used will vary depending on the amount of target antigen and various other factors. All you have to do is make the quantity exist. Further, the time, flow rate, etc. during the contact may be selected and changed as appropriate to select conditions under which the antigen can be adsorbed well. Then, if the contact is carried out in a batch method, the bottom liquid may be recovered using a conventional solid-liquid separation method, and if the contact is carried out in a column method, the effluent may be recovered; The resulting solution is a solution that successfully removes only the antigen and contains the antigen-antibody conjugate without loss. The present invention is a method for easily obtaining an antigen-antibody conjugate with high purity, which is extremely useful in immunotherapy as described above.

Next, the present invention will be described in detail with reference to Examples and Reference Examples.
The present invention is not limited to these. Example 1 1 m9 of swine insulin (2 meds/Mg) to 1 m1 of purified water
and add an equal amount of Complete Freund's adjuvant (product name: Complete Freund''
SAdjuvant (manufactured by DIFCO) was added and sufficiently emulsified, and 0.25 ml of this was injected subcutaneously at four locations on the backs of guinea pigs at a total of 1 m five times every 12 weeks to immunize them.

Two weeks after the final injection, blood was then collected from the guinea pigs to obtain antiserum. Separately, 50 μg of porcine insulin, chloramine TlOOpg, and 1 mc1 of iodine isotope (atomic weight 125) used in the above method were dissolved in 0.05 M phosphate buffer (PI (7.5) 1 Tr.t,
After that, 200 pg of sodium thiosulfate was added to stop the reaction, and then 7 g of Cefatex G-1 was added to the solution.
A column (diameter 2 x 20 cm) packed with 00 was charged to obtain purified labeled 1125-insulin.

Next, 1125-insulin (5
0000DPM/ml) 0.7rn11 antiserum (1:5
0.1 ml (corresponding to 45% of 100% 1125-insulin) was mixed in 0.2 ml of phosphate buffer and left at 4° C. for n hours.

Further, activated carbon microcapsules having selective adsorption ability obtained in Reference Example 2 described later were added to the obtained solution, and 3rC
The supernatant was collected by shaking for 13 minutes, and the 112 in the 1125-insulin-antiserum conjugate in the supernatant was
5-Scintillate insulin. It was measured in a counter (hereinafter referred to as residual 1125-insulin value). Similarly, as a control, instead of activated carbon microcapsules having selective adsorption ability, a dextran solution containing activated carbon dispersed therein used in the conventional method was used. Also, 112 above
5-A solution containing only insulin was also not adsorbed using activated carbon microcapsules that have selective adsorption ability and a dextran solution in which activated carbon was dispersed.
5-insulin was measured (hereinafter referred to as residual 115-insulin value).

As a result, the vertical axis shows activated carbon microcapsules with selective adsorption ability (represented by a - knee index) and dextran solution in which activated carbon is dispersed (represented by a - index).
The horizontal axis shows the amount of activated carbon in activated carbon microcapsules with selective adsorption ability and the dextran solution in which activated carbon is dispersed, and the curve when antiserum is used is shown as a solid line (-).
If the curve for the case of only 115-insulin without using antiserum is shown by the broken line (...), then the first
As shown in the figure.

As a result, in the method of the present invention using activated carbon microcapsules having selective adsorption ability, when antiserum is used, the amount of activated carbon used is about 20 to 80 mg.
The theoretical value of residual 1125-insulin (45%
), that is, unreacted 1125-
It can be seen that insulin is efficiently adsorbed and that the conjugate bound to antiserum shows specific adsorption to 1125-insulin, which does not adsorb at all.

Furthermore, in the case of using only 1125-insulin without using antiserum, the amount of activated carbon used is approximately 40 to 80 m9.
It can be seen that the remaining 1125-insulin was almost 0%, that is, 100% adsorbed to 1125-insulin.Also, for the activated carbon-dispersed dextran solution used as a control, antiserum was used. In this case, 1125-
The insulin value is 25%, i.e. this is the unreacted 1
It can be seen that it exhibits nonspecific adsorption to 1125-insulin, adsorbing not only 125-insulin but also 1125-insulin-antiserum conjugates. Furthermore, the adsorption of 1125-insulin alone shows that the residual 1125-insulin value is 25% (that is, the adsorption rate is 52%) on 40 m9 of activated carbon, and 80 mg
The residual 1125-insulin value was 7% (ie, the adsorption rate was 93%), which did not indicate good adsorption. Furthermore, the influence of serum (serum protein) was measured under the above conditions with the passage of time (5, 10, 20, and 3 doses).

The amount of activated carbon used was 100 mg, and the amount of serum used was 0.2 ml. (As mentioned above, the theoretical value of the 1125-insulin value remaining in activated carbon upon addition of antiserum is 45%.

