JP3045646B2 - Chromatography column, serum pretreatment device and serum pretreatment method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chromatography column suitable as a mini-column, a pretreatment device for serum, and a pretreatment method for serum.

[0002]

BACKGROUND OF THE INVENTION Since calcium phosphate compounds such as apatite have an adsorbing effect on proteins, it is already known to use calcium phosphate compound particles as a packing material for chromatography columns. However, it is cumbersome to uniformly fill the column with calcium phosphate-based compound particles, and it has been particularly difficult to fill the mini-column. In addition, in order to perform a serum test utilizing an antigen-antibody reaction, a chromatography column packed with calcium phosphate compound particles is conventionally used. , Cell lysates,
In addition to removing intracellular particles and the like, it was necessary to remove interfering substances that hinder the test, which required a great deal of time and trouble. Therefore, development of a method capable of removing interfering substances in a short time by a simple operation is required.

[0003]

An object of the present invention is to provide a separation column and a pretreatment method for serum capable of solving the above-mentioned problems of the prior art, using a filler which can be easily loaded and removed, and capable of easily removing substances interfering with a serum test. It is intended to provide.

[0004]

SUMMARY OF THE INVENTION The present invention has achieved the above object by using a functional sheet containing a calcium phosphate compound as a column filler. That is, the chromatography column of the present invention has a perforated plate that allows the passage of fluid into the flow path of the chromatography column main body, and one or more functional sheets on the perforated plate, in the flow direction of the fluid. The functional sheet is placed in the orthogonal direction, and the functional sheet is a functional paper or a functional nonwoven fabric containing 10 to 80% by weight of a calcium phosphate compound having a Ca / P ratio of 0.8 to 2.0 . In the periphery of the sheet and in the upstream channel
The lower end of the sleeve fitted to the inner wall of the channel of the column body
It is characterized in that a side leakage prevention ring is installed between them.

[0005] In the chromatography column of the present invention, in order to prevent the sample liquid from leaking to the outside from the periphery of the functional sheet placed on the perforated plate through the space between the sleeve and the column body. A side-leakage prevention ring is installed between the peripheral edge, the lower end of the sleeve, and the inner wall of the flow path of the column body, so that when the chromatography column is assembled, the inside of the column can be sealed by pressing the sleeve from above. Is preferred.

When the column of the present invention is configured as a mini column, the inlet side of the liquid flow path of the sleeve has a relatively large diameter, and the liquid flow path is tapered from the inflow side to the outflow side. Is preferred. Further, in that case, it is preferable to provide a cross-shaped groove passing through the front end of the flow path at the bottom of the sleeve so that a small amount of liquid can quickly diffuse on the functional sheet.

[0007] In the chromatography column of the present invention, the perforated plate is not particularly limited in its material, pore size, etc., as long as it supports the functional sheet and can pass the liquid, and is made of, for example, stainless steel or ceramics. And the like.

The functional sheet usable in the present invention is C
It is a functional paper or functional nonwoven fabric containing 10 to 80% by weight of a calcium phosphate compound having an a / P ratio of 0.8 to 2.0. Specific examples of the calcium phosphate compound include hydroxyapatite, various apatites such as fluorapatite, monobasic calcium phosphate, dibasic calcium phosphate, tricalcium phosphate, tetracalcium phosphate and the like, and these may be used alone or as a mixture. can do. For such functional papers, see Japanese Patent Application Laid-Open No. 2-1.
The detailed description is omitted since it is described in Japanese Patent No. 04623, but there are two types of filler addition methods when producing functional paper: an internal addition method and a coating method. May be. However, it is necessary that the compound is not completely coated in order to exert the adsorption effect of the calcium phosphate compound. It is preferable to use a calcium phosphate compound as a powder composed of secondary particles having an average particle size of 0.1 to 30 μm. Average particle size 0.1
If it is smaller than μm, it will fall along with water from the paper machine net during papermaking, and if it exceeds 30 μm, it will be too heavy and difficult to disperse uniformly. Such secondary particles are
It is formed by agglomeration or sintering of primary particles having a particle size of 100 to 10,000. Further, it is desirable that the particles used as the filler have a large surface area. When the coating method is adopted, it must be applied on a base paper in combination with an adhesive for forming a three-dimensional network structure.

