JP3086812B2 - Method for transferring fat-soluble compound from fixed layer to resin film - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for transferring a fat-soluble compound from a fixed layer of an analytical element to a synthetic resin film. Specifically, a method and a transfer device for transferring a fat-soluble compound developed on a fixed layer of an analytical element such as a thin-layer chromatography plate to a synthetic resin film, and further, if necessary, detecting the fat-soluble compound on the transfer film Or a method for separating a fat-soluble compound from a transfer membrane.

[0002]

2. Description of the Related Art Lipids, glycolipids,
Lipid-soluble compounds such as phospholipids and glycoproteins transmit their information to organs inside the cell membrane in response to stimulation from outside the cell membrane, and play an important role in cell proliferation, differentiation, canceration or metastasis Have emerged from recent studies. However, it is difficult to separate and purify the liposoluble compound that constitutes the cell membrane, and the analysis requires a complicated operation. That is, extraction of lipids with an organic solvent,
Separation by chromatography, identification using gas chromatography, and the like are required. For this reason, research on fat-soluble compounds has been distant from researchers in other fields, and research has been delayed. Therefore, development of a method for simply and quickly analyzing and measuring the activity of fat-soluble compounds and lipid metabolizing enzymes has been desired.

[0003] Thin-layer chromatography is widely used, particularly in the field of handling compounds of natural products (plants and marine products), such as in the fields of medicines, fragrances, cosmetics and livestock. In the analysis by thin-layer chromatography, first, extraction with an organic solvent is performed, and after separation by thin-layer chromatography, the target substance is scraped off from the thin-layer chromatography plate, the target substance is extracted with a solvent, and further, Purification, structural analysis, or measurement of bioactivity. These operations are also complicated, and since there are many parts depending on the experience of the operator, there is a drawback that individual differences easily appear.

[0004] In particular, thin-layer chromatography is generally used for qualitative purposes, and is rarely used for quantitative purposes. This is because in thin-layer chromatography, the fixed layer supporting the target substance is thick, so that the detection sensitivity only on the surface of the fixed layer is lower than in other chromatography, and is not suitable for quantitative analysis. Furthermore, it was also difficult to quantitatively recover the target substance from the fixed layer.

[0005]

SUMMARY OF THE INVENTION The present inventors have sought to solve the above-mentioned drawbacks in analytical methods such as thin-layer chromatography and to develop a method for simply and rapidly analyzing and measuring fat-soluble compounds. As a result of intensive research, it was possible to transfer the separated fat-soluble compound to a synthetic resin membrane at a high recovery rate by using a thin-layer chromatography plate or the like, and the transferred fat-soluble compound was thin. It has been found that handling is easier than operating on a layer chromatography plate, and the target substance can be detected with high sensitivity. The present invention is based on such findings.

[0006]

Therefore SUMMARY OF THE INVENTION The present invention, after impregnating the transfer organic solvents in the fixed layer of the analytical element carrying a lipophilic compound, synthesized the fixed layer surface carrying the lipophilic compound The present invention relates to a method for transferring a fat-soluble compound from an analytical element, wherein a resin film is brought into contact with the substrate under heating to transfer the fat-soluble compound from the fixed layer to the synthetic resin film. Further, the present invention provides (1) a press having a smooth flat surface.
An upper lid portion provided in the center of the inner side of the pressure plate portion, (2) a fixing layer of the analysis element and a synthetic resin film are brought into contact with each other.
The analysis element and the synthetic resin film are placed in an overlapping state.
A mounting plate that can be placed at the center of the upper surface
(3) connecting means for connecting the upper lid portion and the mounting table,
And (4) from the fixed layer impregnated with the organic solvent for transfer,
The fat-soluble compound supported on the fixed layer to the synthetic resin film
Including temperature setting means capable of controlling a heating temperature for transfer, a pressing plate portion of the upper lid portion or a mounting plate of the mounting table.
At least one of the parts is connected to a heating source to form a heating plate
The present invention also relates to a transfer device characterized by the above.

Hereinafter, the present invention will be described in detail. In the present specification, the “lipid-soluble compound” is an organic compound that is insoluble or poorly water-soluble and is soluble in an organic solvent or an aqueous organic solvent, and includes, for example, natural products (particularly, biological substances and drug metabolites), pharmaceuticals, and cosmetics , Food additives, or substances present in the environment (eg, pesticides, pollutants). These compounds include, for example, terpenes, steroids, benzvilenes, or tars. Representative lipid-soluble biological substances include lipids, glycolipids, phospholipids, lipid-soluble vitamins, hormones, and glycoproteins.

In the present specification, the analytical element is a support and a fixed layer carried thereon (or a fixed layer which is also a support).
And is an element that can be individually separated in the fixed layer due to differences in various parameters (for example, distribution coefficient and electric charge) included in the multi-component mixed system.
In the present invention , since the fixed layer is brought into surface contact with the synthetic resin film, the analytical element must have a structure capable of surface contact and have the fixed layer.

A typical analytical element is a flat plate (support) made of glass, aluminum, plastic, or the like.
A test piece for thin layer chromatography (that is, a thin layer chromatography plate) having a fine powder thin layer (fixed layer) such as silica gel, alumina, or cellulose supported thereon, and a paper chromatograph using a filter paper as a support and a fixed layer. Examples include a test piece for chromatography (that is, filter paper), a test piece for zone electrophoresis using a gel as a support and a fixed layer (that is, a gel for electrophoresis), and the like. This onset bright, when applied to thin-layer chromatography, and improved detection sensitivity than the conventional method, the operation also becomes easy, further, it allows for quantitative analysis was extremely difficult in the conventional method.

