JPH07300499A - Protein separator and method for removing protein using the same protein separator - Google Patents

Abstract

PURPOSE:To provide the subject separator capable of simply. rapidly and precisely separating a protein in a small amount of a sample by installing a membranous filter at the bottom on the outside of a cartridge container having a connecting part of a Luer type structure. CONSTITUTION:This protein separator 1 is obtained by installing a membranous filter 5 at the bottom on the side of a liquid outlet 6 in the interior of a diskllke or a cylindrical cartridge container, made of a plastic and having a liquid inlet 3 and the liquid outlet 6 of a Luer type structure, filling an adsorbent 4 comprising granules having 2-400mum average particle diameter such as hydroxyapatlte on the filter 5 and filling a sealing filter 7 thereonto. Furthermore, this method for simply and rapidly removing a protein in a small amount of a sample is to take the sample containing the protein in an injection syringe, connect the injection syringe to a connecting part 3 of the Luer type structure in a cartridge container 2 of the protein separator l, pass the sample containing the protein through the interior of the cartridge 2, adsorb the protein on the adsorbent 4 and recover the solution from which the protein is removed from the outlet 6 of the Luer slip type outlet 6.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a disposable protein separator and a method for removing a protein from a protein-containing sample using the protein separator, and more particularly, a disc cartridge container filled with a granular adsorbent. The present invention relates to a protein separator and a method for adsorbing and removing a small amount of protein in a sample.

[0002]

2. Description of the Related Art When analyzing a sample in the technical fields of environment, medicine, agricultural chemicals, cosmetics and biotechnology, it is necessary to remove proteins contaminated in the sample as a pretreatment. Conventionally, in order to remove such proteins, a method of precipitating a protein using a saturated ammonium sulfate solution, perchloric acid, trichloroacetic acid, zinc sulfate, etc., a method using a centrifugal ultrafiltration method, or a membrane filtration method is adopted. Has been done. However, in the conventional method, in addition to equipment problems such as the need for equipment such as a centrifuge or an aspirator for suction, the protein contained in a small amount in the sample can be removed efficiently and reliably by a simple operation. However, a technique capable of efficiently removing a small amount of protein by a simple operation has been demanded in the fields of environment, medicine, pesticides, analysis fields and the like.

[0003]

SUMMARY OF THE INVENTION Therefore, the present invention has been made in view of such a demand, and an object thereof is to provide a simple operation without requiring a device such as a centrifuge and a suction aspirator. It is an object of the present invention to provide a protein separator capable of efficiently removing proteins in a sample and a method for removing proteins using the separator.

[0004]

In order to achieve the above object, the inventors of the present invention have made various studies and as a result, have a disc or cylindrical cartridge container having a liquid inlet / outlet of a luer type structure to which a sample injection syringe can be connected. A deproteinizing separator having an adsorbent filled therein was completed. That is, according to the present invention, a membrane filter is laid on the bottom of a disc cartridge container having a luer structure liquid inlet / outlet, and an average particle diameter of 2 μm is provided on the membrane filter.
The present invention relates to a protein separator characterized by being filled with a particulate adsorbent of -400 μm. As a preferred embodiment, (2) a membrane filter is laid on the inner bottom of a disk cartridge having a liquid inlet / outlet of a luer type structure to which a syringe for sample injection can be connected, and an average particle diameter of 2 μm-400 μ is provided on the filter.
The above-mentioned (1) in which the granular adsorbent or the crushed adsorbent of m is filled and a sealing membrane filter is installed at the inlet if necessary.
Item 3. The protein separator according to paragraph (3), wherein the granular adsorbent is at least one selected from the group consisting of hydroxyapatite, fluoroapatite, silica, carbon, activated carbon, anion-based ion exchange resin, and cation-based exchange resin. (1) or the protein separator according to (2), (4) the adsorbent filled portion has an inner diameter of 5 mm-100 mm and a thickness of 1 mm-5.
Item (1) to (4) above, wherein the protein separator according to item (1) or (2) above, and (5) the disk-shaped or cylindrical cartridge in the range of 0 mm, are made of plastic. The protein separator and the like described in any one of 1. The present invention also provides (a) a protein-containing sample in an injection cylinder, which is connected to a luer-type structure connecting portion of a disc-shaped or cylindrical cartridge filled with an adsorbent,
(B) an adsorbent as a preferred embodiment of a method for removing a protein by a protein separator, characterized in that the sample is passed through the cartridge to adsorb the protein on the adsorbent to recover a solution from which the protein has been removed. The base material of (a) is at least one selected from the group consisting of hydroxyapatite, fluoroapatite, silica, carbon, activated carbon, anion-based ion exchange resins, and cation-based exchange resins, and the method for removing proteins according to item (a) above, (C) After removing the protein described in (a) above, an eluent is passed through the cartridge container,
Contacting the adsorbent to which the protein in the container is adsorbed,
Examples thereof include a method for removing a protein by eluting the protein from the adsorbent.