The functional non-woven fabric is prepared by impregnating the non-woven fabric with a slurry of the above-mentioned calcium phosphate compound using a thermoplastic polymer fiber for at least a part of the raw material fiber,
It can be produced by a papermaking technique or a coating method in the same manner as the functional paper.

In the present invention, a functional sheet subjected to high-temperature and high-pressure steam sterilization can be used as the functional sheet. This sterilization is generally performed at a temperature of 100 ° C. (1.1 kg / cm ² , absolute pressure) to 200 ° C. (15.9 kg / cm ² , absolute pressure).
It is preferably performed for 60 minutes. If the temperature is lower than 100 ° C., a sterilizing effect cannot be obtained, and if it exceeds 200 ° C., the paper or nonwoven fabric of the functional sheet may be deteriorated. Sterilization with high-temperature and high-pressure steam is usually performed in an autoclave, and a medical autoclave is particularly preferable as the autoclave. Sterilization conditions are generally 121 ° C. and 2.2 kg / cm.
Conditions of ^{2 to} 132 ° C., 3.0 kg / cm ² apply.

[0011] One or more functional sheets as described above can be placed on a perforated plate as a filler depending on the purpose. By selecting the number of functional sheets, the adsorption ability can be easily adjusted from a weak stage to an advanced stage capable of adsorbing many proteins. By using the column of the present invention, dust, crushed cells, intracellular particles and the like can be removed even with a single functional sheet, and further, by setting the conditions appropriately, an interfering substance to a serum test utilizing an antigen-antibody reaction. In particular, high-density lipoprotein (HDL cholesterol) can be adsorbed and removed.

[0012] Therefore, the chromatography column of the present invention is useful as a pretreatment device for serum. Further, the functional sheet used as a filler in the present invention has a high adsorption ability to proteins, but has a pH of 6.9 to 10
Does not adsorb proteins in serum diluted with
It was found to adsorb cholesterol and oily substances.

Accordingly, the present invention further provides for the
The present invention provides a serum pretreatment method comprising diluting with a buffer solution having a pH of 6.9 to 10, preferably pH 8 to 10, and then injecting the solution through the inlet of the chromatography column of the present invention and passing through a functional sheet. I do. PH buffer
Various substances can be used as long as they are 6.9 to 10, but for example, sodium phosphate buffer, potassium phosphate buffer, tris phosphate buffer, and the like are preferable.

Further, since the functional sheet has a high adsorptivity to proteins as described above, serum proteins (immunoglobulin) can be adsorbed to the functional sheet by bringing serum into contact with the functional sheet. . At that time, antigen-antibody reaction interfering substances such as HDL cholesterol and oily substances also remain on the functional sheet, but it was found that these interfering substances were removed from the functional sheet by washing and dehydrating operations. Therefore, if the serum protein remaining on the functional sheet after the washing and dehydration is eluted using a buffer, the eluate contains an antigen or an antibody. It can be suitably used for various serum tests.

[0015] That is, the present invention further comprises contacting serum with a functional sheet to adsorb serum proteins to the functional sheet, and removing the antigen-antibody reaction inhibitor from the functional sheet by washing and dehydrating. A method for pretreating serum, wherein protein components remaining in the functional sheet are eluted with a buffer having a pH of 6.0 to 6.8, and the eluate obtained by this method is used as a sample for an antigen-antibody reaction. And a method for detecting an antigen or antibody in serum.

The functional sheets used in these methods are the same as those described in detail for the chromatography columns. When a functional sheet subjected to high-temperature and high-pressure steam sterilization is used, an accurate test result can be obtained without the risk of contamination by microorganisms, particularly when used in a test of a culture system. Various buffers can be used as long as they have a pH of 6.0 to 6.8. For example, a sodium phosphate buffer, a potassium phosphate buffer, a trisphosphate buffer, and the like are suitable. It is.

The serum test to which the method of the present invention can be applied is not particularly limited as long as it utilizes an antigen-antibody reaction. Examples thereof include RA test (latex agglutination test), ASO test, CRP test, Examples of such tests include an HBs antigen-antibody test, a syphilis serum test, and an AIDS test, which are also performed on a human dock, an hemagglutination test, a hemagglutination inhibition test, and a bacterial agglutination test.

