JP2001208755A - Method for screening physiological active material utilizing surface plasmon resonance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple screening method by which a physiological active material can be discovered efficiently for the development of pharmaceuticals, diagnostic medicines, agricultural chemicals, etc. SOLUTION: In this screening method in which the interaction between a ligand immobilized on the surface of a metallic thin film stuck to glass is detected by surface plasmon resonance, a cell membrane is used as the ligand and a material which interacts to the membrane is selected by immobilizing the membrane on the surface of the thin film under the presence of a phospholipid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention belongs to the technical field of screening for a physiologically active substance useful as a pharmaceutical or the like, and more particularly to a simple screening method for a new type of physiologically active substance by utilizing surface plasmon resonance.

[0002]

BACKGROUND OF THE INVENTION The ultimate interface of life activity at the cellular level is the cell membrane. Specific molecular recognition and permeation and release of substances according to each cell, binding to other cells and specific substances, etc. are all performed by cell membrane functions. Therefore, substances (bioactive substances) having some activity on living organisms, such as various pharmaceuticals, diagnostics, and agricultural chemicals, have some interaction with the cell membrane surface and exhibit their functions. By monitoring the interaction between the cell membrane and the substance on the cell membrane, a useful physiologically active substance can be efficiently screened and utilized for the development of various drugs, diagnostic agents, agricultural chemicals, and the like.

Conventionally, in order to examine the interaction between a cell and a substance in order to screen for a physiologically active substance, it is necessary to measure antagonistic inhibition using a radioactive substance. Measurements had to be performed at a dedicated facility. For this reason, simple screening is practically impossible, and screening is performed using experimental animals and cultured cell systems. Laboratory animals are expensive and require appropriate facilities and breeding positions without holidays, and cultured cell lines do not have all the necessary cell lines. For rapid and low-cost development of pharmaceuticals, diagnostics, pesticides, and the like, a simple screening method capable of efficiently finding a physiologically active substance has been desired.

[0004]

SUMMARY OF THE INVENTION The present inventor has developed a surface plasmon resonance device (surface plasmon resonance biosensor) which has been recently developed to directly detect the interaction between a cell membrane and a substance which did not exist before. The present invention was devised by devising a means capable of detecting the substance as a physiologically active substance that can be detected easily and easily.

Thus, according to the present invention, in a method for detecting the interaction between a substance immobilized on the surface of a metal thin film adhered to glass and a substance by surface plasmon resonance, a cell membrane is used as the ligand, A method for screening for a physiologically active substance is provided, which comprises immobilizing the substance on the surface of a metal thin film in the presence thereof and selecting a substance that interacts with the cell membrane.

[0006]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Surface plasmon resonance (SPR), which is an object of the present invention, means that light travels through glass on which a metal thin film is adhered, and the entire surface of the glass at the interface. When the light is reflected, the resonance occurs when the wave number of the evanescent wave generated at the interface and the surface plasmon wave generated in the metal thin film coincide with each other. When this resonance occurs, the intensity of the reflected light decreases. Since surface plasmon waves are generated on the surface of a metal thin film, if there is a concentration change or a mass change in a medium in the immediate vicinity, the surface plasmon waves change,
Accordingly, the angle of incidence that reduces the intensity of the reflected light changes.

[0007] A surface plasmon resonance biosensor was developed in the early 1990s utilizing such a phenomenon. In a typical SPR biosensor, a ligand is immobilized on the surface of a metal thin film (generally, a gold thin film) adhered (coated) to glass, and a flow cell for accommodating a substance to be measured is provided so as to be in contact with the ligand. Use a disposable measuring chip (sensor chip). This sensor chip is inserted (mounted) into the apparatus main body, near-infrared p-polarized light is incident on the glass, and the change in the incident angle that reduces the intensity of the reflected light according to the principle described above is determined as a resonance signal [1].
0 ^-4 degree of change is called a 1RU (resonance unit)].
For such an SPR biosensor, for example,
It is described in detail in "Protein Nucleic Acid Enzyme Vol37 (1992)", pp. 53-60 and 81-87.

[0008] By using the SPR biosensor as described above, it is possible to perform quantification of substances that specifically react with or bind to the ligand in accordance with the ligand immobilized on the surface of the metal thin film, or to measure the interaction. Is possible. But,
In practice, in many cases, it is difficult to immobilize a ligand on a metal thin film, and the types of ligands used are limited. For example, a cell membrane is immobilized and used as a ligand in an SPR biosensor. No examples are found.

Recently, a maker of an SPR biosensor has provided a metal thin film of a measurement chip to which an alkyl chain is bonded and coated, as a material for immobilizing liposomes or the like as a ligand. But,
Even with such a chip, cell membrane components cannot be stably immobilized on a metal thin film.