The compounds used as a transfer organic solvent used in this onset bright do not dissolve virtually fat-soluble compound is at room temperature,
As the compound is heated to the transfer temperature, the fat-soluble compound starts to dissolve to some extent, and can itself evaporate during the transfer step at the transfer temperature, and a compound having amphiphilicity is particularly preferable. Representative organic solvents for transfer are, for example, lower alcohols, especially lower alcohols having 1 to 4 carbon atoms, such as methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol or mixtures thereof. Further, an aqueous solution of the organic solvent or a mixture thereof can also be used as the organic solvent for transfer.

[0011] In this onset bright, kind of analytical element can be suitably select an appropriate transfer organic solvents depending on the type of fat-soluble compounds to be transferred, miss lipophilic compounds transferred to the transfer solvent It is particularly preferable to use an aqueous solution of a lower alcohol or a mixture of lower alcohols in view of the absence. When an aqueous alcohol solution is used, the alcohol concentration is preferably about 30 to 80 (v / v)%.

The amount of the transfer organic solvent used can be appropriately selected depending on the analysis element. For example, in the case of thin-layer chromatography, the organic solvent only needs to be immersed in the entire fixed layer, and in the case of paper chromatography, the organic solvent need only be immersed in the entire filter paper. In the case of electrophoresis, an organic solvent (transfer solvent) may be immersed in an electrophoresis membrane (cellulose acetate).

It is preferable to add a cation to the above-mentioned organic solvent for transfer because transfer efficiency is improved. The cation is preferably a metal ion, and can be added to the transfer organic solvent in the form of a salt with an inorganic acid. As metal ions, for example, magnesium, calcium, cadmium,
Aluminum, tin, antimony, or cobalt ions can be used. From the viewpoint of environmental pollution, it is preferable to use calcium or magnesium.

As the synthetic resin film, any resin film capable of supporting a fat-soluble compound to be transferred and having organic solvent resistance can be used. In particular, it further has heat resistance and / or water resistance. It is preferable to use a film made of a synthetic resin. Here, the respective resistances may be such that they do not denature when they come into contact with an analytical element containing an organic solvent for transfer under heating (about 200 ° C. or lower). According to the confirmation by the present inventors, specifically, a fluororesin, particularly polyvinylidene difluoride (PVDF); a cellulose resin, particularly nitrocellulose; a saturated polyester resin,
In particular, polyethylene terephthalate; vinylidene chloride resin, particularly polyvinylidene chloride; polyamide resin, particularly nylon 6,6, can be suitably used. It is preferable to use a fluororesin, particularly polyvinylidene difluoride (PVDF), because it is excellent in heat resistance and organic solvent resistance. Although the thickness of the synthetic resin film is not particularly limited,
The thinner the better the transfer efficiency.

In the present invention, generally, a multi-component mixture system test sample by a conventional analytical techniques, by conventional mobile phase (developing solvent) or voltage, the fixed layer of the analytical element is separated to expand the fixed layer An organic solvent for transfer is immersed. In addition,
Oite this onset bright, the supporting method of lipophilic compounds to the fixed layer is not particularly limited to the embodiments, it may be immersed transfer organic solvents in the pre-deployed state. In the case of a test piece for thin layer chromatography (plate) and a test piece for paper chromatography (filter paper), after the developing solvent is air-dried, for example,
The plate or the filter paper is immersed in the transfer organic solvent tank, or the transfer organic solvent is sprayed and immersed in the fixed layer. In the case of electrophoresis test pieces, put the transfer organic solvent in a thin container,
Immediately immerse the membrane in a small amount of transfer solvent. In the immersion step using the transfer organic solvent, it is preferable to treat the analysis element in the horizontal direction so that the fat-soluble compound in the fixed layer does not move.

The test piece treated with the transfer organic solvent is brought into contact with the synthetic resin film before the solvent is dried. It is preferable that the analytical element test piece is placed horizontally while preventing movement of the fat-soluble compound in the fixed layer, and a synthetic resin film is placed thereon. In the present invention, this contact step is heated.
It carried out under preferably you conducted under pressure. The degree of heating is preferably a temperature at which the organic solvent for transfer is vaporized and the fat-soluble compound to be transferred is sublimated, melted or eluted. Therefore, the heating temperature is appropriately selected depending on the organic solvent for transfer and the test sample to be used.However, when an aqueous lower alcohol is used as the organic solvent for transfer and thin layer chromatography in which a biological sample is developed is processed, about It is preferred to heat at 120-200 ° C. for about 20 seconds to about 5 minutes. In addition, the degree of pressure guarantees perfect contact between the fixed layer and the synthetic resin film,
It is sufficient that no non-contact portion is generated.

In the heating method, sufficient heat is supplied to the contact surface between the fixed layer and the synthetic resin film, so that the organic solvent for transfer is vaporized and the fat-soluble compound to be transferred is sublimated, melted or eluted,
Furthermore, there is no particular limitation as long as it does not substantially affect other than the contact surface. When the thickness of the analysis element is thin and the analysis element can efficiently conduct heat to the contact surface, transfer from the synthetic resin film or from the analysis element can be performed. There is no significant effect on efficiency. However, when the thickness of the analysis element is large, for example, in the case of a test piece for thin-layer chromatography or electrophoresis, it is preferable to heat from the outside of the synthetic resin film.

