JP2794592B2 - Method for concentrating polymer substances in biological fluids - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to the concentration of biological fluids in the fields of biochemistry and medicine in order to analyze biological macromolecules such as proteins, DNA, lipids, etc. In doing so, use a water-absorbing resin that is a crosslinked polyacrylamide that allows the concentration of these components by absorbing only the aqueous solution of the low-molecular components in the solution without deteriorating the target product. The present invention relates to a method for concentrating a polymer substance in a biological fluid as a treatment.

[Conventional technology]

Conventionally, blood and blood have been used for biological research and diagnosis purposes.
It is widely practiced to collect body fluids such as lymph and analyze and identify high molecular components such as proteins therein. On this occasion,
It is well known that the sensitivity and accuracy of analysis are increased by separating and removing water and low molecular components other than the target substance from the test sample in advance and increasing the concentration of the high molecular components.

Conventionally, as described above, several methods have been used to increase the concentration of the polymer component. For example, the one achieved by bringing a test solution into contact with an ultrafiltration membrane under pressure and passing only moisture, for example, Japanese Patent Application Laid-Open No. 60-145069.
And JP-A-61-12284. Further, a water-absorbing resin comprising a crosslinked product of sodium polyacrylate has been conventionally known, and the water-absorbing resin is usually commercially available in a powder state.

[Problems to be solved by the invention]

However, several problems have been pointed out with respect to the above-mentioned conventional technology. For example, for a concentration method using an ultrafiltration membrane, special equipment is required for filtration, and the operation also requires skill. Generally, biological fluids for analysis and identification to which the present invention is directed are several cc.
It is very important that the operation adopted since a large number of samples are subjected to a test in parallel at the same time is simple and reliable. For example, it is easily understood by those skilled in the art that the above-mentioned water-absorbing resin is introduced into a test solution, only water is absorbed, and the target substance is concentrated in the remaining solution. However, the water-absorbing resin cannot be applied to the present invention for the purpose of concentrating proteins and the like, because the water-absorbing resin does not have the ability to select water molecules from polymers such as protein molecules.

An object of the present invention is to solve the above-mentioned problems by using a water-absorbing resin for concentrating biological fluid that absorbs only low-molecular component aqueous solutions while excluding lipids and / or high-molecular substances in the biological fluid. An object of the present invention is to provide a method for concentrating a polymer substance in a biological fluid as a pretreatment for analysis in which a lipid and / or a polymer substance in a body fluid is concentrated by a simple operation without deteriorating.

[Means for solving the problem]

The present invention is configured as follows to achieve the above object. That is, according to the present invention, the equilibrium water absorption ratio in physiological saline is 5 to 10, the water content of the polymer crosslinked product containing 50 to 100 mol% of acrylamide monomer units is 20% by weight or less, and the diameter is 0.1 to 1 mm. A pretreatment for analysis characterized in that a water-absorbent resin consisting of spherical particles is immersed in a biological fluid to be tested, and the gel that has absorbed water is filtered to concentrate proteins and / or DNA with a molecular weight of 10,000 or more. The present invention relates to a method for concentrating a polymer substance in a biological fluid as a method.

Hereinafter, the water absorbing resin for concentrating biological fluid according to the present invention will be described in detail.

The crosslinked nonionic acrylic polymer used in the present invention must have a water absorption capacity of 4 to 20 times its own weight in physiological saline, and particularly, a desirable water absorption capacity is 5 to 5.
The range is 10 times. If the water absorption capacity is 20 or more, the separation characteristics of the aqueous solution of the low-molecular component from lipids and / or high-molecular substances deteriorate, and the high-molecular substances are absorbed into the gel together with water. is there. Further, a low water absorption ratio of the crosslinked resin means that the water absorption per resin unit amount is reduced, and naturally the concentration efficiency is reduced.

In addition, the surface of the water-absorbing resin for concentrating biological fluid must be as smooth as possible in order to minimize the adhesion of the liquid. However, if the surface of the resin is porous, the contained concentrate is discharged out of the system together with the water-absorbing gel, and the recovery efficiency is reduced. The surface of the resin is a portion in which the high molecular substances removed at the time of water absorption are particularly concentrated, and the surface of the resin is separated from the free water portion so as to be redistributed into the liquid before separating the water-absorbing gel. In the meantime, it is desirable that liquid exchange be performed smoothly and promptly and efficiently by liquid flow or diffusion.
If irregularities are present on the gel surface, the irregularities tend to hinder liquid exchange. However, it goes without saying that there may be large irregularities that do not hinder the liquid flow or diffusion.

