JPH0975430A - Method for removing contaminant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simplify removal of microorganisms, etc., which have contaminated a cell culture medium and physiologically activating substance by forming an unsoluble carrier while an antibiotic is used as a constituent. SOLUTION: An unsoluble carrier is formed from a film, fibers, etc., in the form of Petri dish or the like, for example from polytetrafluoroethylene. As constituent of the carrier, an antibiotic is used consisting of penicillin, cephem monobactum or tetracyclin antibiotic. Binding of the antibiotic with the carrier is made by the carrier binding method, bridging method, comprehensive method, graft method, etc., the first named method being used usually. It may also be accepted that the unsoluble carrier is formed from magnetic particles.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a cell (mainly suspension cell and semi-adsorptive cell) suspension using an insoluble carrier characterized by containing an antibacterial agent as a constituent. The present invention relates to a method for adsorbing and removing a microorganism (including virus and mycoplasma) lipopolysaccharide contaminated with a physiologically active substance solution, and a nucleic acid. Further, the present invention relates to a method for detecting and quantifying microorganisms, lipopolysaccharides and nucleic acids in cell suspensions and physiologically active substance solutions, using an insoluble carrier characterized by containing an antibacterial agent as a constituent. Secondly, it relates to a method for adsorbing and removing microorganisms, lipopolysaccharides and nucleic acids in a cell suspension and a physiologically active substance solution using magnetic particles characterized by containing an antibacterial agent as a constituent. Further, the present invention relates to a method for detecting and quantifying microorganisms, lipopolysaccharides and nucleic acids in cell suspensions and physiologically active substance solutions, using magnetic particles characterized by containing an antibacterial agent as a constituent.

[0002]

2. Description of the Related Art When a cell culture medium is contaminated with microorganisms, (1) the microorganisms contaminating the cell culture medium are identified. (2) Isolate contaminated cell culture media from other cell culture media. (3) Incubator and l
Clean the aminal flow hood with disinfectant,
Check the HEPA filter. (4) In order to prevent the cultured cells from being killed by the antibacterial agents (antibiotics and antifungal agents), a doserisponse test is performed. Contamination microorganisms are removed by following the procedure.

Next, antibacterial agents (antibiotics and antifungal agents)
In order to determine the toxic concentration of Escherichia coli and remove microbial contamination of the cell culture medium, (1) cells are dispersed in a medium containing no antibacterial agent, and the number of cells is counted and diluted to a concentration for cell passage.
(2) The cell suspension was added to the multiwell culture.
Dispense into plate or small flask. F
Anti-bacterial agents such as unzone, tyrocine, etc. at a constant concentration of multi well culture plate
Dispense in each well of e or each flask. For example, in the case of Fungizone, 0.25, 0.50,
Concentrations of 1.0, 2.0, 4.0, 8.0 ug / ml are selected. (3) multi wellculture
The cells in the plate or flask were observed every day during the culture, and the cell contents were lost, the vacuoles of the cells appeared, the confluency of the cells was reduced,
Observe signs of toxicity such as rounded cells. (4)
The harmful antibacterial agent concentration is determined, and the contaminated cells are subcultured for 2 to 3 passages in a medium containing an antibacterial agent at a concentration of 1 to 1/2 of the concentration. (5) The cells subjected to the operation of (4) are subjected to primary culture in an antimicrobial-free medium. (6) The cells subjected to the operation of (5) are subcultured for 4 to 6 passages in the medium of (4) to confirm the removal of contamination. I have to take the steps. That is, the conventional method has a drawback that the operation procedure for removing the contamination is extremely complicated.

Lipopolysaccharide
ide: LPS) is a toxic substance that constitutes the outer membrane of the cell wall of Gram-negative bacteria and causes fever and shock symptoms when entering the living body. It is not allowed to mix lipopolysaccharide into medical products such as injectables and medical devices such as artificial kidneys. In order to remove lipopolysaccharide from pharmaceuticals and the like, methods such as ion chromatography, gel chromatography and affinity chromatography have been proposed. However,
These methods are not practical methods because the equilibrium adsorption amount is small and it is difficult to set conditions such as pH, ionic strength, and regeneration conditions.

In the production of physiologically active substances by genetic engineering, genes are incorporated into Escherichia coli, Bacillus subtilis, cultured animal cells and the like to cause the bacteria or cells to produce physiologically active substances. In this case, a problem is the separation of the produced physiologically active substance from the gene (nucleic acid) and endotoxin produced by Gram-negative bacteria such as Escherichia coli.

[0006]

The object of the present invention has been made in view of the above circumstances, and the procedure for removing microorganisms, lipopolysaccharides and nucleic acids contaminated with a cell culture medium and a physiologically active substance solution is extremely simple. Another object is to provide a suitable carrier. Another object of the present invention is to provide a method for detecting and quantifying lipopolysaccharide, a microorganism and a nucleic acid using the carrier. Furthermore,
It is an object of the present invention to provide a method for removing lipopolysaccharide, a microorganism and a nucleic acid, and a method for detecting and quantifying a lipopolysaccharide, which uses magnetic particles characterized by containing an antibacterial agent according to the present invention as a constituent component.