FIG. 1 is a perspective view schematically showing one embodiment of the transfer device according to the present invention. The transfer device 10 includes an upper lid 1
And the mounting table 2. The upper lid 1 and the mounting table 2 are connected at their respective ends 3 by an appropriate fixing tool 4 and are connected at their opposite ends 5 so as to be openable and closable. The method of connecting the upper lid 1 and the mounting table 2 is not limited to the above-described embodiment. For example, elastic members (for example, springs) connected to the upper lid 1 and the mounting table 2 are provided at four inner corners of the upper lid 1 and the mounting table 2. Place the entire lower surface of the upper lid 1 on the mounting table 2
May be able to move up and down in parallel with the upper surface.

A pressing plate 11 having a smooth surface is provided at the center of the inside of the upper lid 1. The pressing plate portion 11 may be made of an inelastic material, but is preferably made of an elastic material such as a synthetic resin foam because an appropriate contact pressure can be provided at the time of the contacting step. When the elastic pressing plate 11 is used, it is preferable to provide a stopper 12 near the open / close end 5 of the upper lid 1 (or the mounting table 2) to control the contact pressure. Pressing plate 11 to a suitable heating source (not shown)
It is preferable that the heating plate is connected to the heating plate, and a temperature setting means (not shown) for controlling the heating temperature is provided. On the upper surface of the mounting table 2, a mounting plate portion 21 on which an analysis element is placed
Is provided at the center. In some cases, a guide fixing part 22 may be provided around the mounting plate part 21. The mounting plate 21 may be connected to an appropriate heating source (not shown) to form a heating plate, and a temperature setting means (not shown) for controlling a heating temperature may be provided.

When transfer is performed by using the transfer device 10, an appropriate heating temperature is set by a temperature setting means, and the upper cover 1 is opened. Subsequently, the analysis element previously impregnated with the organic solvent for transfer is placed on the mounting plate portion 21 of the mounting table 2 with the fixing layer facing upward, and a synthetic resin film is further placed on the fixing layer. Overlap, preferably a glass fiber filter paper is overlaid on the synthetic resin membrane and the top lid 1 is closed. The analysis element, the synthetic resin film, and the glass fiber filter paper are pressed between the mounting plate portion 21 and the pressing plate portion 11.

At this time, the glass fiber filter acts as a buffer against the pressing pressure. Further, since the heating is performed from the pressing plate 11 side, the fat-soluble compound moves from the fixed layer to the synthetic resin film together with the transfer organic solvent, and at that time, the transfer organic solvent evaporates. The evaporated organic solvent for transfer is discharged from between the glass fiber filter papers. The fat-soluble compound moves in a sublimation state, a melting state, or an elution state, and aggregates on the synthetic resin film. When the synthetic resin film has an appropriate thickness, the fat-soluble compound aggregates on the surface where the synthetic resin film comes into contact with the glass fiber filter paper. After a predetermined time has elapsed, the upper lid 1 is opened, and the synthetic resin film is taken out. When performing the transfer using the same analysis element continuously, a new synthetic resin film is placed on the fixed layer, and the same operation as described above can be repeated.

When the mounting plate portion 21 is connected to an appropriate heating source (not shown) to form a heating plate, a glass fiber filter is placed on the mounting plate portion 21 of the mounting table 2. A synthetic resin film may be stacked thereon, and an analysis element impregnated with an organic solvent for transfer may be placed on the synthetic resin film with the fixed layer facing down. By the contact treatment, substantially only the fat-soluble compound can be transferred onto the synthetic resin film in a pure form. In addition, the position on the fixed layer can be preserved and transferred. Further, by appropriately selecting the transfer organic solvent, transfer from the fixed layer to the synthetic resin film can be performed extremely quantitatively.

The lipophilic compound thus transferred can be detected, analyzed or measured on the synthetic resin film by various methods. These detection, analysis or measurement operations are easier to handle on a synthetic resin film than on the fixed layer of the analysis element. In particular, since fat-soluble compounds that can generally be handled only in an organic solvent can be handled on a synthetic resin membrane in a state separated from the organic solvent, many experiments and analyzes that have been difficult until now Will be provided, and a new tool for researching fat-soluble compounds will be provided. Further, since proteins, bacteria, viruses, lectins and the like that bind to the transcribed fat-soluble compound can be detected, they can be used for analysis of antibacterial agents, antiviral agents and lipid binding proteins. Further, purification and separation from now on can be performed easily.

When a fat-soluble compound is detected on a synthetic resin film, for example, a known color former can be used.
As the color former, a normal color former used for detecting a fat-soluble compound on a fixed layer of an ordinary analytical element can be used as it is. The inventor of the present invention has proposed that, for glycolipids and sialic acid-containing glycolipids, for example, for orcinol reagents or resorcinyl reagents, and for phospholipids, the Dittmer reagent contains about 40 to 60% of methyl alcohol. It has been found that the use of the prepared color former improves the affinity for the transfer film and also improves the color stability.

As described above, since the transferred fat-soluble compound can be colored with the color-forming reagent, it can be quantitatively analyzed with very high reproducibility by an image analyzer. In addition, the detection of fat-soluble compounds on a synthetic resin film, particularly a fluororesin film, is more sensitive than the detection on thin-layer chromatography, paper chromatography, etc. It is effective for analyzing lipids in medium and cancer tissues, cancer-associated glycolipids, lipid peroxides, bioactive lipids, drug metabolites, and the like.

When a fat-soluble compound is detected on a synthetic resin film, it can be detected with high sensitivity using an enzymatic reaction and an immunostaining method. Also in this case, the enzyme reaction and the immunostaining method used for detecting a fat-soluble compound on the fixed layer of an ordinary analytical element can be used to directly detect enzyme activity in tissues and serum. , And can also be applied to serodiagnosis.