The present invention will be described in more detail below. The feature of the present invention is, firstly, that the protein separator according to the present invention is a disposable container, which is a disc-shaped or cylindrical cartridge container having a connectable luer-type connecting portion of a syringe. Secondly, it is a method of removing proteins by using an adsorbent, and the present invention has been completed based on such an idea.

The disc-shaped or cylindrical cartridge container according to the present invention has a shape and structure as illustrated in FIGS. 1, 2, 3 and 4, and the liquid inlet / outlet has a luer structure. It is possible to connect an injection cylinder for sample injection. 1 and 3 show that the inlet is a female luer type and the outlet is a luer slip. The size of the cartridge container is not particularly limited, but a container having an adsorbent filled portion having an inner diameter of 5 mm-100 mm and a thickness of 1 mm-50 mm is used.

Since the cartridge container is disposable, a material such as plastic having good biodegradability is suitable. The pore size of the membrane filter installed at the bottom of the adsorbent filling section inside the cartridge container is about 0.1.
The thickness is preferably 30 μm to 30 μm to prevent the adsorbent from being washed away and also to remove large debris such as cell debris. It has been found that the combined use of the adsorbent and the membrane filter synergistically increases the protein removal effect. For the membrane filter, it is preferable to use a hydrophilic material when removing the water-soluble protein and a hydrophobic material when removing the hydrophobic protein. Preferred materials are olefinic polymers, polyvinylidene fluoride, cellulose acetate and the like for water-based and ionic-based materials, and polypropylene, polytetrafluoroethylene and the like for non-aqueous-based materials.

If the adsorbent adsorbs a protein,
Hydroxyapatite, fluoroapatite, C ₄ silica, C ₈ silica, C ₁₈ silica, but not limited to
C ₄ polymer, _{C 8 polymer, C 18} polymer, graphite carbon, activated carbon, anionic ion exchange resins, can be used a cationic ion exchange resin. As the adsorbent, granules or crushed particles having an average particle size of 2 μm to 300 μm are used, and a preferable average particle size is 2 μm-3
It is 00 μm. Particularly preferred average particle size is 10 μm −1
It is 00 μm. Hydroxyapatite has the chemical formula Ca ₁₀
It is represented by (PO ₄ ) ₆ · (OH) ₂ , and fluoroapatite is represented by the chemical formula Ca ₁₀ (PO ₄ ) ₆ · F ₂ . The filling rate of the adsorbent in the cartridge container (the volume occupied by the adsorbent with respect to the total volume in the container) is 50% or more,
A preferable filling rate is 70% or more, and a particularly preferable volume rate is 90% or more. A membrane filter or quartz wool is installed at the upper entrance of the packing section to prevent the adsorbent from dissipating.

