JPH0612994B2 - Bioactive substance-immobilized magnetic particles - Google Patents

Description

TECHNICAL FIELD The present invention relates to magnetic fine particles having a physiologically active substance immobilized thereon, which is useful as a means for medical treatment, analysis and the like.

[Prior Art] It has been reported and reported by RP Blakemove that magnetotactic bacteria contain magnetic fine particles composed of magnetite in a state of being arranged in a chain shape (Science, 190 , 337-379 (197).
Five)). The size of these magnetic particles is as small as about 500-1500Å and has a single domain structure.The ones obtained from the same type of bacteria are very uniform in size and shape, making it difficult to artificially synthesize them. Is.

[Problems to be solved by the invention]

Conventionally, no attempt has been made to use magnetic fine particles obtained from such magnetotactic bacteria as a carrier for a physiologically active substance such as a drug.

An object of the present invention is to use the magnetic fine particles obtained from such magnetotactic bacteria as medical or analytical means.

[Structure of the Invention] The present inventors have found that the fine particles obtained from magnetotactic bacteria are extremely useful as carriers for physiologically active substances.

That is, the present invention provides magnetic fine particles that are separated from magnetotactic bacteria and have a physiologically active substance immobilized thereon.

The magnetotactic bacterium is a bacterium having a property of swimming along the magnetic field lines, and contains magnetic fine particles made of magnetite in the body, which are arranged in a chain form in one or two rows. As such magnetotactic bacteria, it is known that various bacteria such as bacilli, cocci, and helicobacteria inhabit in freshwater and seawater, and a specific example is deposited as ATCC31632. The bacterial strain (MS-1) can be mentioned. Such a magnetotactic bacterium can be easily collected from fresh water and seawater using a magnet, for example, by the method described in Example 1 described later.

The magnetic particles contained in magnetotactic bacteria are about 50 as described above.
With a size of 0 to 1500Å, the size and shape are highly uniform when the bacteria from which they are derived are of the same species. The magnetic fine particles are present in the cells in a state of being covered with a coating film mainly made of protein, and are called magnetosomes.

The magnetic fine particles used as the carrier of the physiologically active substance in the present invention are those separated from the above-mentioned magnetotactic bacteria, and can be used both in the state of being covered with the coating film and in the state of removing the coating film. You can In general, magnetic fine particles having a coating film have an advantage of being highly compatible with a living body.

In order to separate the magnetic particles from the magnetotactic bacteria collected by culturing or culturing, for example, after concentrating with a microcentrifuge, the cell wall can be dissolved with a lysozyme solution and the centrifugation can be repeated again. Is obtained. Further, to remove the coating film, for example, it may be treated with alkali,
Magnetic particles of magnetite are obtained by washing.

There is no particular limitation on the kind of the physiologically active substance immobilized on the magnetic fine particles separated from the magnetotactic bacterium in this manner, and the following can be exemplified.

Enzymes: hydrolases such as amylase, protease, cellulase, hemicellulase, lipase, pectinase,
Lysozyme, urease, invertase, dextrase, peptidase, lytic enzyme (mold, yeast), etc .; oxidoreductase, for example, glucose oxidase, catalase, lipoxynase, cytochrome C, peroxidase, isomerase, for example, glucose isomerase, etc .; transferase A releasing enzyme such as aspartase or hyaluronidase; and various restriction enzymes.

○ Immune related substances; lymphokines such as interleukin 2 (IL-2),
Lymphotoxin (LT), cancer-destroying factor (CBF), etc., monokine, for example, TNF (tumor necrosis factor), and other cytokines; interferon, that is, α-interferon, β-
Interferon, γ-interferon; Antigens such as IgG, IgA, IgM, IgE, albumin, HCG, AF
P, cardiolipin antigen, blood group substance, concanavalin A, DNT, prostaglandin, CRP, HBs, human growth hormone, steroid hormone, etc .; antibodies such as anti-albumin antibody, anti-HCG antibody, anti-IgG
Antibody, anti-IgA antibody, anti-IgM antibody, anti-IgE antibody, anti-IgC antibody,
Anti-AFP antibody, anti-DNT antibody, anti-prostaglandin antibody, anti-human coagulation factor antibody, anti-CRP antibody, anti-HBs antibody, anti-human growth hormone antibody, anti-steroid hormone antibody, and serum containing them, and monoclonal antibody; Growth factors ○ Nucleic acid related substances ○ Plant-derived bioactive substances ○ Other bioactive substances such as prostaglandins
(PG), human tissue plasminogen activator (TPA), etc.