[0007]

[Means for Solving the Problems] To achieve the above object,
As a result of diligent studies, the inventor of the present invention has found that (1) an insoluble carrier characterized by containing an antibacterial agent as a constituent easily removes microorganisms contaminated with a cell suspension and a physiologically active substance solution. . (2) An insoluble carrier characterized by containing an antibacterial agent as a constituent component easily removes lipopolysaccharide contaminated with a cell suspension and a physiologically active substance solution. (3) An insoluble carrier characterized by containing an antibacterial agent as a constituent component easily removes nucleic acid contaminated with a cell suspension and a physiologically active substance solution. And completed the present invention. The carrier relating to the present invention is
It has a large adsorption capacity and can easily set conditions such as pH, ionic strength, and regeneration conditions, and can easily adsorb microorganisms, lipopolysaccharides, and nucleic acids. Furthermore, by using the carrier, the substance can be quantified. Furthermore, the carrier can be easily regenerated by washing with the solvent according to the present invention.

The insoluble carrier of the present invention means a carrier to which the antibacterial agent of the present invention can be bound or blended. The shape of the insoluble carrier may be a membrane, a fiber, a hollow fiber, a granular material or the like. Specific examples of the insoluble carrier of the present invention include (1) polytetrafluoroethylene, (2) polyethylene,
(3) polyisoprene, (4) polybutadiene, (5)
Polystyrene, (6) polymethyl acrylate, (7) polyvinyl chloride, (8) cellulose resin (regenerated cellulose, acetyl cellulose, nitrocellulose, etc.),
(9) Polyethylene terephthalate, (10) Polyacrylonitrile, (11) Polyvinyl alcohol, (1
2) Aminopropyl glass beads and the like can be mentioned, but the present invention is not limited thereto. Specific product names include petri dishes, films, test tubes, tubes, syringes, catheters, flasks, gels and the like.

The binding method of the antibacterial agent and the carrier according to the present invention may be a carrier binding method, a cross-linking method, an encapsulation method, a graft method or the like, but the carrier binding method is usually used. The carrier and the antibacterial agent may be directly bonded or may be bonded via a spacer, in order to prevent nonspecific adsorption of cells and physiologically active substances to the carrier according to the present invention, activation of complement, etc. As a spacer, it is desirable to introduce a hydrophilic group in advance. As a method of binding the carrier and the antibacterial agent, covalent bond, ionic bond, hydrophobic bond, coordinate bond and the like can be considered, but covalent bond which is less likely to peel off the antibacterial agent is desirable. Examples of covalent bond include amide bond, ester bond, ether bond, amino bond,
Examples thereof include imino bond, sulfide bond and disulfide bond. A desirable method for binding the antibacterial agent or the spacer to the carrier is to activate the carrier with a cyanogen halide, an epoxy compound, a halogeno organic halide, dialdehyde, benzoquinone, etc., and then bond the antibacterial agent or the spacer. Epoxidation method, reductive amination method, thiol activation method and the like can be considered as the method of binding the ligand via the spacer. Further, a kneading method in which an antibacterial agent is kneaded into a carrier is also included in this method. Furthermore, a method of applying an antibacterial agent to the surface of the carrier is also included in this method.

[0010]

【The invention's effect】

(1) An insoluble carrier characterized by comprising an antibacterial agent according to the present invention as a constituent component can efficiently adsorb and remove microorganisms, lipopolysaccharides and nucleic acids contaminated with cell suspensions and physiologically active substance solutions. It will be possible. (2) Detection and quantification of microorganisms, ribopolysaccharides and nucleic acids contaminated with cell suspensions and physiologically active substance solutions, using an insoluble carrier characterized by comprising an antibacterial agent according to the present invention as a constituent. It becomes possible. (3) The magnetic particles, which are characterized in that the antibacterial agent according to the present invention is a constituent component, can efficiently adsorb and remove microorganisms, lipopolysaccharides, and nucleic acids that contaminate cell suspensions and physiologically active substance solutions. It will be possible. (4) Detection and quantification of microorganisms, lipopolysaccharides, and nucleic acids contaminated with cell suspensions and physiologically active substance solutions, using magnetic particles characterized by containing an antibacterial agent according to the present invention as a constituent component It becomes possible.