Mass spectrometry can be directly performed on the fat-soluble compound on the synthetic resin film. Also in this case, the method used for directly mass-analyzing a fat-soluble compound on a fixed layer of an ordinary analytical element can be used as it is. The fat-soluble compound transferred onto the synthetic resin film can be separated from the synthetic resin film. Also in this case, the method used for separating a fat-soluble compound from a fixed layer of a normal analytical element can be used as it is. As described above, since the fat-soluble compound transferred in a substantially pure state can be easily extracted from the synthetic resin membrane with an organic solvent, the separation and purification of trace components, which has been extremely complicated, has been achieved. It can be easily performed.

[0028]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention. Abbreviations were used for the names of the saccharides used in the following examples. These abbreviations and the names of the saccharides are described in “The New Chemistry Experiment Course, Vol. 4, Lipid III Glycolipid”, edited by The Biochemical Society of Japan (Tokyo Kagaku Dojin, 1990 Table 1 below shows a comparison with the common names and abbreviations described on July 10).

[0029]

Table 1 Terms used in the present specification Common names Abbreviations used in this specification GM1 ganglioside GM1 glucosylceramide glucocereproside GlcCer lactosylceramide cytolipin H (CDH) LacCer ceramide trihexoside P antigen (CTH) Gb ₃ Cer globoside globoside Gb ₄ Cer paragloboside paragloboside nLc ₄ Cer GM3 ganglioside GM3 GM2 ganglioside GM2 GDla ganglioside GDla GDlb ganglioside GDlb GT ganglioside GTla Gg3Cer asialo GM2 Gg3Cer Gg4Cer asialo GM1 Gg4Cer I3SO3-GalCer sulfatide I _³ SO ³ -GalCer S2-6nLc4Cer sialyl para Globo Sid IV ⁶ NeuAcα-nLc ₄ Cer SphM Sufin Myelin SM Phc phosphatidylcholine PC amino CTH amino CTH Lc ₃ Cer

Example 1 Cation Effect in Transfer Solvent 300 μg of purified GM1 ganglioside (GM1) was mixed with chloroform / methanol (2: 1, v / v).
0 ml and dissolve in a thin layer chromatography plate (M
erck: HPTLC-plate)
A fine linear spot was spotted with a 0 μl micro syringe. After air-drying well, the HPTLC-plate was washed with isopropanol / methanol (40:20, v / v) [hereinafter referred to as transfer solvent A] or isopropanol / methanol /
0.2% CaCl ₂ (40: 20: 7, v / v / v)
[Hereinafter referred to as transfer solvent B]. Immediately after immersion, the HPTLC-plate is placed on a flat surface, and polyvinylidene difluoride (manufactured by ATTO; Clear)
(Blat Membrane-P) was placed on an HPTLC-plate, and another glass filter paper (Whatman; glass microfiber filter (GF / A)) was further placed on the PVDF membrane, and the plate was heated at 180 ° C. for 30 seconds using a heat plate. Pressed. After 30 seconds, the PVDF membrane was taken out, and an orcinol reagent (200 mg of orcinol was dissolved in 11.4 ml of sulfuric acid, and distilled water was added to GM1 ganglioside transferred to the PVDF membrane, and distilled water was added to make the whole 100 m).
1 and diluted 2 times with methanol at the time of use), and the result of measurement with a chromatography scanner is shown in Table 2. In Table 2, the measured values are the actually measured values of each of the six experiments, and the recovery is the average value. X + SD means average value + standard deviation,
CV means the coefficient of variation. The addition of cations increased recovery and reduced errors.

[0031]

TABLE 2 transcription solvent amount ([mu] g) measurements ([mu] g) recovery (%) X + SD CV = % 1.4,1.0 0.93 ± 0.344 A 3.0 0.9,1.2 31.1 0 6,0.5 36.99% 2.7,2.9 2.55 ± 0.217 B 3.0 2.5,2.3 85.0 2.5,2.4 8.510%

Example 2: 300 μg of purified GM1 ganglioside (GM1) in a transfer solvent was purified using a mixed solvent of chloroform / methanol (2: 1, v / v).
0 ml and dissolve in a thin layer chromatography plate (H
(PTLC-plate) was spotted with a 10 μl microsyringe in the form of a thin line. Air dry well before HPTLC
-The plate was immersed in a transfer solvent prepared by dissolving 0.2% of various cations in the transfer solvent A for 20 seconds. Immediately after immersion, the HPTLC-plate was placed on a flat surface, and the PVDF membrane used in Example 1 was placed on the HPTLC-plate,
One glass filter paper used in Example 1 was further placed on the PVDF membrane, and the heat was adjusted to 180 ° C. with a heat plate.
Pressed for seconds. After 30 seconds, the PVDF membrane is taken out and
GM1 ganglioside transferred to the VDF membrane was developed with the orcinol reagent used in Example 1 and the result of measurement by a chromatography scanner is shown in Table 3.

[0033]

Table 3 Amount of cation added (μg) Measured value (μg) Recovery (%) No cation added 3.0 1.03 34.33 AlCl ₃ 6H ₂ O 3.0 1.83 61.00 CaCl ₂ 3.0 2.37 79.00 SnCl ₂ 3.0 2.08 69.33 CdCl ₂ 3.0 2.25 75.00 SbCl ₃ 3.0 2.18 72.67 CoCl ₂ 3.0 03 67.67 MgCl ₂ 3.0 2.20 73.33

Example 3 Influence of Temperature During Transfer 300 μg of purified GM1 ganglioside (GM1) was mixed with chloroform / methanol (2: 1, v / v).
0 ml and dissolve in a thin layer chromatography plate (H
(PTLC-plate) was spotted with a 10 μl microsyringe in the form of a thin line. After air-drying well, the thin layer chromatography was immersed in the transfer solvent B for 20 seconds, the HPTLC-plate was quickly placed on a flat surface, and the PVDF membrane used in Example 1 was placed on the HPTLC-plate.
One glass filter paper used in Example 1 was further placed on the DF membrane, and pressed with a heat plate adjusted to various temperatures for 30 seconds. After 30 seconds, the PVDF membrane is taken out and PVDF is removed.
The color of the GM1 ganglioside transferred to the membrane was developed with the orcinol reagent used in Example 1, and the result of measurement with a chromatography scanner is shown in FIG.