As a method for immobilizing these physiologically active substances on the magnetic fine particles, a technique already known as a method for immobilizing an enzyme or the like can be used.

〔Example〕

 Hereinafter, the present invention will be specifically described with reference to examples.

Example 1 (1) Collection of magnetotactic bacteria Bacterial bacteria inhabiting a pond were collected using the magnetotactic bacteria collector shown in FIG. The container of this sampler is composed of a cylindrical side wall 2 (length 8 cm) and an upper end wall 3 (diameter 8 cm), and the lower end is open. A paper (No. 2) 4 is detachably fixed to the lower end by a fixture 5, and the S pole side of a samarium-cobalt magnet 6 is fixed to the center of the outside of the upper end wall. The container 1 of this sampler was filled with distilled water, and the paper filter 4 was attached to the container 1 to be submerged in the pond (7: bottom sediment). After being left for 10 hours, when collected from the pond, magnetotactic bacteria adhered to the central part (magnet fixing part) inside the upper wall of the container 1 in a darkened state,
It was collected.

(2) Immobilization of anti-α-fetoprotein antibody on magnetic microparticles, detection of α-fetoprotein by the immobilized antibody The collected magnetotactic bacteria were concentrated with a microcentrifuge, separated, and then 0.2
% Lysozyme solution for 1 hour to dissolve the cell wall, and centrifugation was repeated again to separate and wash the magnetic fine particles.
The protein coating the magnetic particles was removed by treatment with 5M-NaOH for 12 hours. After reacting 10 mg of these magnetic fine particles with γ-aminopropyltriethoxysilane stock solution for 10 minutes, 2.5%
The mixture was reacted with a glutaraldehyde solution (pH 7.0) for 1 hour. And anti-AFP (α-fetoprotein) antibody solution (5
1 mg (containing mg / ml antibody) was added to immobilize the anti-AFP antibody on the magnetic fine particles. In addition, the anti-AFP antibody was immobilized on the magnetic fine particles that were still covered with the protein film without the γ-aminopropyltriethoxysilane treatment. Next, AFP solution (5mg / ml, pH8.0) 3ml, 25% glutaraldehyde solution 3
Mix μl and catalase 20mg (40,000 units) at 25 ℃,
After leaving for 1 hour, AFP was labeled with catalase. Anti-AFP
10 mg of the antibody-immobilized magnetic fine particles were mixed with 1 ml of a catalase-labeled AFP solution and 1 ml of an unlabeled AFP solution (sample) and then competitively reacted at 37 ° C. for 2 hours. Then, the magnetic fine particles were collected with a magnet and washed. Magnetic particles were added to 0.05M phosphate buffer (pH 7.0) to measure the amount of bound catalase-labeled AFP, magnetically stirred without using a stirrer bar, and 100 μl 3%, H ₂ O ₂ was added. The oxygen produced was measured with an oxygen electrode. Then, the AFP concentration in the sample was estimated from the calibration curve prepared in advance. The response of the oxygen immunity center became steady in about 30 seconds after H ₂ O ₂ was added, and AFP could be measured in a short time.

Example 2 Glucose oxidase-immobilized magnetic fine particles 0.2 mg of magnetic fine particles from which the coating film of proteins and the like was removed in the same manner as in Example 1 was reacted with 1 ml of γ-aminopropyltriethoxysilane for 10 minutes and washed to obtain 2.5% It was reacted with 1 ml of a phosphate buffer containing glutaraldehyde for 1 hour.
Add this to 1 ml of glucose oxidase solution (250 μg / m
The reaction was carried out for 12 hours at 4 ° C. in step 1) and washing.