A feature of the present invention is that when a target cell membrane is immobilized using a measuring chip having an alkyl chain bonded to a metal thin film, a phospholipid is added and immobilization is performed in the presence of the phospholipid. In this way, the cell membrane is very stably immobilized on the metal thin film surface measurement chip. Usually, when a cell membrane component is extracted, it is extracted as an aggregate of a lipid membrane constituting a cell membrane and a membrane protein such as a channel or a surface antigen present on the membrane. When trying to immobilize an alkyl chain with such an aggregate on a metal thin film, the gap tends to be formed in the membrane, where the alkyl chain is exposed, or when the cell membrane is unstable and the sample is flowed. It becomes easier. Then, a uniform and stable lipid component is added, and the cell membrane component is reconstituted on the surface of the metal thin film to obtain a cell membrane-immobilized measurement chip. The cell membrane-immobilized measurement chip thus obtained contains channels and surface antigens on the cell membrane while maintaining the fluidity of the cell membrane, and retains a cell membrane function close to the natural state. It can be used as a suitable measurement chip.

In the present invention, a phospholipid suitable for immobilizing a cell membrane as a ligand on the surface of a metal thin film can be generally represented by the following general formula (I).

[0012]

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In the formula (I), X forms a hydrophobic part of a phospholipid and can be represented by R—O— or R—CO—O—. Here, R is a hydrocarbon group having 5 to 20 carbon atoms, and is preferably a saturated hydrocarbon group (alkyl group) having 12 to 18 carbon atoms. If a double bond is present in this portion, it is liable to be decomposed from the double bond. Therefore, in order to stably immobilize the cell membrane, it is preferable to use a phospholipid having no double bond.

In the formula (I), Z forms a hydrophilic portion of a phospholipid together with a phosphate residue, and preferred examples include choline, ethanolamine, inositol, and glycerol. Y forms a connector part connecting the hydrophobic part and the hydrophilic part, and a preferable example is a residue such as glycerol, glutamic acid, and aspartic acid.

The phospholipid used in the present invention preferably has a neutral charge. Positive or negative charges may cause non-specific binding, and the added phospholipid must be neutral so as not to impair the charge of the cell membrane to be measured. Is preferred.

Furthermore, the phospholipid used for immobilizing a cell membrane in the present invention preferably has a phase transition temperature not too low (about 5 ° C. or higher) and not too high (depending on the cell type). When the phase transition temperature is low, micelles are easily formed, not as a lipid membrane, but a surfactant-like property becomes strong, which is not suitable for immobilizing a cell membrane. On the other hand, if the phase transition temperature is high, the fluidity is lost at the activity temperature of the cell, and the original activity of the cell membrane may be inhibited. In general, it is considered that the phase transition temperature of the phospholipid is preferably lower than the body temperature during the activity of the species, and differs between insect cells and animal cells.

In the present invention, various phospholipids obtained from the above synthetic or natural products can be used. FIG. 1 illustrates the chemical formulas of some phospholipids suitable for use in the present invention. FIG.
Wherein the alkyl chains represented by R and R 'are generally 5 to 2
It has a carbon number of 0 (preferably 12 to 18). In order to immobilize the cell membrane on the surface of the metal thin film of the measurement chip using such a phospholipid, first, the cell membrane extracted from the target biological species and the phospholipid are mixed in an appropriate buffer solution, and then mixed. Prepare a uniform dispersion by sonication or the like. At this time, the concentration of the protein in the cell membrane is generally adjusted to 3 to 10% by weight of the phospholipid, but the optimal value varies depending on the concentration of the receptor contained in the cell membrane, and therefore the optimal value may be outside this range. possible. Next, a measuring chip (commercially available) having a metal thin film surface covered with an alkyl chain as described above is inserted into the main body of the SPR measuring apparatus, and the cell membrane / phospholipid mixed dispersion is flowed into the flow cell. If this is the case (generally 1-2 hours), the cell membrane is immobilized on the metal thin film surface. With the cell membrane securely immobilized as a ligand in this way, the interaction between the cell membrane and the substance to be measured is detected by surface plasmon resonance, and the resonance signal (RU) is detected.
Value) to determine the presence or absence of interaction or the strength of the interaction,
The target physiologically active substance may be selected accordingly.

According to the present invention, a system for examining the interaction of a physiologically active substance on a cell membrane has been developed. For example, the cell membrane of a cancer cell is immobilized on the surface of a metal thin film of a measurement chip, It becomes possible to screen for a detection reagent that specifically binds to cells or a cancer therapeutic agent that destroys only cancer cells. In addition, Bacillus thuringiensis, a microbial pesticide produced by Bacillus thuringiensis, which has recently attracted attention, is mainly composed of proteins, and is mainly composed of proteins. It is known that Bacillus thuringiensis protein that specifically binds to Bacillus thuringiensis produced protein is immobilized on the target membrane of the intestinal epithelial cell of the insect on the metal thin film surface of the measurement chip. In addition, microbial pesticides can be rapidly developed. If the interaction on the cell membrane can be detected, not limited to these two examples, the field of application is wide, and great demand is expected mainly in the fields of pharmaceutical and agricultural chemical development.