According to the present invention, there is further provided a serum containing at least one functional sheet, a plastic tube having one end sealed, and a spacer tube inserted into the tube to support the functional sheet. Provide a pretreatment kit. The functional sheet used here is the same as that described in detail for the chromatography column, and may be one subjected to high-temperature and high-pressure steam sterilization. Using this kit, insert a spacer tube into a plastic tube, place a functional sheet on the spacer tube, drop serum onto the functional sheet, centrifuge, wash and dehydrate, and use a buffer solution. The above-mentioned pretreatment of the serum according to the present invention can be performed by eluting the serum.

[0019]

Next, the present invention will be described in more detail with reference to the drawings, but the present invention is not limited thereto. FIG. 1 is an exploded view of a chromatography column showing one embodiment of the present invention, and FIG. 2 is a cross-sectional view showing an assembled state of the column shown in FIG. This chromatography column includes a column main body 1, a sleeve 2, and a holding nut 3. Column body 1
Has an upstream flow path 1a and a downstream flow path 1b, and a step 1c is formed at the bottom of the upstream flow path 1a. A male screw portion 1d is formed on the outer periphery of the upstream flow path 1a. The perforated plate 4, the functional sheet 5, and the lateral leakage prevention ring 6 surrounding the periphery of the functional sheet 5 are inserted into the upstream side channel 1 a. The presser nut 3 is the male thread 1 of the column body 1.
d has a female screw portion 3a and a shaft hole 3b which are screwed into the column body 1 and presses the sleeve 2 toward the step 1c of the column body 1 when screwed into the column body 1. Therefore, when the holding nut 3 is strongly tightened, the side leakage prevention ring 6 and the functional sheet 5 are pressed against the perforated plate 4 (the stepped portion 1c) by the bottom surface 2a of the sleeve 2 inserted into the upstream flow path 1a. And fixed.

In the illustrated chromatography column, the liquid flow path of the sleeve 2 is tapered from the inflow side to the outflow side. Further, the bottom (outflow end) 2a of the sleeve 2 has a cross-shaped groove 2 passing through the tip of the flow path.
b is provided so that the liquid can be satisfactorily diffused.

When a desired number of functional sheets are loaded into the illustrated chromatography column and the column is assembled, the functional sheet 5 is placed on the perforated plate 4 described above.
A lateral leakage prevention ring 6 is fitted around the outer periphery of the functional sheet 5,
The assembly is completed by screwing the presser nut 3 to the column body 1.

In the chromatography column of the present invention, the material of each part is not particularly limited, but the material of the sleeve and the side leakage prevention ring includes, for example, tetrafluoroethylene copolymer, and the material of the column body and the nut. Is stainless steel.
Examples of the perforated plate include a stainless sintered filter.

Reference Example 1 Softwood pulp and hardwood pulp were mixed at a weight ratio of 7: 3 and beaten to a Canadian standard freeness of 360 cc. The pulp is dispersed in water to prepare a concentration of 0.3% by weight of the pulp slurry, the synthesized Ca / P ratio 1.67 by a wet method, a specific surface area of 45 m ² / g, the hydroxyapatite with an average particle diameter of 2 [mu] m 0 0.7% by weight, JIS-
Papermaking was performed according to P8209. The hydroxyapatite content of the obtained product paper is 70% by weight, and the basis weight is 40 g.
/ M ² . Hereinafter, this functional paper is referred to as HAp paper.

Reference Example 2 An aqueous dispersion containing 10% by weight of porous hydroxyapatite granules having an average particle size of 0.55 μm, a specific surface area of 55 m ² and a Ca / P ratio of 1.67, and 0.05% by weight of sodium dodecyl sulfate was prepared. did. After impregnating a non-woven fabric made of polyethylene having a thickness of 0.4 mm with this dispersion, it was dried with hot air at 130 ° C. The hydroxyapatite carrying rate of the obtained nonwoven fabric was 20% by weight. Hereinafter, this nonwoven fabric is described as a HAp nonwoven fabric.