Thus, the glucose oxidase-immobilized magnetic fine particles according to the present invention were obtained.

In addition, artificial magnetic particles of Zn-ferrio (5000
Similarly, the glucose oxidase was also immobilized on Å). Glucose oxidase was immobilized only on glutaraldehyde on the magnetic fine particles of the magnetotactic bacterium which had not been coated with the protein film, but without γ-aminopropyltriethoxysilane treatment.

When the glucose oxidase-immobilized amount and activity of each of the thus-obtained glucose oxidase-immobilized fine particles were measured, the following results were obtained.

The magnetic fine particles separated from the magnetotactic bacteria of the present invention include 200
μg / mg glucose oxidase was immobilized. In contrast, 1.8 μg / mg glucose oxidase was immobilized on the Zn-ferrite particles. The magnetic fine particles separated from the magnetotactic bacteria have a large particle surface area, which is 1/10 of that of Zn-ferrite particles. Therefore, the enzyme was immobilized 100 times or more with respect to Zn-ferrite particles. Further, the magnetic fine particles of this example exhibited a very high enzyme activity because of the large surface area and the large amount of immobilized enzyme. That is, when glucose oxidase was immobilized, 40% of glucose oxidase-immobilized Zn-ferrite was used.
Double enzyme activity was seen. In addition, the immobilized enzyme reaction using Zn-ferrite particles as a carrier was observed to be inhibited by the metal of Zn-ferrite, and when EDTA was added, the usual non-inhibition type Michaelis
s Returned to reaction. On the other hand, no inhibition was observed when the magnetic fine particles coated with the protein separated from the magnetotactic bacterium were used. It was also shown that the enzyme-immobilized magnetic particles have the ability to reuse the enzyme at least 6 times.

Example 3 Glucose sensor Immobilized amount of glucose oxidase prepared in the above example
200μg / mg glucose oxidase-immobilized magnetic particles
Add 10 g to 30 ml of 0.1 M phosphate buffer (pH 7.0), stir magnetically without using a stirrer bar, turn on the oxygen electrode, and the current value becomes steady, then add the glucose solution as a sample. When added, oxygen was consumed by the action of glucose oxidase and the current value decreased. Then, the glucose concentration could be estimated from the calibration curve prepared in advance.

Example 4 E. coli antibody-immobilized magnetic fine particles In the same manner as in the example, magnetic fine particles (500 to 1500Å) separated from magnetotactic bacteria and having a coating film such as protein removed were γ-
It was reacted with aminopropyltriethoxysilane for 10 minutes and washed, and then reacted with 0.1 M phosphate buffer (pH 7.0) containing 2.5% glutaraldehyde for 1 hour and washed. Further, it was reacted with triamine for 1 hour, washed, and again reacted with a glutaraldehyde solution for 1 hour. The magnetic particles were reacted in an E. coli antibody solution at 4 ° C. for 12 hours to immobilize the antibody. Similarly, the E. coli antibody was also immobilized on Zn-ferrite (5000 Å), which is an artificial magnetic particle. In addition, the E. coli antibody was immobilized on the magnetic fine particles of the magnetotactic bacterium as it was coated with the protein film without γ-aminopropyltriethoxysilane treatment.

With respect to each of the thus-immobilized antibody microparticles, an antigen-antibody reaction was performed using Escherichia coli cultivated until the stationary phase. That is, magnetic fine particles having an antibody immobilized thereon are added to an E. coli suspension, stirred and reacted to bind E. coli to the magnetic fine particles, and then the magnetic fine particles are magnetically separated to measure the number of remaining bacteria in the E. coli suspension. did. In addition, the measurement was performed in the same manner for magnetic fine particles on which bovine serum albumin was immobilized instead of the antibody. From these, the following results were obtained.