Example 4: Operability and recovery with the conventional operation method
Comparison of rates (1) Conventional operation method 300 μg of purified GM1 ganglioside (GM1) was mixed with chloroform / methanol (2: 1, v / v).
0 ml and dissolve in a thin layer chromatography plate (H
(PTLC-plate) was spotted with a 10 μl microsyringe in the form of a thin line. Air dry well before HPTLC
-Plate is chloroform / methanol / 0.2% Ca
It was placed in a developing solvent of Cl ₂ (60: 35: 8, v / v / v) and developed for about 15 minutes. After deployment, air-dry well,
Mark the spot of purified GM1 ganglioside,
The mark of the spot was scraped off, chloroform / methanol (2: 1, v / v / v) was added, and the mixture was mixed vigorously for 20 minutes to extract a fat-soluble compound. After completion of the mixing, the mixture was centrifuged with a refrigerated centrifuge (3000 rpm, 15 minutes), the extraction operation was performed twice, the extract was collected, concentrated in vacuo, dried, and developed with the orcinol reagent used in Example 1. Quantified.

(2) Method of the Present Invention 300 μg of purified GM1 ganglioside (GM1) was mixed with chloroform / methanol (2: 1, v / v).
0 ml and dissolve in a thin layer chromatography plate (H
(PTLC-plate) was spotted with a 0.1 ml microsyringe in the form of a thin line. Air dry well before HPTL
Plate C-plate with chloroform / methanol / 0.2% C
The solution was developed in a developing solvent of aCl ₂ (60: 35: 8, v / v / v) for about 15 minutes. After the development, air-dry well and then purimulin reagent [0.1 g of purimulin is dissolved in 100 ml of water to make a stock solution. 1.0 ml of this stock solution is mixed with 100 ml of acetone-water (4: 1, v / v) mixture. Prepared by spraying. A band of GM1 ganglioside was detected under ultraviolet irradiation, and this was marked with a colored pencil.
Next, it was immersed in the transfer solvent B for 20 seconds. Immediately after the immersion, the HPTLC-plate was placed on a flat surface, and the PVDF membrane used in Example 1 was placed on the HPTLC-plate.
One glass filter paper used in Example 1 was further placed on the VDF membrane, and pressed with a heat plate adjusted to 180 ° C. for 30 seconds. After 30 seconds, the PVDF membrane is taken out and PVDF is removed.
The portion of the mark was cut off with a cutter knife using a color pencil transferred with the GM1 ganglioside transferred to the membrane, and chloroform / methanol (2: 1, v / v) was added thereto to add 2 parts.
Extraction was performed for 0 minutes, and the extracts were collected, concentrated in vacuo, dried, and quantified by coloring with the orcinol reagent used in Example 1. Table 4 shows the results.

[0037]

Table 4 Method Addition amount (μg) Measured value (μg) Recovery (%) Conventional method 3.00 1.3, 1.5, 1.4 1.0, 0.8, 0.7 38.52 0.5, 1.4, 1.8 The method of the present invention 3.00 2.6, 2.7, 2.5 2.5, 2.4, 2.8 86.67 2.7, 2.6, 2.6

Example 5: Transcription of glycolipid and glucosylceramide (GlcCer) and lactosylceramide (LacCe) extracted from a chromogenic tissue by a conventional method and separated and purified by silica bead column chromatography.
r), ceraside trihexoside (Gb ₃ Cer), globoside (Gb ₄ Cer), paragloboside (nLc ₄ C)
er), GM3 gangliooxide (GM3), GM1 ganglioside (GMI), bovine brain ganglioside fraction (g
angliosides) and sulfatide (sul)
3 μg of each sample of the compound (fatide) in a thin-layer chromatography plate (HPTLC-plate) using chloroform: methanol: 0.2% CaCl ₂ (60:35:
8, v / v / v). The developing solvent was air-dried, and the HPTLC-plate was immersed in the transfer solvent B for 20 seconds. Immediately after immersion, the HPTLC-plate was placed on a flat surface, the PVDF membrane used in Example 1 was placed on the HPTLC-plate, and one glass filter paper used in Example 1 was placed on the PVDF membrane, and the temperature was raised to 180 ° C. Pressed on the adjusted heat plate for 30 seconds. After 30 seconds, the PVDF membrane was taken out. The transferred PVDF membrane was immersed in the orcinol reagent used in Example 1 for 2 seconds, and heated on a heat plate (100 ° C.).
Color was allowed to develop on top for 5 minutes. After color development, it was washed twice with water. H
The glycolipid color developed on the PTLC-plate gradually faded over time, but the color developed on the PVDF membrane did not fade.