Example 1 Normal human blood was allowed to stand at room temperature for 60 minutes.
and centrifuged at 10 m for 10 minutes to obtain a sample serum. This serum was diluted to twice the volume with 5 mM sodium phosphate buffer (pH 8.0), and 0.5 ml thereof was injected into the chromatography column shown in FIG. One of the two chromatography columns is loaded with a piece of HAp paper and the other is loaded with H
The Ap nonwoven fabric was loaded as two sheets. Two-dimensional electrophoresis was performed on the serum, the serum after passing through the HAp paper, and the serum after passing through the HAp nonwoven fabric, and the obtained electrophoretic images are shown in FIGS. From these figures, the serum protein was hardly changed (almost not adsorbed), but spots 1, 2, 3 and 5 in FIG. 3 showing HDL cholesterol were significantly reduced in FIGS. 4 and 5. It can be seen that the serum after passing through the HAp paper and the serum after passing through the HAp nonwoven fabric became suitable for use in various serum tests.

Example 2 In a chromatography column having the structure shown in FIGS. 1 and 2, one piece of HAp paper was placed on a stainless sintered filter having a pore size of 2 μm to prevent lateral leakage having a thickness and height of 1 mm. Set up the ring, assemble the column, and attach the column to the Waters chromatograph.
(600E, 490 and Pantos U-228). As an initial eluent, 40 mM of 1 mM sodium dihydrogen phosphate buffer (pH 6.8, 0.30 mM CaCl ₂ ) was used.
0 mM disodium hydrogen phosphate buffer (pH 6.8,
High-performance liquid chromatography was performed by a linear gradient method for 5 minutes to 0.11 mM CaCl ₂ ). The flow rate was 1 ml / min, and the detection of each protein was performed by measuring the absorbance at 280 nm.

FIG. 6 shows an elution curve of serum albumin obtained by performing the above-mentioned chromatography using an aqueous solution of 12 μg / 3 μl of bovine serum albumin as a sample solution. FIG. 7 shows an elution curve of lysozyme obtained by performing the above-mentioned chromatography using an aqueous solution of 20 μg / 5 μl of lysozyme as a sample solution. Cytochrome c 20μg / 5μ
FIG. 8 shows an elution curve of cytochrome c obtained by performing the above-mentioned chromatography using 1 aqueous solution as a sample solution.

Example 3 High-performance liquid chromatography was performed under the same conditions as in Example 2 except that 10 pieces of HAp paper were set. FIG. 9 shows an elution curve of serum albumin obtained by performing the above chromatography using an aqueous solution of bovine serum albumin 20 μg / 5 μl as a sample solution. Lysozyme 20μg / 5μ
FIG. 10 shows an elution curve of lysozyme obtained by performing the above-mentioned chromatography using 1 aqueous solution as a sample solution. FIG. 11 shows an elution curve of cytochrome c obtained by performing the above-described chromatography using an aqueous solution of 20 μg / 5 μl of cytochrome c as a sample solution.

According to the above high performance liquid chromatography, when one sheet of HAp paper was used, most of the serum albumin was not absorbed, but when ten sheets were used, approximately half of the load was adsorbed. In addition, lysozyme adsorbed most of the loaded paper even with one HAp paper,
When 10 sheets of paper were used, the entire amount of the load was adsorbed. As for the cytochrome c, the entire load amount was adsorbed by one sheet of HAp paper. For each of the proteins, almost the same results as those obtained when a column packed with spherical hydroxyapatite or spherical fluorapatite were used could be obtained.

Example 4 FIG. 12 is an explanatory view showing an example of the serum pretreatment kit of the present invention. This kit contains 1 plastic tube
0, a spacer tube 11 and a functional sheet 12, and a cap 1
3 are provided.

H produced in Reference Example 1 as a functional sheet
Pretreatment for measurement of Japanese encephalitis antibody in swine blood by hemagglutination was performed using one piece of Ap paper (2.5 cm square). For comparison, filter paper of the same size (AD
VANTEC S1 B) was used. After washing them with water, 0.4
Washed in M sodium phosphate buffer (pH 6.8), washed again with water and equilibrated. Pig serum 1 on each of these
0 μl was added dropwise. As pig sera, those known to be positive by a conventional measurement method and those known to be negative were used. After washing with water for 1 minute, 5 minutes, or 10 minutes, respectively, place it on the spacer tube 11 inserted in the plastic tube 10 shown in FIG. 12, cover with the cap 13, and centrifuge at 1000 rpm for 3 minutes. Threw away. The concentrations of immunoglobulins (IgG, IgM) and cholesterol in the filtrate were measured, the amounts remaining after washing with water were calculated, and the changes over time in the residual ratios are shown in FIG. In addition, the value of the positive serum pretreated by the conventional method was used as the value of the IgG and IgM washings at 0 minute. For the measurement of cholesterol, a test kit [Determiner TC555, Kyowa Medex Co., Ltd.]
It was used.