The magnetic fine particles separated from the magnetotactic bacteria have a particle diameter as small as 1/10 that of Zn-ferrite, and therefore have a very large surface area per unit magnetic fine particle. Therefore, the amount of immobilized antibody was large and the surface activity was very high. When 50 mg of magnetic microparticles immobilized with E. coli antibody were used to bind E. coli by an antigen-antibody reaction, it was bound to about 8 × 10 ⁶ E. coli in 2 hours. On the other hand, when 50 mg of magnetic microparticles immobilized with bovine serum albumin was used instead of the antibody, it did not bind to E. coli even after 2 hours. Furthermore, when 50 mg of the magnetic fine particles on which the E. coli antibody was immobilized again on the magnetic fine particles on which albumin was immobilized, 2 × 10 ⁶ Escherichia coli was bound in 2 hours. Then, when the magnetic microparticles to which Escherichia coli was bound were immobilized on the oxygen electrode, there was a respiratory activity of Escherichia coli, and it was shown that Escherichia coli was bound to the magnetic microparticles to which the Escherichia coli antibody was immobilized. This indicates that E. coli can be separated by immobilizing the E. coli antibody on the magnetic fine particles. In addition, by washing the magnetic microparticles bound with E. coli with 0.2 M glycine-HC1 buffer (pH 2.3), the bound E. coli can be separated by the antigen-antibody reaction, and the magnetic microparticles with immobilized antibody can be reused. It was possible to

Example 5 Magnetic Microparticles Immobilized with Restriction Enzyme In the same manner as in Example 1, the restriction enzyme Bam HI was immobilized on the magnetic particles from which the coating film of protein or the like was removed. Bam HI is an enzyme that cleaves at one site of pBR322 in E. coli plasmid. 16 μl distilled water, 10 μl pBR322 (0.1 μg / μl),
Magnetic microparticles on which Bam HI was immobilized were added to a mixture of 3 μl of a 10 × 10 reaction buffer, and the mixture was magnetically stirred and incubated at 37 ° C. for 1 hour to cleave pBR322. Cleavage of pBR322 could be confirmed by agarose gel electrophoresis after adding a dye solution. Furthermore, magnetic particles with Bam HI immobilized can be recovered and reused by performing the same operation, and pBR can be used again.
It was shown that 322 was truncated.

〔The invention's effect〕

The bioactive substance-immobilized magnetic fine particles of the present invention have a particle size of magnetic fine particles separated from a carrier, a magnetotactic bacterium.
Since it is as small as 500-15000Å, it has a large surface area and a very high amount of physiologically active substance loaded. Therefore, the obtained physiological activity is extremely high. In addition, since it is extremely small as described above, it can be moved inside blood vessels without any trouble, and since the magnetic fine particles have a single domain structure, they have a large magnetic moment and move in a relatively weak magnetic field. Can be controlled. In addition, since a physiologically active substance can be locally concentrated at a desired location by applying a magnetic field in this way, the dose of a drug or the like can be significantly reduced, and the drug can be easily collected and recovered. It can be used.

The bioactive substance-immobilized magnetic fine particles of the present invention are thus used as a drug, or as a sensor material for analysis,
It is useful as a biocatalyst for bioreactors and a material for cell separation.

[Brief description of drawings]

 FIG. 1 shows an example of a magnetotactic bacteria collector. 1 ... Container 4 ... Paper filter 6 ... Magnet 7 ... Water bottom deposit

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location G01N 33/553 9015-2J

Claims (3)

[Claims] 1. An isolated from a magnetotactic bacterium,
Magnetic fine particles on which a physiologically active substance is immobilized. 2. The magnetic fine particles according to claim 1, which have a coating film provided in bacteria. 3. The magnetic fine particles according to claim 1 or 2, wherein the physiologically active substance is an enzyme, an immune-related substance, a growth factor, a nucleic acid-related substance or a plant-derived physiologically active substance. .