[0011]

The present invention will be described in more detail with reference to examples.
The present invention is not limited to the embodiments. << Embodiment 1. -Preparation of polymyxin B-immobilized glass membrane-1) Poring glass membrane (Na ₂ O-B ₂ O manufactured by Corning Incorporated)
_(3- SiO ₂ type glass film) is immersed in a 0.5% aqueous solution of γ-glycidoxypropyltrimethoxysilane and stirred for 2 hours to obtain silanol groups on the surface of the porous glass and γ-glycidoxypropyltrimethoxysilane. Reacted with silane. 2) Remove the water content of the porous glass membrane subjected to the operation of 1),
It was dried at 120 ° C. for 3 hours. 3) Perform the operation of 2) in a solution prepared by dissolving polymyxin B at a ratio of 2% in a carbonate buffer of pH 9.5 at 30 ° C., immerse the porous glass membrane returned to room temperature, gently shake, and The reaction was carried out at ℃ for 24 hours. 4) The porous glass membrane subjected to the operation of 3) was immersed in purified water, gently shaken and washed. 5) The porous glass membrane subjected to the operation of 4) was immersed in a 0.2 M NaOH solution dissolved in a 20% ethanol solution, gently shaken and washed. 6) The porous glass membrane subjected to the operation of 5) was immersed in pyrogen-free purified water, and while the purified water was being exchanged, it was gently shaken and washed until the pH of the washing solution reached 7.0. 7) After drying the porous glass membrane subjected to the operation of 6), aseptic,
Stored in a pyrogen-free plastic bag.

Example 2 -Removal of Pseudomonas aeruginosa by polymyxin B-immobilized porous glass membrane-Namalva cells were added to a medium containing 10% bovine serum containing RPMI-1640 as a main component at 1.2 × 10 ⁴ cells / ml.
20 ml was suspended in a petri dish having a volume of 30 ml and sterilized with ethylene oxide gas. Next, RPMI-16 containing Pseudomonas aeruginosa was added to the petri dish.
10% bovine serum-supplemented medium ( P. aer
uginosa 5.0 × 10 ² / ml) (2 ml) was added. The polymyxin B-immobilized porous glass membrane prepared in Example 1 was placed in a newly prepared petri dish having a capacity of 30 ml. Then, the whole amount of 10% bovine serum-containing medium containing RPMI-1640 containing Pseudomonas aeruginosa and Namalwa cells as a main component was added, and the mixture was added at 37 ° C. in a carbon dioxide incubator at 6 ° C.
Cultured for hours. After gently shaking the petri dish, the supernatant was further added with a newly prepared polymyxin B-immobilized porous glass membrane prepared in Example 1 and having a volume of 30 ml.
Was collected into a petri dish of and cultured at 37 ° C. for 6 hours. After 6 hours, the supernatant was collected in a cell culture flask having a volume of 50 ml and examined under a light microscope. As a result, Pseudomonas aeruginosa was not observed in the cell culture medium. In addition, the Namalpa cells were alive at 1.0 × 10 ⁴ cells / ml.

Example 3 -Removal of various microorganisms by polymyxin B-immobilized porous glass membrane-In place of Pseudomonas aeruginosa of Example 2, Staphylococcus aureus, Escherichia coli,
Bacillus, Klebsiella, Enterobacter, Salmonella, Citrobacter, morphobacterium,
The same operation was performed using any one of Enterobacter bacteria, virus and mycoplasma (the amount of the added microorganism was 10 ³ cells in each case). as a result,
As in Example 2, no bacterium was observed in the final cell culture medium.

Example 4 -Removal of LPS derived from Pseudomonas aeruginosa by polymyxin B-immobilized porous glass membrane-1.2 x 10 ⁴ cells / ml of Namalwa cells in 10% bovine serum-containing medium containing RPMI-1640 as a main component
20 ml was suspended in a petri dish having a volume of 30 ml and sterilized with ethylene oxide gas. Next, RPMI-16 containing Pseudomonas aeruginosa was added to the petri dish.
40% 10% bovine serum supplemented medium ( Paeru
² ml of ginosa ( containing 5.0 × 10 ² / ml) was added. The polymyxin B-immobilized porous glass membrane prepared in Example 1 was placed in a newly prepared petri dish having a capacity of 30 ml. Then, the whole amount of 10% bovine serum-containing medium containing RPMI-1640 containing Pseudomonas aeruginosa and Namalwa cells as the main components was added, and the mixture was cultured in a carbon dioxide incubator at 37 ° C. for 6 hours. After gently shaking the petri dish, the supernatant was further aliquoted into a freshly prepared polymyxin B-immobilized porous glass membrane prepared in Example 1 and having a volume of 30 ml, and the mixture was placed at 37 ° C. for 6 hours. Incubated for hours. After 6 hours, the supernatant was dispensed into a polystyrene test tube sterilized with ethylene oxide gas having a volume of 15 ml, and 100 μm thereof was collected.
Pseudomonas aeruginosa-derived endotoxin (LP
S) was quantified. As a result, Pseudomonas aeruginosa-derived endotoxin (LPS) was not detected in the cell culture medium. Moreover, the Namalwa cell has 1.0 × 10 ⁴ cells.
s / ml was alive.