FIG. 3 shows the results of color development (a) on the HPTLC-plate and the results of color development transferred to the PVDF film (b) according to the conventional method. 4 and 5 show the transfer efficiency of glycolipids. That is, FIG. 4 shows the results of transfer to a PVDF membrane after separation on an HPTLC-plate and measurement of the amount of transferred glycolipids for four types of glycolipids using a chromatography scanner. In FIG. 4, ●
Is globoside, は is lactosylceramide, ▲ is GM3 ganglioside, and △ is sialylparagloboside. As is evident from FIG. 4, all four glycolipids described above showed good linearity, and thus the method of the present invention has high reliability regarding quantitativeness. FIG. 5 shows the HPTL
Lactosylceramide (LacCer) on C-plate
After separating the lactosylceramide (LacCer) from the lactosylceramide (LacCer) transferred to a PVDF membrane, the lactosylceramide (LacCer) was transferred to a PVDF membrane. The detection sensitivity (●) measured by the scanner is shown. As is evident from FIG. 5, the transfer obtained by the method of the present invention had much higher detection sensitivity and better linearity.

Example 6 Transcription of Sialic Acid-Containing Glycolipids and Chemical Coloring GM3 ganglioside (GM3), GM2 ganglioside (GM2), G
M1 ganglioside (GM1), GDla gallaglioside (GDla), GDlb gallaglioside (GDlb)
And GT ganglioside (GT), respectively, at 0.05 μg, 0.1 μg, 0.5 μg, 1.0 μg,
Alternatively, spot an amount of 3.0 μg on a thin layer chromatography plate (HPTLC-plate), and use chloroform: methanol: 0.2% CaCl ₂ (60:35:
8, v / v / v), and the developed solvent was air-dried. Subsequently, the thin-layer chromatography plate was immersed in the transfer solvent B for 20 seconds. Immediately after immersion, the HPTLC-plate was placed on a flat surface, and the PVDF membrane used in Example 1 was HP
The glass filter paper used in Example 1 was placed on the TLC-plate, and the glass filter paper used in Example 1 was placed on the PVDF membrane, and pressed on a heat plate adjusted to 180 ° C. for 30 seconds.

After 30 seconds, the PVDF membrane was taken out. The PVDF membrane after the transfer was dissolved in a resorcinol reagent (200 mg of resorcinol in 10 ml of water, and concentrated hydrochloric acid 8
0 ml and 0.25 ml of 0.1 M copper sulfate were added, and the whole was adjusted to 100 ml with water, and diluted twice with methanol at the time of use) for 2 seconds, inserted into two glass plates, and placed on a heat plate. (100 ° C.) for 5 minutes. After color development, it was washed twice with water. According to the above method,
The result of coloring the sialic acid-containing glycolipid is shown in FIG. 6 (PVDF described on the right side of FIG. 6). In addition, similarly to the above, each sample was spotted on an HPTLC-plate, developed with the same solvent system as described above, and then a test in which the resorcinol reagent was sprayed on the HPTLC-plate to form a color was performed for comparison. The results are also shown in FIG. 6 (HPTLC-plate described on the left side of FIG. 6).

In FIG. 6, GM3 is GM3 ganglioside, GM2 is GM2 ganglioside, GM1 is GM1
Ganglioside, GDla is GD1a gallaglioside,
GDlb gallaglioside and GT are GT gangliosides. Concentration (μg)
Is the amount of spot on the HPTLC plate.

Example 7: Phospholipid transfer and chemical coloring method Phosphatidylethanolamine (P) which is a commercially available standard phospholipid ( Phospholipit kit manufactured by Funakoshi)
E), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC)
And 3 μg of each sample of sphingomyelin (SM) were applied to a thin-layer chromatography plate (HPTLC-plate).
And chloroform: methanol: 0.2% C
After developing with a solvent system of aCl ₂ (60: 35: 8, v / v / v), the developing solvent was air-dried. Then, HPTLC-
The plate was immersed in the transfer solvent B for 20 seconds. Immediately after immersion, the HPTLC-plate was placed on a flat surface, the PVDF membrane used in Example 1 was placed on the HPTLC-plate, and one glass filter paper used in Example 1 was placed on the PVDF membrane, and the temperature was raised to 180 ° C. Pressed on the adjusted heat plate for 30 seconds. After 30 seconds, the PVDF membrane was taken out, and the transferred phospholipid was immersed in a Dittmer reagent described later for 5 minutes. After color development, it was washed twice with water. The results are shown in FIG. 7 (b on the right). In addition, similarly to the above, each sample was spotted on the HPTLC-plate, developed with the same solvent system as described above, and then a test in which the Dittmer reagent was directly sprayed onto the HPTLC-plate to form a color was also performed for comparison. The results are also shown in FIG. 7 (a on the left). The color of the phospholipids developed on the HPTLC-plate faded over time, but the color developed on the PVDF membrane did not fade.

Color development with the Dittmer reagent was carried out in the following manner. That is, 4.01 g of molybdenum trioxide
Was added to 100 ml of 25N sulfuric acid and dissolved by boiling. This solution is designated as solution A. Further, 180 mg of powdered molybdenum was added to 50 ml of solution A, and the mixture was slowly boiled for 15 minutes. After allowing to cool, the supernatant is decanted to liquid B. A
The liquid and the B liquid were mixed in equal volumes to obtain a spraying reagent.
PVD after transfer treatment diluted twice with methanol at the time of use
The F film was immersed for 5 minutes. After color development, it was washed twice with water.