FIG. 1 is a sectional view taken along the line AA ′ in FIG. 2 of the disc-shaped protein separator (1) of the present invention. Disk (2)
There is a luer lock type inlet (3) at the top of which a syringe can be connected. The disk is filled with an adsorbent (4) having a particle size of 2 μm to 400 μm. A membrane filter (5) having a pore size of 0.2 μm to 2 μm is laid on the bottom of the disk so that the adsorbent does not flow out. The lower outlet (6) of the disc has a luer slip type connection to which a needle can be connected. In the deproteinization treatment, a small amount of sample in a syringe is injected from the inlet (3), the sample is brought into fluid contact with the adsorbent inside the disc, and the deproteinized sample is eluted from the outlet. Reference numeral 7 in FIG. 1 denotes a sealing filter. The disc 2 can be manufactured by integrating the upper half portion and the lower half portion by fusion bonding or any other method. FIG. 2 shows a plan view of the disc type protein separator seen from above. FIG. 3 is a cross-sectional view taken along the line BB ′ of FIG. 4, which is a shape effective for increasing the adsorption of proteins in the disc-shaped protein separator of the present invention. Basically, it is the same as the disc type, but since the adsorbent can be filled more than the disc type, the deproteinization amount of the cylindrical sample can be increased. The numbers in the figure are the same as those in FIG. FIG. 4 is a plan view of the cylindrical protein separator seen from above.

Example 1 A membrane filter having a pore diameter of 0.45 μm was laid on the bottom of an inner diameter of 25 mm inside a plastic cartridge container showing the sectional structure taken along the line AA ′ of FIG. As the above, a protein separator was prepared in which hydroxyapatite granules having an average particle size of 30 μm were filled and enclosed. The filling amount of the absorbent was 0.25 mg, and the filling rate was 92%. To evaluate the performance of this protein separator, bovine serum albumin 10 mg / ml and lysozyme 1.2 were used as acidic proteins.
mg / ml and basic protein cytochrome C 1.9 m
A mixed aqueous solution of g / ml was prepared as a sample. Fig. 5 shows the results of high performance liquid chromatography analysis using a hydroxyapatite column on the mixed aqueous solution before deproteinization. The chromatogram in Fig. 5 shows bovine serum albumin,
The measurement results are shown as peaks for lysozyme and cytochrome C. As analysis conditions for high performance liquid chromatography, flow rate 1 ml / min, UV detection wavelength 280 n
adopted m. The above-mentioned sample was sucked with a syringe, and the syringe was connected to the above-mentioned protein at the luer structure portion and passed.
When the eluate was collected and analyzed, the results shown in Table 1 and FIG. 6 were obtained. FIG. 6 is a chromatogram obtained by analyzing the eluate by high performance liquid chromatography. The conditions of a flow rate of 1 ml / min and an ultraviolet ray detection wavelength of 280 nm were adopted for the analysis, and the sensitivity of the detector was set before deproteinization. This is four times higher than that of the sample analyzed. In the chromatogram of FIG. 6, the protein was adsorbed to the adsorbent and the entire amount was removed from the sample, as is clear from the disappearance of the peaks of the three proteins present in the chromatogram of FIG. Next, 0.4 mol of sodium phosphate solution was passed through the separator having adsorbed the protein using a syringe to elute the protein. The result of analyzing the eluate by high performance liquid chromatography is shown in FIG. The peaks of bovine serum albumin, lysozyme, and cytotrochrome C appeared, which proved that the protein was eluted from the adsorbent and could be recovered. The adsorption amount (mass capacity) of bovine serum albumin on the apatite granule adsorbent in the cartridge is 0.62 m.
and the amount of adsorbed Titrochrome C was 2.5 mg.

Example 2-6 A protein separator having the same structure as in Example 1 was prepared except that hydroxyapatite granules having an average particle size of 5 μm, 10 μm, 100 μm, 200 μm and 350 μm were filled in a cartridge at the filling rate shown in Table 1. Prepared. Using these protein separators, the effect of removing proteins was evaluated under the same conditions and operations as in Example 1. The results are shown in Table 1.

Comparative Example 1 A protein separator having the constitution shown in Table 1 was prepared using hydroxyapatite granules having an average particle size of 400 μm. The protein removing effect was evaluated in the same manner as in Example 1, and the results are shown in Table 1.