Example 5 -Synthesis of resin in which phosphonium residue is introduced into glycidyl methacrylate-divinylbenzene spherical copolymer-Method of Nonaka et al. (Journal of Japan Chemical Society, 1097-, 1994)
(A) Trioctylphosphine (TOP) -introduced phosphonium by reacting glycidyl methacrylate-1,4-divinylbenzene spherical copolymer with hydrogen chloride and then reacting with alkylphosphine according to Resin, (b) alkyldimethylphosphine (having 10 carbon atoms
˜16) introduced phosphonium resin, and (c) (3-aminopropyl) phosphine (TAPP) introduced phosphonium resin were synthesized. That is, the following (1) to (3)
It was incorporated according to the procedure of. (1) Synthesis of glycidyl methacrylato 1,4-divinylbenzene spherical copolymer 50 ml of a 1 wt / wol% gelatin aqueous solution, 6 g of anhydrous sodium sulfate, and 450 ml of purified water were placed in a pressure-resistant glass autoclave and gently stirred to form a dispersion bath. Prepared. The dispersion bath, a mixed solution of a glycidyl methacrylate monomer prepared in advance, a 1,4-divinylbenzene monomer and isobutyl acetate were replaced with nitrogen for 1 hour. Subsequently, 0.5 g of benzoyl peroxide dissolved in the monomer solution was added to the solution. Subsequently, the solution was stirred for 30 minutes at a rotation speed of 280 rpm without increasing the temperature. Then, the temperature of the said solution was raised to 78 degreeC over 1 hour, and it superposed | polymerized at the said temperature for 1 hour. Further, the temperature of the solution was raised to 90 ° C. over 1.5 hours, and aging was carried out at 90 ° C. for 2 hours to complete the polymerization. Next, the solution was allowed to cool and suction filtered, the polymer was immersed in a large amount of purified water, and boiled in a draft to dissolve gelatin. Then, it was suction filtered, immersed in methanol to remove the diluent, and dried. Subsequently, a spherical polymer having a size of 32 to 60 mesh was separated by a sieve. (2) Synthesis of Resin Having 2-Chloro-3-hydroxypropyl Group 1 g of glycidyl methacrylate-1,4-divinylbenzene spherical copolymer sufficiently dried under reduced pressure in a pressure test tube.
And 0.4 M HCl-1,4-dioxane (20 ml) were left overnight and allowed to swell sufficiently, then the container was tightly sealed and the mixture was kept at 70 ° C. for 3 times.
The mixture was reacted in an oil bath for an hour and washed thoroughly with purified water. This was dried sufficiently to obtain a resin having a 2-chloro-3-hydroxypropyl group. (3) Synthesis of Resin Having Phosphonium Residue 2-Chloro-3 fully dried under reduced pressure in a pressure-resistant test tube.
-Add 1 g of resin having hydroxypropyl group and add Cl
4 times the amount of trioctylphosphine (TOP)
Was added under a nitrogen atmosphere, the bottle was tightly capped, and the mixture was placed in an oil bath at 100 ° C.
The reaction was allowed for 0 hours. Subsequently, it was washed with methanol and purified water, immersed in 1M HCl, and then sufficiently washed with purified water until the methyl orange did not discolor. Further, it was extracted with a Soxhlet extractor for 15 hours. Next, it was air-dried and sufficiently dried under reduced pressure to synthesize a resin having trioctylphosphine (Cl type resin). The obtained Cl type resin is 1M
After immersing in NaOH and exchanging the 1M NaOH solution 5 times, it was washed with purified water until the pH of the washing solution became neutral.
Subsequently, the resin was air-dried and further sufficiently dried in a vacuum dryer to obtain a resin having trioctylphosphine (OH type resin). Further, by carrying out the same operation, a resin having an alkyldimethylphosphine (having 10 to 10 carbon atoms) is obtained.
16), (3-aminopropyl) phosphine (TAP
P) introduced phosphonium resin (Cl type resin and O
H type resin) was synthesized.

Example 6. >>-Removal of Staphylococcus aureus by phosphonium resin-Ball-1 cells were added to RITC57-1 medium by 1.2x.
Suspended at a concentration of 10 ⁴ cells / ml, 20 ml thereof
Was collected in a petri dish with a capacity of 30 ml sterilized with ethylene oxide gas. Next, RITC57-1 medium ( S. aureu) containing Staphylococcus aureus was added to the petri dish.
s : IFO 13276: 5.0 × 10 ² / ml included)
2 ml was added. 2 g of a phosphonium-type floating resin (OH-type resin) introduced with (3-aminopropyl) phosphine (TAPP) prepared in Example 5, which had been sterilized with ethylene oxide gas in advance, was placed in a newly prepared cell culture flask with a capacity of 150 ml. I put it in. Subsequently, RITC57 containing S. aureus and Ball-1 cells as described above.
-1 The total amount of the medium was added, and the mixture was cultured in a carbon dioxide incubator at 37 ° C for 6 hours while gently shaking. After 6 hours, the supernatant was collected in a cell culture flask having a volume of 150 ml and examined under a light microscope. As a result, in the cell culture medium,
No Staphylococcus aureus was observed. In addition, Ball-
One cell was alive at 1.1 × 10 ⁴ cells / ml.

Example 7. -Removal of various microorganisms by phosphonium resin-In place of Pseudomonas aeruginosa of Example 6, Staphylococcus aureus, Escherichia coli,
Bacillus, Klebsiella, Enterobacter, Salmonella, Citrobacter, morphobacterium,
The same operation was performed using any one of Enterobacter bacteria, virus and mycoplasma (the amount of the added microorganism was 10 ³ cells in each case). as a result,
As in Example 6, no bacterium was observed in the final cell culture medium.