Example 8: Direct mass fraction of transfer fat-soluble compound
A 3 μg GM3 ganglioside (GM3) sample separated and purified from the analyzed erythrocytes by a conventional method was spotted on a thin-layer chromatography plate (HPTLC-plate), and chloroform: methanol: 0.2% CaCl ₂ (60:35:
8, v / v / v), and the developing solvent was air-dried, and the primulin reagent used in Example 4 was sprayed on an HPTLC-plate. The band of GM3 ganglioside was confirmed by UV irradiation and marked with a pencil. continue,
The HPTLC-plate was immersed in the transfer solvent B for 20 seconds. Immediately after immersion, the HPTLC-plate was placed on a flat surface, the PVDF membrane used in Example 1 was placed on the HPTLC-plate, and one glass filter paper used in Example 1 was placed on the PVDF membrane, and the temperature was raised to 180 ° C. Pressed on the adjusted heat plate for 30 seconds. After 30 seconds, the PVDF membrane is taken out, the band of GM3 ganglioside transferred to the PVDF membrane is cut out, and the structure can be directly determined by mass spectrometry. The identification of fragment ions can also be performed using a tandem mass spectrometer.

FIGS. 8 and 9 show the results of mass spectrometry of GM3 ganglioside transferred to the PVDF membrane. FIG. 8 shows the results of secondary ion mass spectrometry (SIMS), and FIG. 9 shows the results of dandem mass spectrometry (MS-MS). 8 and 9, 1151 is a molecular ion, and 860 is -O
-Glc-O-Cer, 698 is -O-Glc-OC
er and 290 indicate NeuAc-Hz0 fragments.

Example 9 Separation and Purification of Lipid Neutral glycolipid sample 2 extracted from human meconium by a conventional method
00 μg of a thin layer chromatography plate (HPTL)
C: plate), chloroform: methanol: 0.2% CaCl ₂ (60: 35: 8, v / v /
v) After primary development in a solvent system, air-dry and further chloroform: methanol: 0.2% CaCl ₂ (60:25:
4, v / v / v). After air-drying, the primulin reagent used in Example 4 was sprayed, glycolipids were detected by UV irradiation, and the band was marked with a pencil. continue,
The HPTLC-plate was immersed in the transfer solvent B for 20 seconds. Immediately after immersion, the HPTLC-plate was placed on a flat surface, the PVDF membrane used in Example 1 was placed on the HPTLC-plate, and one glass filter paper used in Example 1 was placed on the PVDF membrane, and the temperature was raised to 180 ° C. Pressed on the adjusted heat plate for 30 seconds. After 30 seconds, the PVDF membrane was taken out, the lipid transferred to the PVDF membrane was cut off with a cutter knife, and chloroform: methanol (2: 1, v / v) was used.
Lipids were extracted from the solution. This extract is again used for HPTLC
-Separation on a plate revealed that the lipids extracted from the meconium were each separated and purified, and could be rapidly purified and separated without being affected by extraction and transcription.

FIG. 10 shows a two-dimensional thin-layer chromatogram (FIG. 10A) of glycolipids obtained by directly coloring the glycolipids separated and developed secondarily on the HPTLC-plate with a primulin spraying reagent (FIG. 10A). The results of transferring the glycolipids that were secondarily developed and separated on the plate to a PVDF membrane, extracted from the PVDF membrane, and confirmed the degree of purification on an HPTLC-plate (FIG. 10B) are shown. FIG. 11 shows the yield of glycolipid purified and separated from the PVDF membrane by transferring the glycolipid developed and separated on the HPTLC-plate to the PVDF membrane by the above method.

In FIG. 10, 1 is glucosylceraside-I, 2 is glucosylceraside-II, 3 is glucosylceraside-III, 4 is lactosylceraside-I, 5 is lactosylceraside-II, 6 Is lactosylceramide-III,
7 is ceramide trihexoside-I, 8 is ceramide trihexoside-II, 9 is globoside-I, and 10 is globoside-II. Also, in FIGS. 10A and 10B, the primary development is performed from the bottom to the top, respectively, and the secondary development is further performed from the right to the left. Therefore, the movement of the sample 1 is the largest, and the movement of the sample 20 is the least. In addition, as shown in FIG. 11, the recovery rate was 88.5% for GlcCer and La
72.6% of cCer, 82.5% of Gb3Cer, G
86.2% for b4Cer, 77.0% for Lc3Cer,
nLc4Cer 68.5%, Gg3Cer 82.2%
%, Gg4Cer is 83.6%, I3SO3-GalC
er is 73.8%, GM3 is 92.0% and GM1 is 9
3.1%, S2-6nLc4Cer 75.2%, GD
73.1% of la, 84.3% of SphM, and 8 of PhC
8.2%, with an average value of 81.4%.

Example 10: Enzyme production for fat-soluble compounds
10 μg of a sample obtained by extracting aminoCTH (aminoceramide trihexoside) and sialyl paragloboside from erythrocytes for use in a conventional manner was spotted on a thin-layer chromatography plate (HPTLC-plate) in a thin line and air-dried well. And transfer the HPTLC-plate to transfer solvent B
After immersion for 0 second, the HPTLC-plate was quickly placed on a flat surface, the PVDF membrane used in Example 1 was placed on the HPTLC-plate, and one glass filter paper used in Example 1 was further placed on the PVDF membrane. And pressed on a heat plate adjusted to various temperatures for 30 seconds.

After 30 seconds, the PVDF membrane was taken out, and for amino CTH, UDP-galactosyltransferase (manufactured by Boehringer) using it as a substrate and galactose transferase derived from bovine milk (manufactured by Sigma) or cancer patients The serum was allowed to act, and the resulting reaction product paragloboside was allowed to act on the antibody H-11 against paragloboside (prepared by the present inventors), followed by the action of a peroxidase-labeled second antibody (manufactured by Kappel) to develop color. Was detected. For sialyl paragloboside, a sialidase using the same as a substrate (manufactured by Sigma; C. perf derived from Clostridium pufflinsens) is used.
ringenns), the resulting reaction product paragloboside was allowed to act on the antibody H-11, and the same peroxidase-labeled second antibody was allowed to act thereon, whereby paragloboside was detected.