In FIG. 13, ◎ indicates IgG and IgM remaining on the HAp paper, ○ indicates cholesterol remaining on the HAp paper, ◇ indicates IgG and IgM remaining on the filter paper, and △ indicates remaining on the filter paper. Indicates cholesterol.

Next, 0.40 M sodium phosphate buffer (pH 6.8, 0.12 mM CaCl 2) was placed on HAp paper.
₂ ) 25 μl was added dropwise and centrifuged in the same manner. This operation was performed twice to collect a total of 50 μl of the filtrate. This filtrate was used as a sample for a hemagglutination inhibition reaction test. The operation of this test was in accordance with the infectious disease epidemiological prediction survey and inspection method (May 1986) of the Ministry of Health and Health.

For comparison, the conventional pretreatment method, ie,
Non-specific agglutinin was removed by acetone extraction and addition of goose blood cells. This pretreatment required more than 24 hours. Using the serum obtained by this pretreatment as a sample, the hemagglutination inhibition test was performed.

The antibody titer of the solution separated from the serum using the HAp paper was the same as that of the serum obtained by the conventional pretreatment method in the case of the HAp paper washed for 5 minutes. As is clear from FIG. 13, in the case of filter paper, no immunoglobulin remained after 10 minutes of washing, but in the case of HAp paper, 100% of immunoglobulin remained, and cholesterol was 50% and 10 minutes after washing with water for 5 minutes. Only about 40% remained by washing with water. In addition, non-specific agglutinin was not detected on the HAp paper for one minute after washing with water, so it is considered that it is not adsorbed on the HAp paper. Therefore, the serum pretreated by the method of the present invention is suitable for various tests utilizing an antigen-antibody reaction, and can be tested in a small amount, and the pretreatment of the serum according to the present invention is performed in about 30 minutes. It can be easily performed in a significantly shorter time than the conventional method.

Example 5 The 1 × 3 cm HAp paper produced in Reference Example 1 was washed with water, then washed in a 0.4 M sodium phosphate buffer (pH 6.8), washed again with water, and equilibrated. After drying, put in a glass petri dish and put in an autoclave, 121 ° C, 1.2
The solution was steam-sterilized at atmospheric pressure for 20 minutes, moistened with water immediately before use, and 10 μl of swine serum was dropped thereon. As pig sera, positive and negative samples of Japanese encephalitis virus antibody according to the conventional measurement method were used. After washing with water for 5 minutes, a plastic tube for centrifugation (made of polystyrene, capacity 1.
5 ml) and centrifuged (r = 5.5 cm, 10
00 rpm, 3 minutes). After collecting this filtrate (referred to as filtrate 1), 35 μl of 0.40 M sodium phosphate buffer (pH 6.8) was added to HAp paper, and the mixture was centrifuged in the same manner to obtain a filtrate (hereinafter referred to as filtrate 2). collected. further,
0.40 M sodium phosphate buffer (pH
6.8) An operation of adding 35 μl and centrifuging in the same manner was repeated to obtain a filtrate (hereinafter, referred to as filtrates 3, 4 and 5).
Collected. 25 μl each of 35 μl of these filtrates
l was a sample of the hemagglutination inhibition reaction. As a control,
Serum from which nonspecific agglutinin was removed by a conventional pretreatment method, that is, acetone extraction and goose blood cell addition, was used. The hemagglutination inhibition reaction was operated in accordance with the infectious disease epidemiological prediction survey and inspection technique (May 1986) of the Ministry of Health and Health. The remaining filtrate (10 μl) was subjected to electrophoresis to examine the adsorbed protein. The electrophoresis method used was the method of Manabe et al. For detection, a silver staining method was used.