Example 8. >>-Removal of Pseudomonas aeruginosa-derived LPS by phosphonium resin-Ball-1 cells were added to RITC57-1 medium at 1.2x.
Suspended at a concentration of 10 ⁴ cells / ml, 20 ml thereof
Was collected in a petri dish with a capacity of 30 ml sterilized with ethylene oxide gas. Next, RITC57-1 medium ( P. aeruginos) containing Pseudomonas aeruginosa was added to the petri dish.
a containing 5.0 × 10 ² / ml) 2 ml was added. In a cell culture flask with a capacity of 150 ml newly prepared, 2 g of phosphonium floating resin (OH type resin) introduced with (3-aminopropyl) phosphine (TAPP) prepared in Example 5, which had been sterilized with ethylene oxide gas in advance, was placed. It was Subsequently, the whole amount of RITC57-1 medium containing the above-described Pseudomonas aeruginosa and Ball-1 cells was added, and the mixture was cultured in a carbon dioxide incubator at 37 ° C for 6 hours while gently shaking.
After 6 hours, the supernatant was aliquoted into a polystyrene test tube sterilized with ethylene oxide gas having a volume of 15 ml, and 100 μl of the supernatant was used to determine the endotoxin (LPS derived from Pseudomonas aeruginosa) by the endotoxin quantitative reagent Pyrodick manufactured by Seikagaku ) Was determined. As a result, erythodotoxin (LPS) derived from Pseudomonas aeruginosa was not detected in the cell culture medium. In addition, Ball-1 cells have 1.0 ×
10 ⁴ cells / ml were alive.

Example 9 -Removal of LPS derived from Escherichia coli by phosphonium resin-A column having a capacity of 15 ml was filled with 10 ml of a phosphonium resin (OH type resin) into which (3-aminopropyl) phosphine (TAPP) prepared in Example 5 was introduced, and 1% was charged. It was washed with 100 ml of sodium deoxycholate solution and 100 ml of distilled water for injection. Then, E. c
LPS solution (10 EU / ml) derived from oli 0111 8
Apply ml, close the cock, and in at room temperature for 2 hours
I cubated. Subsequently, 10 ml of distilled water for injection was applied to the column and allowed to flow down at a flow rate of 1 ml / min, and the outflow fractions were fractionated up to 15 ml by 1 ml. For each fraction, the endotoxin (LP
S) was quantified. As a result, endotoxin (LPS) derived from Escherichia coli was not detected in the cell culture medium.

Example 10 >>-(3-aminopropyl) phosphine covalently bonded polyethylene terephthalate film-derived LPS from E. coli
Quantification- (3-aminopropyl) phosphine (TAPP) dissolved in ethyl methyl ketone was applied to the surface of a polyethylene terephthalate film having a thickness of 1 mm to form a film of 5 um. Next, the film was cut into a size of 1 cm × 1 cm. Subsequently, the film was sterilized with ethylene oxide gas and dispensed into a polystyrene test tube sterilized with ethylene oxide gas having a volume of 5 ml. 3 ml of purified water containing 1% pyrogen-free albumin and LPS (derived from E. coli 0111) was taken into the polystyrene test tube, and gently shaken to give 3
Incubate for 30 minutes at 7 ° C. After 30 minutes, the polystyrene test tube subjected to the above operation was washed 3 times with 3 ml of a pyrogen-free phosphate buffer solution. Into a newly prepared polystyrene test tube sterilized with ethylene oxide gas and having a capacity of 5 ml, 100 μl of each of the endotoxin quantification reagent (pyrodic reagent) manufactured by Seikagaku Kogyo was collected. The polyethylene terephthalate film that has been subjected to the above operation is put into the test tube by aseptic operation, and incu
I batted. After 30 minutes, each 1 ml of 0.6N acetic acid was added to the polystyrene test tube, and the absorbance at 405 nm was measured. The same operation was performed using the standard LPS to draw a calibration curve. The amount of LPS in the sample was calculated from the drawn calibration curve.

Example 11 -Preparation of amikacin-bonded magnetic particles-Volume 5 sterilized with ethylene oxide gas in a draft
In a 0 ml polystyrene flask, BioMag (magnetic particles: Perseptive Biosystems
10 ml was collected. Subsequently, 35 ml of coupling buffer was added to the flask, stirred, and then Mag
The washing operation for separating the magnetic particles with a net separator was repeated 5 times. In a flask containing the magnetic particles that had been subjected to the operation, 20m of a 5% glutaraldehyde solution
1 was added and stirred vigorously for 5 minutes. Subsequently, the flask was gently stirred for 3 hours. After stirring Magnet
The magnetic particles were separated with an ic Separator. Subsequently, 35 ml of the coupling buffer was added to the flask containing the magnetic particles that had been subjected to the operation, and the mixture was stirred.
The washing operation for separating magnetic particles with a tic Separator was repeated 5 times. Then, in the flask containing the magnetic particles that had been subjected to the operation, a solution of amikacin dissolved in a coupling buffer (concentration of amikacin: 5 mg / ml)
10 ml was added and stirred vigorously for 5 minutes. Subsequently, the flask was gently stirred at room temperature for 24 hours. After stirring,
The magnetic particles were separated with a Magnetic Separator. In the flask containing the magnetic particles that were operated,
35 ml of 5% glycine solution was added and vigorously stirred for 5 minutes. Subsequently, the flask was gently stirred for 1 hour.
After stirring, the magnetic particles were separated with a Magnetic Separator. Subsequently, 35 ml of the washing buffer was added to the flask containing the magnetic particles that had been subjected to the above operation, and after stirring, the washing operation of separating the magnetic particles with a Magnetic Separator was repeated 5 times to prepare amikacin-bound magnetic particles. Amikacin-bonded magnetic particles were stored at 4 ° C.