The water-absorbing resin particles having such a smooth surface are produced by copolymerizing a nonionic acrylic monomer aqueous solution dispersed in an organic liquid with a suitable dispersant with a polyvalent vinyl compound. The polymerization is initiated by adding a water-soluble radical generator or irradiating ultraviolet rays to generate radicals in the monomer aqueous solution. As the water-soluble radical generator, a persulfate, a peroxide, a combination of these with a reducing agent, a water-soluble azo compound, or the like is used.

Specifically, as the water-soluble radical generator, oxidizing agents such as ammonium persulfate, potassium persulfate, hydrogen peroxide, and peracetic acid, and / or sodium hydrogen sulfite,
N, N, N ', N',-tetramethylethylenediamine (abbreviation,
TEMED) or water-soluble azo-based polymerization initiators such as azobisamidinopropane hydrochloride and azobisisocyanovalerate, and other water-soluble radical generators. It is also possible to use them or to use various kinds of the above-mentioned initiators in combination. The water-absorbent resin for concentrating biological fluid used in the present invention is obtained by copolymerizing a polyvalent vinyl compound in an amount of 0.1 to 10 mol% with respect to the acrylic monomer in order to impart a water absorption ratio of luminance.

As the nonionic acrylic monomer used in the present invention, acrylamide is most awarded, but 50 mol%
In the following range, by copolymerizing another nonionic acrylic monomer, hydrogen bonds between polymer chains can be reduced, and the water absorption rate can be improved. Nonionic acrylic monomers used for such purposes include:
Examples thereof include diacetone / acrylamide, N-alkyl / acrylamide, N, N-dialkyl / acrylamide, N, N-morpholine / acrylamide, hydroxyethyl (meth) acrylate, and polyethylene glycol / mono (meth) acrylate.

In addition, the ionic acrylic monomer can be copolymerized within the range of 10 mol% or less. Examples of the ionic acrylic monomer applied for this purpose include acrylamide / alkyl sulfonate, (meth) acryloyloxyalkyl / trialkylammonium salt and the like.

The polyvalent vinyl compound to be copolymerized with these nonionic monomers can be arbitrarily selected from crosslinking agents used in the production of ordinary superabsorbent resins, and N, N-methylenebisacrylamide, polyethylene glycol Examples thereof include (meth) acrylate and triacrylformal.

A technique of adding a small amount of a crosslinking agent that reacts with the polymer functional group to the dispersion medium after the polymerization to increase the crosslinking density on the resin surface is also known as a superabsorbent resin production technique, and can be used in the present invention.

As an organic liquid for dispersing an aqueous monomer solution, all can be used in principle as long as they are substantially insoluble,
Usually, liquid hydrocarbons such as cyclohexane, isoparaffin, kerosene and medium oil are used. As a dispersant added to these organic liquids, a low HLB surfactant, ethyl cellulose, or the like is used. The polymer after polymerization is in the form of a gel containing water, and the water is removed by azeotropic dehydration or by filtration, followed by ventilation drying or heat drying. The dried resin thus obtained is spherical particles, and the surface of the dried resin is smoothed by a liquid flow or diffusion or the like so that the liquid exchange can be performed smoothly and quickly and efficiently. I have.

The diameter of the spherical particles suitable for use in the present invention is 0.1
A range of about 1 mm is desirable. Spherical particles with a large diameter have a slow water absorption rate and a long operation time, whereas spherical particles with a small diameter can form powder in the liquid when immersed in the biological fluid to be tested. Or, there is a drawback that it easily becomes dust and scatters. The operation of concentrating a biological fluid using the water-absorbent resin for concentrating a biological fluid is performed by putting the water-absorbent resin into a subject to be inspected, immersing it for a certain period of time, and filtering off the remaining liquid. It is desirable to perform a stirring operation such as vibration of the container during immersion so that the polymer substance is recovered in the residual liquid as much as possible without remaining on the surface of the water-absorbing gel, but it can withstand practical use even with standing immersion. Suction filtration, centrifugal filtration or the like is used for filtering the residual liquid.
Suction filtration is suitable for large-volume liquid treatment, and centrifugal filtration has little water adhering to the gel surface and high recovery. Coating the resin surface with an antistatic agent or mixing an inert fine powder such as silica, alumina, titania, etc. to coat the surface with the antistatic agent to prevent static electricity that is easily generated in the dried resin particles is required at the time of weighing. Adhesion and scattering are prevented and work becomes easy. The water-absorbing resin used in the present invention has a water content of 20% by weight or less,
Used after drying to 5% by weight or less.