The antibody titer of the solution separated from the serum using unsterilized HAp paper was the same as the antibody titer of the solution separated from the serum using sterilized HAp paper. When sterilized, as in the case without sterilization, the antibody is
A total of 98% of the filtrate 2 and the filtrate 3 could be recovered. Filtrate 1
Was 0%, and filtrates 4 and 5 each had a recovery rate of 1%. The results of the electrophoresis are shown in FIGS. FIG.
FIG. 15 is an electrophoresis pattern showing a two-dimensional electrophoresis image of Japanese encephalitis virus antibody-positive swine serum. FIG. 15 shows filtrate 2 when non-sterilized HAp paper was used, and FIG. 16 used sterile HAp paper. 5 is an electrophoresis pattern showing a two-dimensional electrophoresis image of a filtrate 2 in a case. When these electrophoresis images were compared, albumin was hardly adsorbed on the HAp paper. The electrophoresis image of the adsorbed serum protein was the same in the case of autoclaving and in the case of not sterilizing. From these results, it was proved that the HAp paper maintained its performance sufficiently even at high temperature and high pressure steam sterilization.

[0038]

According to the present invention, the adsorptive capacity of the filler can be easily adjusted by appropriately selecting the number of functional sheets, and the chromatography having the adsorptive capacity suitable for each use. It can be a column. Further, the chromatography column of the present invention has a pH of 6.9 or more.
By injecting a serum diluted with 10 buffers, for example, a sodium phosphate buffer, a potassium phosphate buffer, a trisphosphate buffer, or the like, a high-density liposome which is an interfering substance in a serum test using an antigen-antibody reaction is injected. Since the protein can be selectively adsorbed and removed, the chromatography column of the present invention is suitable for pretreatment of serum for a serum test, and is useful as a pretreatment device for serum. Also, since a functional sheet is used as a filler for the column, loading and removal are extremely easy. Furthermore, since serum test interfering substances such as cholesterol attached to the functional sheet can be removed to a negligible degree by washing with water, the serum is brought into direct contact with the functional sheet to adsorb serum proteins, and then washed with water. , A serum suitable for a serum test can be easily obtained by eluting a serum protein with a buffer solution. That is, according to the present invention, serum pretreatment can be performed in a very short time with a simple operation. Further, the functional sheet used in the present invention is sterilized by high-temperature and high-pressure steam and maintains its adsorption characteristics, and thus can be suitably used for antibody separation or antibody test in a culture system.

[Brief description of the drawings]

FIG. 1 is an exploded view of a chromatography column showing one embodiment of the present invention.

FIG. 2 is a sectional view when the chromatography column shown in FIG. 1 is assembled.

FIG. 3 is an electrophoresis pattern diagram showing a two-dimensional electrophoresis image of untreated serum.

FIG. 4 is an electrophoresis pattern diagram showing a two-dimensional electrophoresis image of serum after passage through HAp paper in Example 1.

FIG. 5 is an electrophoresis pattern diagram showing a two-dimensional electrophoresis image of serum after passage through HAp paper in Example 1.

FIG. 6 is an elution curve diagram of serum albumin obtained in Example 2.

FIG. 7 is an elution curve diagram of lysozyme obtained in Example 2.

FIG. 8 is an elution curve diagram of cytochrome c obtained in Example 2.

FIG. 9 is an elution curve diagram of serum albumin obtained in Example 3.

FIG. 10 is an elution curve diagram of lysozyme obtained in Example 3.

FIG. 11 is an elution curve diagram of cytochrome c obtained in Example 3.

FIG. 12 is an explanatory view showing one example of a serum pretreatment kit of the present invention.

FIG. 13 is a graph showing changes over time in the residual ratio of immunoglobulin and cholesterol after washing with water performed in Example 4.

FIG. 14 is an electrophoresis pattern diagram showing a two-dimensional electrophoresis image of swine serum positive for Japanese encephalitis virus antibody.

FIG. 15 is an electrophoresis pattern diagram showing a two-dimensional electrophoresis image of the filtrate 2 when non-sterilized HAp paper is used in Example 5.