Example 12 -Removal of Pseudomonas aeruginosa by amikacin-bound magnetic particles-The EB virus mutant human B lymphoid cell line was suspended in RITC57-1 medium at a concentration of 1.2 x 10 < ⁴ > cells / ml, and 20 ml thereof was added to ethylene. It was dispensed into a petri dish with a volume of 30 ml that had been sterilized with oxide gas. Next, on the petri dish, the RI TC57-1 medium containing Pseudomonas aeruginosa ( P.
aeruginosa 5.0 × 10 ² / ml included) 2m
1 was added. 5 ml of amikacin-bonded magnetic particles prepared in Example 11, which had been previously washed with a 1% sodium deoxycholate solution and pyrogen-free purified water, was collected in a newly prepared cell culture flask having a capacity of 150 ml. Then, the whole amount of RITC57-1 medium containing the above-mentioned Pseudomonas aeruginosa and EB virus mutant human B lymphoid cell line was added, and the cells were cultured in a carbon dioxide incubator at 37 ° C. for 6 hours while gently shaking. 6 hours later Magnetic
The magnetic particles were separated with a Separator. The separated solution was dispensed into a cell culture flask with a volume of 150 ml, and examined under a light microscope. As a result, Pseudomonas aeruginosa was not observed in the cell culture medium. In addition, the EB virus mutant human B lymphoid cell line was 1.0 × 10 ⁴ cells.
/ Ml was alive.

Example 13 -Removal of LPS derived from Escherichia coli by amikacin-bonded magnetic particles-Namarba cells were added to a medium containing 10% bovine serum containing RPMI-1640 as a main component at 1.2 × 10 ⁴ cells / ml.
20 ml was suspended in a petri dish having a volume of 30 ml and sterilized with ethylene oxide gas. Next, a 10% bovine serum-containing medium containing E. coli-containing RPMI-1640 as a main component ( E. coli 5.
² ml (containing 0 × 10 ² / ml) was added. In a newly prepared cell culture flask with a capacity of 150 ml, add 1% in advance.
5 ml of amikacin-bonded magnetic particles prepared in Example 11, which had been washed with a sodium deoxycholate solution and pyrogen-free purified water, were collected. Subsequently, the whole amount of 10% bovine serum-containing medium containing RPMI-1640 containing the above-mentioned Escherichia coli and Namalwa cells as a main component was added, and the mixture was cultured in a carbon dioxide incubator at 37 ° C. for 6 hours while gently shaking. 6 hours later Magnetic Separato
The magnetic particles were separated by r. Volume of separated solution is 150m
1 cell culture flask. 100 μl of the solution
For, the endotoxin was quantified by the endotoxin quantitative reagent Pyrodic manufactured by Seikagaku. As a result, endotoxin derived from Escherichia coli was not detected in the cell culture medium. In addition, 1.0 for Namalwa cells
× 10 ⁴ cells / ml were alive.

Example 14 -Quantification of LPS derived from Escherichia coli by amikacin-bonded magnetic particles- 1 ml of amikacin-bonded magnetic particles prepared in Example 11, which had been washed with 1% sodium deoxycholate solution and pyrogen-free purified water, was sterilized with ethylene oxide gas in a volume of 5 ml. It was collected in a polystyrene test tube. Into the polystyrene test tube, 3 ml of purified water containing 1% albumin and LPS (derived from E. coli 0111) was collected and shaken gently at 37 ° C for 3 times.
It was incubated for 0 minutes. 30 minutes later Magneti
Magnetic particles were separated with a c Separator. next,
The operated amikacin-bonded magnetic particles were washed 3 times with 3 ml of pyrogen-free water. Next, 100 μl each of the endotoxin quantitative reagent (Pyrodic reagent) manufactured by Seikagaku Kogyo Co., Ltd. was taken into a polystyrene test tube containing the amikacin-bonded magnetic particles that had been subjected to the operation, and the mixture was stored at 37 ° C. for 30 minutes
I cubated. After 30 minutes, 1 ml each of 0.6N acetic acid was added to the polystyrene test tube subjected to the above operation, and 405
The absorbance at nm was measured. The same operation was performed using the standard LPS to draw a calibration curve. The amount of LPS in the sample was calculated from the drawn calibration curve.