Before drying, the polyacrylamide gel may be washed with water to remove trace amounts of soluble components remaining in the polyacrylamide gel.

[Functions and effects of the invention]

The crosslinked polyacrylamide for biofluid concentration used in the method for concentrating polymer substances in biological fluid according to the present invention has the property of absorbing only low molecular component aqueous solution in a short time while excluding lipids and / or polymer substances. Is a water-absorbing resin having
Such properties can be achieved by imparting an appropriate crosslink density and a specific surface area to a water-absorbent resin composed of a crosslinked nonionic acrylic polymer mainly composed of acrylamide.

That is, by setting the water absorption ratio to 20 or less, the high molecular substance and the like cannot enter the inside of the crosslinked resin, and only the low molecular component aqueous solution is absorbed. As a result of the hydrophilicity of the polymer functional group mainly composed of acrylamide and the high specific surface area of 50 cm ² / g or more, this polyacrylamide gel can complete water absorption in a short time and give a constant concentration rate .

Further, the water-absorbing resin for concentrating biological fluid used in the method for concentrating a high-molecular substance in a biological fluid according to the present invention has a smooth surface, and therefore has little adsorption of high-molecular substances and a high recovery rate. As described above, the lipid and / or the high molecular substance can be separated and concentrated from the aqueous solution of the low molecular component by a simple operation. Therefore, the present invention is used as a pretreatment method for analyzing lipids, DNA, proteins, etc. in biological fluids. Optimal.

The above concentration and separation operation can be performed again on the residual liquid, and the dilution operation can be used in combination.

For example, when removing low-molecular components from biosynthetic high-molecular substances labeled with a radioisotope, the low-molecular components are absorbed and separated into water with the water-absorbent resin for concentrating biological fluid, and the remaining liquid is diluted and concentrated again. By repeating the above, the object can be achieved, and the amount of radioactive substances to be discarded can be reduced by drying the water-absorbing gel.

〔Example〕

Next, an embodiment of a method for concentrating a high molecular substance in a biological fluid according to the present invention will be described. However, the present invention is not limited to the technical idea constituted by the matters described in the claims. Unless otherwise described, it is needless to say that the present invention is not limited to the following embodiments.

Synthesis Example-1 50 equipped with a stirrer, thermometer, reflux condenser, and nitrogen inlet tube
In a 0 ml 5-neck separable flask, add cyclohexane 2
Then, 1 g of ethylcellulose (T-100 manufactured by Hercules) was added and dissolved by heating to 60 ° C., and oxygen was removed by passing nitrogen gas. To 100 g of a 20% aqueous solution of acrylamide, 1 cc of a 10% aqueous solution of N, N-methylenebisacrylamide and 2,2'-azobis (2-amidinopropane)
1.6 g of a 10% aqueous solution of hydrochloride was added to a dropping funnel, and oxygen was removed through nitrogen gas. This was gradually dropped into cyclohexane with stirring to carry out polymerization.

After polymerization at 60 ° C. for 3 hours, the reflux condenser was replaced with an azeotropic water separator, and azeotropic dehydration was performed in a water bath at an external temperature of 80 to 90 ° C. while stirring in a flask. After sufficient dehydration, the polymer particles are separated by filtration and the cyclohexane is removed by drying, whereby a bead-shaped superabsorbent resin can be obtained.

Particles having a diameter of 0.1 to 1 mm obtained by sieving this resin and having a water content of 12% are referred to as Sample-1. Sample-1 absorbed physiological saline 7.6 times its own weight.