FIG. 16 is an electrophoresis pattern diagram showing a two-dimensional electrophoresis image of the filtrate 2 when using sterilized HAp paper in Example 5.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Column main body 2 Sleeve 3 Presser nut 4 Eye plate 5 Functional sheet 6 Side leakage prevention ring 10 Plastic tube 11 Spacer tube 12 Functional sheet 13 Cap

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Tetsuro Ogawa 2-36-9 Maenocho, Itabashi-ku, Tokyo Asahi Optical Industry Co., Ltd. (72) Inventor Tsuneo Hiraide 2-36, Maenocho, Itabashi-ku, Tokyo No. 9 Asahi Optical Co., Ltd. (72) Inventor Yoshiya Yoshida 4699-1 Totsuka-cho, Totsuka-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Hiroshi Nakayama 2-7-6 Nishiwaseda, Shinjuku-ku, Tokyo (56) References Special Kaihei 4-232852 (JP, A)

Claims (14)

(57) [Claims] 1. A perforated plate for allowing a fluid to pass through a flow channel of a chromatography column main body, and one or more functional sheets are placed on the perforated plate in a direction orthogonal to a flow direction of the fluid. And the functional sheet has a Ca / P ratio =
0.8 to 2.0 of a calcium phosphate compound of 10 to 8
0% by weight containing functional paper or functional non-woven fabric,
A chromatography column, comprising a lateral leakage prevention ring provided between a peripheral edge of a functional sheet, a lower end of a sleeve fitted into an upstream flow path, and an inner wall of a flow path of a column body. 2. A sleeve having a liquid flow path tapering from an inflow side to an outflow side, and a cross-shaped groove passing through a front end of the flow path is provided on an end face of the sleeve on an outflow side.
A chromatography column as described. 3. The chromatography column body has an upstream flow path and a downstream flow path, and a step portion on which a perforated plate is placed is formed at the bottom of the upstream flow path. The chromatography column according to claim 1, wherein a male screw portion is formed in the column, and the male screw portion and the presser nut are combined. 4. The chromatography column according to claim 1, wherein the functional sheet is subjected to high-temperature high-pressure steam sterilization. 5. An apparatus for pretreating serum, comprising the chromatography column according to claim 1. 6. A method comprising diluting serum with a buffer having a pH of 6.9 to 10, followed by injecting the diluted serum into the chromatography column according to claim 1 and passing through a functional sheet. Pretreatment method for serum. 7. The method for pretreating serum according to claim 6, wherein the serum is diluted with a buffer having a pH of 8 to 10. 8. Serum having a Ca / P ratio of 0.8 to 2.0.
Contains 10 to 80% by weight of a calcium phosphate compound
Serum protein is adsorbed to the functional sheet by contacting with a functional sheet made of a functional paper or a functional nonwoven fabric , and after removing the antigen-antibody reaction inhibitor from the functional sheet by washing and dehydrating, the pH is adjusted to 6.0 to 6.0. 6.8 A serum pretreatment method, comprising eluting a protein component remaining in a functional sheet with the buffer of 6.8. 9. The serum pretreatment method according to claim 8, wherein the buffer is a trisphosphate buffer, a sodium phosphate buffer or a potassium phosphate buffer. 10. The pretreatment method for serum according to claim 8, wherein the functional sheet has been subjected to high-temperature and high-pressure steam sterilization. 11. The method according to claim 11, wherein the serum has a Ca / P ratio of 0.8 to 2.0.
10 to 80% by weight of a calcium phosphate compound
Serum sheet on the functional sheet by contact with a functional sheet made of a functional paper or a functional non-woven fabric , and after removing the antigen-antibody reaction inhibitor from the functional sheet by washing and dehydrating operations, pH 6.0. A method for detecting an antigen or an antibody in serum, wherein a protein component remaining in a functional sheet is eluted with a buffer of ~ 6.8, and the eluate is used as a sample for an antigen-antibody reaction. 12. The method for detecting an antigen or antibody in serum according to claim 11, wherein the functional sheet is subjected to high-temperature and high-pressure steam sterilization. 13. A phosphoric acid having a Ca / P ratio of 0.8 to 2.0.
Functional paper containing 10-80% by weight of calcium compound
Alternatively , a serum pretreatment kit comprising one or more functional sheets made of a functional nonwoven fabric , a plastic tube having one end sealed, and a spacer tube inserted into the tube to support the functional sheet. . 14. The pretreatment kit for serum according to claim 13, wherein the functional sheet is subjected to high-temperature and high-pressure steam sterilization.