Example 15 >>-Synthesis of Resin in which Quaternary Ammonium Salt is Introduced into Glycidyl Methacrylate-Divinylbenzene Spherical Copolymer-Method of Nonaka et al. (Journal of Japan Chemical Society, 1097-1994)
Glycidyl methacrylate-1,4-divinylbenzene spherical copolymer, quaternary ammonium salt (3-
A resin having a quaternary ammonium salt introduced therein was synthesized by reacting (trimethoxysilyl) -propyloctadecyldimethylammonium chloride. That is, according to Example 5, Cl type resin and OH type resin were synthesized.

Example 16 >>-Quaternary ammonium salt-bonded glycidyl methacrylate-
L derived from Escherichia coli by divinyl rubenzene spherical copolymer
Removal of PS, DNA and RAN-Prepared in Example 13 in a 15 ml volume column (3-
10 ml of a resin (OH type resin) into which (trimethoxysilyl) -propyloctadecyldimethylammonium chloride was introduced was filled and washed with 100 ml of a 1% sodium deoxycholate solution and 100 ml of distilled water for injection. Then, E. E. coli HB101-derived cell extract (DNA content 30 μg / ml, RAN content 520 μg / ml, LPS content 20 EU / ml) 8 m
1 was applied and incubated at room temperature for 2 hours. Then, add 10 ml of distilled water for injection to the column.
It was plied and allowed to flow down at a flow rate of 1 ml / min, and the outflow fractions were fractionated up to 15 ml by 1 ml. For each fraction, the endotoxin (LPS) derived from Escherichia coli was quantified by the endotoxin quantification reagent Pyrodic manufactured by Seikagaku Corporation. As a result, endotoxin derived from Escherichia coli was not detected in the cell culture medium. Further, the amount of DNA and the amount of RAN were quantified for the fraction, but neither DNA nor RAN was detected in any of the fractions.

Example 17 -Regeneration of amikacin-bound magnetic particles with surfactant and antibacterial agent- 1 ml of amikacin-bound magnetic particles prepared in Example 9 was dispensed into a polystyrene flask sterilized with ethylene oxide gas and having a volume of 50 ml. In the polystyrene flask, 1% albumin and LPS ( E. coli 01
11-derived: 30 m of purified water containing 100 EU / ml)
Take 1 aliquot and incubate for 2 hours at 37 ° C with gentle shaking.
I cubated. 2 hours later Magnetic Se
The amikacin-bound magnetic particles were separated with a parameter.
Next, the magnetic particles were treated with 40 ml of a 1% sodium deoxycholate solution three times and 40 m of pyrogen-free purified water.
1 × 3 times. Next, add the magnetic particles to a volume of 5 ml.
Into a polystyrene test tube sterilized with ethylene oxide gas, the sample was collected by aseptic operation. Next, 100 μl each of the endotoxin quantitative reagent (Pyrodic reagent) manufactured by Seikagaku Kogyo was collected in the polystyrene test tube, and the temperature was 37 ° C.
It was incubated for 30 minutes. After 30 minutes, 1 ml of 0.6N acetic acid was added to the polystyrene test tube which was operated, and the absorbance at 405 nm was measured. As a result, E. coli 0111 derived LPS
Was not detected, confirming the cleaning effect of the sodium deoxycholate solution. When the magnetic particles were washed with a 5% streptomycin solution, the same washing effect was confirmed.

Example 18 -Removal of LPS, DNA, and RAN derived from Escherichia coli by covalently bound pradimachine derivative carrier-A column having a capacity of 15 ml was filled with 10 ml of covalently bound pradimachine derivative (Pradimachine E), and 1% sodium deoxycholate was added. It was washed with 100 ml of the solution and 100 ml of distilled water for injection. Then, E. c
Cell extract from oli HB101 (DNA content 30
8 g (μg / ml, RAN content 520 μg / ml, LPS content 20 EU / ml) was applied, and the column was stopped and incubated at room temperature for 2 hours. Then, 20 ml of distilled water for injection was applied to the column, and the flow rate was 1
Flow out at a flow rate of ml / min, and the effluent fractions will be 1 ml each for 15 m.
Fractionated to 1 For each fraction, the amount of E. coli-derived endotoxin (LPS) was quantified by the endotoxin quantification reagent Pyrodic manufactured by Seikagaku. as a result,
No endotoxin derived from Escherichia coli was detected in the cell culture medium. In addition, regarding the relevant fraction, DNA
Amount and RAN amount were quantified.
A and RAN were not detected.