Example 7 A membrane filter having a pore diameter of 0.2 μm is laid at the bottom of an inner diameter of 25 mm inside a plastic cartridge container having a sectional structure shown in FIG. 1, and an average particle diameter of 70 μm is used as an adsorbent on the membrane filter.
A protein separator was prepared in which m graphite carbon granules were filled and enclosed. The filling amount of the adsorbent was 0.031 mg, and the filling rate was 83%. In order to evaluate the performance of this protein separator, a mixed solution containing 4 mg / ml of human serum albumin, 1 mg / ml of ribonuclease A and 1 mg / ml of α-chymotrypsin hydrophobic protein was prepared. FIG. 8 shows the results of analysis by mixed solution high performance liquid chromatography. In the figure, peaks showing the existence of the above-mentioned three kinds of proteins appear. The mixed solution was used as a sample and sucked by a syringe, and the syringe was connected to the luer structure of the protein separator to pass the solution. When the eluate was collected and analyzed, the results shown in Table 2 and FIG. 9 were obtained. According to FIG. 9, the peak of the chromatogram showing the presence of the protein disappears in FIG. 8, which indicates that the entire amount of the protein was specifically removed. Furthermore, it was possible to remove the organic substances existing in the sample before the deproteinization treatment.

Examples 8-12 and Comparative Example 1 As shown in Table 2, the same procedure as in Example 1 was repeated except that graphite carbon granules having different average particle diameters were used and packed at the packing ratio shown in the table. Was prepared, and the protein removal effect was evaluated under the same conditions and operations as in Example 7, and the results are shown in Table 2.

[Table 1]

[Table 2]

[0015]

EFFECT OF THE INVENTION The protein separator according to the present invention has a unique structure filled with an adsorbent, can remove a small amount of protein by a simple operation, and can be used as a pretreatment for analysis of a small sample. It is useful as an operation.

[Brief description of drawings]

1 is a cross-sectional view of the protein separator according to the embodiment of the present invention taken along the line AA ′ in FIG.

FIG. 2 is a plan view of a protein separator according to an embodiment of the present invention.

FIG. 3 is a sectional view taken along line BB ′ of FIG. 4 of the protein separator according to the embodiment of the present invention.

FIG. 4 is a plan view of a protein separator according to an embodiment of the present invention.

FIG. 5 is a diagram showing analysis results by high performance liquid chromatography of a sample used in the protein removal method according to one example of the present invention.

FIG. 6 is a diagram showing the results of high-performance liquid chromatography analysis of a sample from which proteins have been removed by the protein removal method according to one embodiment of the present invention.

FIG. 7 is a diagram showing the results of analysis by high performance liquid chromatography of proteins eluted and collected from the adsorbent according to one example of the present invention.

FIG. 8 is a diagram showing analysis results by high performance liquid chromatography of a sample used in the protein removal method according to one example of the present invention.

FIG. 9 is a diagram showing a chromatogram of a sample obtained by removing a protein using the protein removing method according to one example of the present invention.

[Explanation of symbols]

 1 ・ ・ ・ ・ Protein Separator 2 ・ ・ ・ ・ Disc 2 '・ ・ ・ ・ Cylinder 3 ・ ・ ・ ・ Lure lock type inlet 4 ・ ・ ・ ・ Adsorbent 5 ・ ・ ・ ・ Membrane filter 6 ・ ・ ・ ・ Lure Slip type outlet 7 ... Sealing filter

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07K 1/22 G01N 30/14 A 30/60 A 33/48 A

Claims (2)

[Claims] 1. A membrane filter is laid on the bottom of the disc-shaped or cylindrical cartridge container having a luer-type structure on the outlet side, and an average particle diameter of 2 μm-40 is provided on the filter.
A protein separator characterized by being filled with an adsorbent of 0 μm. 2. A protein-containing sample is taken in an injection cylinder, and the injection cylinder is connected to a luer-type structural connection part of a disk-shaped or cylindrical cartridge container filled with an adsorbent, and the protein-containing sample is passed through the cartridge. A method for removing a protein by a protein separator, which comprises lysing, adsorbing the protein on an adsorbent, and collecting a solution from which the protein has been removed.