) As a result, as shown in Table 4, the residual 1125-insulin level of the dextran solution in which activated carbon was dispersed increased due to the addition of serum, that is, the adsorption of the antigen 1125-insulin was strongly inhibited by the presence of serum. However, the activated carbon microcapsules with selective adsorption ability used in the present invention have no effect on the adsorption of the antigen 1125-insulin by serum even when antiserum is added or not. It showed specific adsorption to 1125-insulin. As mentioned above, the activated carbon microcapsules with selective adsorption ability used in the present invention are 1125-insulin (i.e., antigen).
1125-insulin-antiserum conjugate (i.e. antigen-antibody conjugate) is not adsorbed at all, and furthermore, its adsorption ability is completely affected by serum (serum protein). It was an excellent product showing specific adsorption to 1125-insulin, which is not regulated.

Example 2 Antiserum was obtained in the same manner as in Example 1 using 1 mg of porcine crystalline glucagon.

Furthermore, porcine crystal glucagon was treated in the same manner as in Example 1 to obtain 112'-glucagon as an index. Antigen 1125-glucagon 0.7 mL and antiserum (
Add 0.1 mL of phosphate buffer (diluted 1:30,000) to 0.1 mL of phosphate buffer.
2 mt, and left at 4°C for 172 hours. Activated carbon microcapsules (activated carbon amount: 20,771 g) having selective adsorption ability obtained in Reference Examples 1 to 5 described later were added thereto, and 3TC13 powder was shaken. The liquid was collected, and 1125-glucagon in the 1125-glucagon-antiserum conjugate in the supernatant was measured.

As a result, these activated carbon microcapsules with selective adsorption ability were able to adsorb well to the antigen V25-glucagon, and no adsorption was observed to the 1125-glucagon antiserum conjugate. The amount of 1125-glucagon was almost 100% of the theoretical value, and these activated carbon microcapsules having selective adsorption ability were excellent in showing specific adsorption to 1125-glucagon. In addition, as a control, in a dextran solution in which activated charcoal was dispersed, the antigen 1
It showed nonspecific adsorption to the antigen 1125-glucagon, which adsorbed not only 125-glucagon but also the 1125-glucagon-antiserum conjugate. Reference Example 1 40 g of dextran with an average molecular weight of 70,000 (manufactured by Seikagaku Corporation) was swollen with 15 mt of water, 75 mt of an aqueous sodium monohydroxide solution was added to dissolve it, and 14 g of activated carbon (Strong Shirasagi) was added to the dextran solution. was uniformly dispersed, and the resulting dispersion was mixed with Lanex (CRODANI).
500ml of pharmacopoeial liquid paraffin containing 1% (manufactured by PPON)
350 r.d. during t. p. m. While stirring, disperse into 5 fine drops, and add 25 mL of epichlorohydrin to this dispersion system.
was added dropwise and continued stirring at 50°C for 2 hours to obtain activated carbon-containing microcapsules in which activated carbon was dispersed in the dextran crosslinked polymer gel. The microcapsules were washed, further washed with water until neutral, and air-dried at 40°C to obtain activated carbon microcapsules having selective adsorption ability in which activated carbon was dispersed in a dextran crosslinked polymer gel.

Reference example 2 60g of 5-dextrin (manufactured by Wako Pure Chemical Industries, Ltd.) in 20ml
The resulting dispersion was swollen in 500 mL of pharmacopoeically liquid paraffin containing 1% of Lanex and stirred (300 r. .p.m.) below 0,
After dispersing into fine droplets, 30 ml of epichlorohydrin was added dropwise under stirring, and a crosslinking reaction was carried out at 50°C for 2 hours to obtain microcapsules in which activated carbon was dispersed in a dextrin crosslinked polymer gel, which was then separated overnight. After thorough washing in the order of n-hexane, acetone, water, 1N hydrochloric acid, and water, it was dried with ventilation at 40°C to obtain activated carbon microcapsules having selective adsorption ability in which activated carbon was dispersed in a dextrin crosslinked polymer gel.

Activated carbon microcapsules coated with cellulose and having selective adsorption ability (the degree of acetylation of cellulose in the wall material is 0.4%).
(below) was obtained.

Reference Example 6 Cellulose acetate (degree of acetylation 54.1%: diacetate)
20g was dissolved in DMSO2OOml, and activated carbon (Strong Shirasagi) was dispersed in this solution.

This dispersion was then dropped into water 20' from a capillary with an inner diameter of about 1 liter to transfer DMSO to the aqueous phase.
By continuing stirring, activated carbon microcapsules coated with cellulose acetate and having selective adsorption ability were obtained. Reference Example 7 Cellulose acetate (degree of acetylation 54.1%: diacetate (book) 2
0g was dissolved in DMSO2OOTfLL, and 20g of activated carbon (Strong Shirasagi) was dispersed in this solution.