Example 19 -Removal of LPS derived from Escherichia coli by quaternary ammonium salt-immobilized nonwoven fabric-Polyester nonwoven fabric (Toyobo Co., Ltd .: 6 denier) is graft-polymerized with a hydrophilic group (methacrylic acid), and further quaternary ammonium salt antibacterial agent , (3- (trimethoxysilyl)
A non-woven fabric having a quaternary ammonium salt immobilized thereon was prepared by binding propyloctadecyldimethylammonium chloride (manufactured by Dow Corning). The nonwoven fabric was cut into a size of 5 cm × 5 cm and sterilized with ethylene oxide gas. In a 10% bovine serum-containing medium containing RPMI-1640 as a main component, 1.
20 × suspended at a concentration of 2 × 10 ⁴ cells / ml
ml is ethylene oxide gas sterilized, volume 30 ml
It was collected in a petri dish. Then, 2 m of a medium containing 10% bovine serum containing E. coli-containing RPMI-1640 as a main component (containing E. coli 5.0 × 10 ² / ml) was added to the petri dish.
1 was added. The above-mentioned non-woven fabric sterilized with ethylene oxide gas in advance was dispensed into a newly prepared cell culture flask having a capacity of 150 ml. Subsequently, the whole amount of 10% bovine serum-containing medium containing RPMI-1640 containing Escherichia coli and Namalwa cells as a main component was added, and the mixture was cultured in a carbon dioxide incubator at 37 ° C for 6 hours while gently shaking. 6
The nonwoven was aseptically removed after a period of time. The solution obtained by separating the non-woven fabric was dispensed into a cell culture flask having a volume of 150 ml. For 100 μl of the solution, endotoxin was quantified by Pyrodick, an endotoxin quantification reagent manufactured by Seikagaku Corporation. As a result, endotoxin derived from Escherichia coli was not detected in the cell culture medium. In addition, Namalwa cells have 1.0 × 10 ⁴ cells /
ml was alive.

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Claims (16)

[Claims] 1. An insoluble carrier comprising an antibacterial agent as a constituent. 2. The antibacterial agent is a penicillin antibiotic, a cephem antibiotic, a monobactam antibiotic, a tetracycline antibiotic, a chloramphenicol antibiotic, a macrolide antibiotic, an aminodaricoside antibiotic, a polypeptide antibiotic. Characterized by being selected from antibiotics, pyridonecarboxylic acid-based synthetic antibacterial agents, antifungal agents, antiviral agents, sulfa agents, pyridinium compounds, phosphonium compounds, quaternary ammonium salts, and pradimycin group antibiotics The insoluble carrier according to claim 1. 3. The insoluble carrier according to claim 1, wherein the insoluble carrier is magnetic particles. 4. A method for adsorbing and removing microorganisms in a cell suspension contaminated with microorganisms and microorganisms in a physiologically active substance solution contaminated with microorganisms, using the insoluble carrier according to claim 1. 5. The lipopolysaccharide in a cell suspension contaminated with lipopolysaccharide and the lipopolysaccharide in a physiologically active substance solution contaminated with lipopolysaccharide are adsorbed and removed by using the insoluble carrier according to claim 1. Method. 6. The lipopolysaccharide in a cell suspension contaminated with lipopolysaccharide and the lipopolysaccharide in a physiologically active substance solution contaminated with lipopolysaccharide are adsorbed to the insoluble carrier according to claim 1. A method for detecting and quantifying lipopolysaccharide by a method for detecting and quantifying lipopolysaccharide. 7. A method for adsorbing and removing microorganisms in a cell suspension contaminated with microorganisms and microorganisms in a physiologically active substance solution contaminated with microorganisms, using the insoluble carrier according to claim 3. 8. The lipopolysaccharide in a cell suspension contaminated with lipopolysaccharide and the lipopolysaccharide in a solution of a physiologically active substance contaminated with lipopolysaccharide are adsorbed and removed using the insoluble carrier according to claim 3. Method. 9. A method for detecting and quantifying a microorganism after adsorbing the microorganism in a cell suspension contaminated with the microorganism and the microorganism in a physiologically active substance solution contaminated with the microorganism to the insoluble carrier according to claim 3. A method for detecting and quantifying microorganisms by the method. 10. The lipopolysaccharide in a cell suspension contaminated with lipopolysaccharide and the lipopolysaccharide in a physiologically active substance solution contaminated with lipopolysaccharide are adsorbed to the insoluble carrier according to claim 3. A method for detecting and quantifying lipopolysaccharide by a method for detecting and quantifying lipopolysaccharide. 11. A method for adsorbing and removing nucleic acid in a cell suspension contaminated with nucleic acid and nucleic acid in a physiologically active substance solution contaminated with nucleic acid using the insoluble carrier according to claim 3. 12. The nucleic acid in a cell suspension contaminated with nucleic acid and the nucleic acid in a physiologically active substance solution contaminated with nucleic acid are adsorbed to the insoluble carrier according to claim 3, and then the nucleic acid is detected and quantified. A method for detecting and quantifying nucleic acid according to the method. 13. The method according to claim 3, wherein a solution containing a surfactant is used.
A method for washing and regenerating the insoluble carrier described. 14. A method for washing and regenerating the insoluble carrier according to claim 3, using a solution containing an antibacterial agent. 15. A solution containing a surfactant for washing and regenerating the adsorption activity of the insoluble carrier according to claim 3. 16. A solution containing an antibacterial agent for washing and regenerating the adsorption activity of the insoluble carrier according to claim 3.