Synthesis Example-2 To the separable flask of Synthesis Example-1, 150 g of isoparaffin (Isopar G, exxon) and 0.5 g of sorbitan monooleate were added, heated to 60 ° C. to dissolve, and oxygen was removed by passing nitrogen gas. 15% aqueous solution of acrylamide
A mixture obtained by adding 0.1 g of N, N-methylenebisacrylamide, 0.1 g of triacrylformal and 1.6 g of a 1% aqueous solution of 2,2'-azobis (2-amidinopropane) hydrochloride to 100 g of the mixture was subjected to nitrogen aeration. While stirring, the mixture was gradually dropped to perform polymerization. After polymerization at 60 ° C for 3 hours, polymer particles were filtered off, the oil was washed off with normal hexane, washed with running water for 24 hours, then dried at 105 ° C with ventilation, and spherical particles having a water content of 12.5% were obtained. I got

Particles having a diameter of 0.1 to 1 mm obtained by sieving this are
Call it 2. Sample-2 absorbed physiological saline 6.9 times its own weight.

Synthesis Example-3 200 g of cyclohexane was charged into the same separable flask as in Synthesis Example-1, 1 g of ethylcellulose (T-100, manufactured by Hercules) was added, and the mixture was heated to 60 ° C. to dissolve, and oxygen was removed through nitrogen gas. .

Acrylamide 8g, diacetone / acrylamide 6g, N,
An aqueous solution obtained by mixing 0.5 g of N-methylenebisacrylamide, 0.5 g of diethylene glycol dimethacrylate and 85 g of water was mixed with an aqueous solution of 2,2′-azobis (2-amidinopropane) hydrochloride.
1.6 g of a 0% aqueous solution was added to prepare a monomer solution. After the monomer aqueous solution was aerated with nitrogen, it was gradually dropped into cyclohexane with stirring to carry out polymerization. After completion of the dropwise addition with stirring at 60 ° C., the mixture was left for 3 hours to complete the polymerization, washed with warm cyclohexane, then washed with running water for 24 hours, and then dried at 105 ° C. with ventilation to give a water content of 12%. Water absorption 8.
Three times the resin was obtained.

Particles having a diameter of 0.1 to 1 mm obtained by sieving this are
Called 3. Particles having a diameter of 0.1 mm or less generated by this sieving are referred to as Comparative Sample-1 and particles having a diameter of 1 mm or more are referred to as Comparative Sample-2.

Example-1 Each water-absorbing resin sample was weighed into a 400 ml centrifugal filtration tube, and BSA (bovine serum albumin) was added to 20 mM phosphate buffer.
After injecting 300 mg of a BSA solution in which 1 ppm was dissolved, the solution was allowed to stand for 30 minutes to absorb water, and then subjected to centrifugal filtration at 2000 G for 5 minutes to separate a mother liquor. The concentration of BSA in the mother liquor was determined by liquid chromatography, and compared with the theoretical concentration ratio based on the concentration ratio.
The recovery of A was calculated. The conditions for liquid chromatography are
It is as follows.

 Column; Clear Pack GFS-3.8 × 300 mm solution; 20 mM phosphate buffer (pH 7.0 0.2 M NaCl detector; UV 215 nm 2.0 AUSF injection amount; 100 μ) The results of each evaluation test are shown in Table-1.

Example-2 By the same operation as in Example-1, the standard protein solution of each molecular weight described in Table-2 was absorbed by 40 mg of each water-absorbing resin sample, and the recovery rate was measured. The result is shown in Table-2.

Example 3 A water-absorbing resin sample-1 was weighed into a centrifugal filtration tube having a content of 400 µm, and the amount described in Table 3 was weighed.
After injecting 350 μm of an aqueous DNA solution containing, the mixture was allowed to stand still for 30 minutes to absorb water, followed by centrifugal filtration at 2000 G for 5 minutes to separate the mother liquor. The DNA concentration in the mother liquor was measured from the absorbance at 260 nm, and the DNA recovery was calculated from the comparison with the concentration ratio. Table-Results
3 is shown.

Example-4 By the same operation as in Example-3, DNA of each molecular weight shown in Table-4 was absorbed by 40 mg of each water-absorbing resin sample, and the recovery rate was measured. The results are shown in Table-4.

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Claims (1)

(57) [Claims] (1) an equilibrium water absorption ratio in a physiological saline solution of 5
The water-absorbing resin consisting of spherical particles having a water content of 20% by weight or less and a diameter of 0.1 to 1 mm is immersed in a biological fluid to be tested. Thereafter, a method of concentrating a high molecular substance in a biological fluid as a pretreatment for analysis, comprising concentrating a protein and / or DNA having a molecular weight of 10,000 or more by filtering a gel that has absorbed water.