This dispersion liquid has an inner diameter of about 1? 0.1M from the capillary of
Add dropwise to phosphate buffer (PH6.O) 15' and add DMSO
was transferred to the buffer phase and continued stirring to obtain activated carbon microcapsules coated with cellulose acetate and having selective adsorption ability. Reference Example 8 30g of ethylene cellulose and 120g of methylene chloride
Dissolve in 0ml and add 90g of activated carbon (Strong Shirasagi) to this solution.
was dispersed.

This dispersion was emulsified and dispersed in a 0.5% aqueous solution of sodium laurylbenzenesulfonate 6'. Further, stirring was continued at room temperature and the solution was stirred. Ren chloride was evaporated, and the precipitate was then collected, washed with water, and dried to obtain activated carbon microcapsules coated with ethylene cellulose and having selective adsorption ability. Reference Example 9 Cellulose acetate (degree of acetylation 54.1%: diacetate 1
50 g was dissolved in 350 ml of 13% aqueous acetone, and 225 g of activated carbon (Strong Shirasagi) was dispersed in this solution.

This dispersion liquid was emulsified and dispersed in 9000 ml of liquid paraffin liquid containing 180 g of Lanex.
Droplets with a diameter of 0p were formed. Further stirring was continued to evaporate acetone, and then the precipitate was filtered out, washed with n-hexane, and dried to obtain activated carbon microcapsules coated with cellulose acetate and having selective adsorption ability.

[Brief explanation of the drawing]

FIG. 1 shows the activated carbon microcapsules having selective adsorption ability obtained in Reference Example 2 and 7 Reference Example 3 acrylamide 200g containing activated carbon dispersed therein. ．． 12 g of N,N''-methylenebisacrylamide and 20 g of ammonium persulfate were dissolved in 500 ml of a 3% sodium carboxymethyl cellulose aqueous solution, and 30 g of activated carbon (strong Shirasagi) θ was dispersed therein. N
1 in which ″,N″-tetramethylenediethylamine was dissolved
% Ranex-containing liquid paraffin at room temperature under stirring in the form of fine droplets, and continued stirring for about 3 hours to form a gel.Activated carbon was dispersed in the steric voids in the high 5 molecular weight cross-linked polymer. Microcapsules having adsorption ability were formed, which were taken out, washed with n-hexane, acetone and water, and vacuum dried at 50 to 600C to obtain activated carbon-containing microcapsules which have crosslinking polymerization selective adsorption ability. Reference example 4 Cellulose acetate (degree of acetylation 54.1%: diacetate)
Dissolve 20g in 24Oml of DMF, uniformly disperse 20g of activated carbon (carbofin) therein, then drop this dispersion into water using an atomizer cup (inner diameter 5Tn!n) to coat it with cellulose acetate. Activated carbon microcapsules with selective adsorption ability were obtained. Next, after washing the microcapsules thoroughly with soft water,
2000 ml of 1% sodium hydroxide aqueous solution was added to this and stirred at about 70°C for 1 hour. After the reaction was completed, the microcapsules were filtered out, thoroughly washed, and activated carbon microcapsules coated with cellulose and having selective adsorption ability The degree of acetylation of cellulose in the wall material was 0.4% or less. Reference Example 5 Cellulose acetate (degree of acetylation 54.5%: diacetate)
40g methylene chloride 1e1 acetone 400ml
Dissolved in a mixed solvent of 40 g of activated carbon (carbofin) was dispersed in this solution, and the resulting dispersion was dissolved in an aqueous solution of 30 g of sodium laurylbenzenesulfonate.
e under stirring at room temperature, and continued stirring to evaporate the solvent.Then, the precipitate was filtered out, washed with water, and dried to obtain activated carbon microcapsules coated with cellulose acetate and having selective adsorption ability. .

Claims (1)

[Claims] 1. Reacting a solution containing an antigen and an antigen-antibody conjugate with a bifunctional or higher crosslinking agent capable of reacting with the hydroxyl groups of a polymeric material having hydroxyl groups while forming an ether crosslinking group on the polymeric material. Microcapsules with selective adsorption ability, in which activated carbon is dispersed in the steric voids of a high molecular weight hydrophilic copolymer obtained by Microcapsules having selective adsorption ability in which activated carbon is dispersed in the steric voids in the high molecular weight crosslinked polymer obtained by subjecting the resulting high molecular weight crosslinking polymerization reaction, and activated carbon having selective adsorption ability using cellulose as a wall material. 1. A method for separating an antigen and an antigen-antibody conjugate, which comprises bringing into contact an activated carbon microcapsule having a selective adsorption capacity selected from the group consisting of microcapsules.