[0019]

[Examples] In the following, the midgut cell membrane extracted from Japanese moth was examined.
By detecting the interaction using SRP,
Bioactive protein derived from Bacillus thuringiensis
An example will be described for the case of cleaning.
Is not limited by this embodiment.Step 1: Preparation of the midgut cell membrane fraction of Japanese moth  Continuously raised in the laboratory, Bacillus thuringiensis ser
ovar kurstaki HD-73
Remove the midgut of the terminal larva, homogenize, and
In the presence of calcium ions, the cell membrane of the midgut epithelium
Centrifugation, taking advantage of the weaker precipitate formation
Purification and extraction were performed. First, add 24 mM salt to the midgut homogenate.
Add the same amount of magnesium chloride solution and 2,500g for 15 minutes
Centrifuge to sediment undesired cell membranes.
Centrifuge at 30,000 g for 30 minutes.
Cell membranes were precipitated and collected. Repeat the same operation
After repetition, the cell membrane was further purified and used as a sample.

[0020]Step 2: A chip for measuring the midgut cell membrane of the diamondback moth
Immobilization on top  Measure the protein concentration of the extracted cell membrane, and
1 mM 2C dissolved in pha₁₄-Gly-PC (synthetic phosphorus
Lipid MW = 650, see FIG. 1)
Protein concentration is 2C by weight₁₄5 of gly-PC
%. Cell membrane and 2C₁₄-Gly-PC
Apply ultrasonic waves to the mixed solution using a sonic bath to evenly disperse.
Scatter (three times a minute). Surface plasmon measurement
Gold thin film surface on BIAcore 1000 manufactured by BIAcore
Chip covered with an alkyl chain (BIAcore
(Commercially available as a sensor chip HPA)
Cell membrane / phospholipid mixed dispersion at 1 μl / min for 110 minutes
It was fixed by flowing.

[0021]Step 3: Having insecticidal activity against Japanese moth
Preparation of proteinaceous toxin  Bacillus thuring with insecticidal activity against Japanese moth
iensis serovar kurstaki HD-73 Inkl-Jombo
Solubilization buffer [50 mM Na_Two
CO_Three(PH 10.0), 10 mM DTT (dithios
(Reitoll), 1 mM EDTA]
Incubated at 37 ° C for 1 hour. 15000rpm
Centrifuge for 5 minutes, take 230 μl of the supernatant, and
7.36 μl of ZeK (1 mg / ml) was added,
Incubated for 0 min. 1N HCl 10.8
Neutralize by adding μl and further add PMSF (proteinase K
Was added to a concentration of 1 mM, and
The sample which stopped the sampling was used as a sample.

[0022]Step 4: The midgut epithelial cell membrane of the diamondback moth
Xin interaction  Immobilized the midgut epithelial cell membrane of the diamondback moth obtained in step 2
Change the concentration of the toxin sample obtained in step 3 to the measurement chip.
Then, the interaction between the cell membrane and the toxin was measured. This
Toxin binds to receptors on the cell membrane and
Destruction of mid-gut epithelial cells by creating pores, killing Konaga
Has the effect of bringing. Surface plasmon measuring device
Binds the cell membrane to the toxin according to the concentration
(Fig. 2).
Syn did not show binding to the cell membrane (FIG. 3).

[0023]

According to the present invention, the interaction between a cell membrane and a physiologically active substance can be measured by utilizing surface plasmon resonance, and screening of a physiologically active substance can be easily performed.

[Brief description of the drawings]

FIG. 1 shows a chemical formula of a phospholipid suitable for use in the present invention.

FIG. 2 is a graph showing resonance signals of surface plasmon resonance in which the interaction between the membrane of the midgut epithelial cell of Japanese moth and the toxin derived from Bacillus thuringiensis is detected as an example of the present invention.

FIG. 3 is a graph showing a resonance signal of surface plasmon resonance in which an interaction between a membrane of the midgut epithelial cell of the Japanese moth and a toxin derived from Bacillus thuringiensis was detected as an example of the present invention. Does not interact with.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/50 G01N 33/50 Z (72) Inventor Eiichi Mizuki 1465-5 Aikawa-cho, Kurume-shi, Fukuoka Fukuoka (72) Inventor Satoko Yamashita 1465-5 Aikawa-cho, Kurume-shi, Fukuoka Fukuoka Prefecture, Japan AA05 BB12 BB15 DD03 EE02 EE05 GG04 HH01 JJ19 KK01

Claims (1)

[Claims] 1. A method for detecting the interaction between a substance immobilized on the surface of a metal thin film adhered to glass and a substance by surface plasmon resonance, wherein a cell membrane is used as the ligand and the surface of the metal thin film is exposed in the presence of a phospholipid. Fixed to
A method for screening a physiologically active substance, comprising selecting a substance that interacts with the cell membrane.