As described above, glycolipids can be reacted with glycolytic enzymes or glycosyltransferases on lipids transferred to PVDF membranes, and phospholipases can be reacted on phospholipids. . After the reaction, each band is cut out and extracted with a chloroform: methanol (2: 1, v / v) solution. When this extract is separated again by thin-layer chromatography, it is possible to know which lipid has undergone the enzymatic action. This method can be used to search for enzymes in cells, tissues or serum.

As the antibody H-11, a monoclonal antibody secreted from the hybridoma was established by extracting spleen cells from a mouse immunized by injection of paragloboside, fusing the spleen cells with mouse myeloma cells, and establishing a hybridoma. .

Example 11: Protein binding to lipid 5 μm of sample obtained by extraction from human meconium by a conventional method
g was spotted on a thin layer chromatography plate (HPTLC-plate) with a thin line, and chloroform: methanol: 0.2% CaCl ₂ (60: 35: 8, v / v).
/ V), and the developed solvent is air-dried.
The TLC-plate was immersed in the transfer solvent B for 20 seconds, the HPTLC-plate was quickly placed on a flat surface, and the PVDF membrane used in Example 1 was placed on the HPTLC-plate. Use 1 glass filter paper
The sheets were placed and pressed for 30 seconds with a heat plate adjusted to various temperatures.

Thirty seconds later, the PVDF membrane was taken out, reacted with an antibody MSG-15 (prepared by the present inventors) against sialyl paragloboside, and detected with the peroxidase-labeled second antibody used in Example 9. The results are shown in FIG. 12 (b in FIG. 12). As a comparative test, after the sample was developed on an HPTLC-plate and air-dried, the antibody MSG-
15 was allowed to act, and detection was carried out using the peroxidase-labeled second antibody used in Example 9. The results are shown in FIG.
A). In FIG. 12, 1 is 0.5 μg and 2 is 1.6.
μg, 3 is 8.0 μg, 4 is 40.0 μg, 5 is 200
μg and 6 are 1000 μg of sialylparagloboside.

As described above, to the lipid transferred to the PVDF membrane, a sample which seems to bind to a lipid component such as bacteria, virus, lectin or antibody is added and reacted, and then a labeled antibody or the like which recognizes the binding component is added. , It is possible to clarify whether or not a substance that recognizes a lipid component is contained in the sample. By this method, a substance that recognizes a lipid component can be efficiently separated.

The above antibody MSG-15 includes:
Splenocytes were removed from mice immunized by injection with sialyl paragloboside, fused with mouse myeloma cells to establish hybridomas, and monoclonal antibodies secreted from the hybridomas were used.

[0058]

According to the present invention, a fat-soluble compound carried in the fixed layer of the analytical element can be easily and quickly (within about 2 minutes).
Thus, it is possible to transfer only the fat-soluble compound onto the synthetic resin film in an extremely high purity state while maintaining the position. In addition, since the fat-soluble compound on the synthetic resin film is separated from the organic solvent, various treatments can be performed without being restricted by the use of the organic solvent. Further, the handleability on the synthetic resin film is better than the handleability of the analytical element on the fixed layer. Therefore, as compared with the case where the fat-soluble compound is directly detected or measured by an analytical element such as a thin-layer chromatography plate, the operation is simplified as a whole and the accuracy is improved. Further, the fat-soluble compound in the multi-component sample can be easily and quickly analyzed and measured.

Further, by appropriately selecting the transfer organic solvent and the transfer temperature, a plurality of replicas can be obtained from the analysis element. Furthermore, for example, for the detection of glycolipids, the reagents used in conventional thin-layer chromatography plates can be used as they are, and the detection sensitivity is higher than that of conventional thin-layer chromatography plates. . Also, transcription of phospholipids is possible. And
There is almost no fading of lipids stained on the synthetic resin membrane.

[Brief description of the drawings]

FIG. 1 is a perspective view of one embodiment of a transfer device according to the present invention.

FIG. 2 is a graph showing the effect of temperature when GM1 ganglioside (GM1) is transferred from a thin-layer chromatography plate to a PVDF membrane by the method of the present invention shown in Example 3.

FIG. 3 is a photograph instead of a drawing, showing a comparison between a color development result after transfer by the method of the present invention shown in Example 5 and a color development result on a thin layer chromatography plate by a conventional method.

FIG. 4 is a graph showing the relationship between the added amount and the transferred amount when four glycolipids are transferred from a thin-layer chromatography plate to a PVDF membrane by the method of the present invention shown in Example 5.

FIG. 5 is a graph showing a comparison of detection sensitivity between the case where lactosylceramide is colored on a thin-layer chromatography plate and the case where color is transferred to a PVDF membrane and then developed by the method described in Example 5. .

FIG. 6 is a photograph instead of a drawing, showing a comparison between a color development result after transfer of a sialic acid-containing glycolipid and a color development result on a thin-layer chromatography plate according to a conventional method by the method of the present invention shown in Example 6. It is.

FIG. 7 is a photograph, instead of a drawing, showing a comparison between a color development result after transfer of phospholipids and a color development result on a thin-layer chromatography plate according to a conventional method by the method of the present invention shown in Example 7.

FIG. 8 shows secondary ion mass spectrometry (SI) of GM3 ganglioside transferred to a PVDF membrane by the method described in Example 8.
9 is a graph showing the results of (MS).

FIG. 9 shows tandem mass spectrometry (MS-MS) of GM3 ganglioside transferred to a PVDF membrane by the method described in Example 8.
9 is a graph showing the results of (MS).

FIG. 10 shows a dimensional thin-layer chromatogram (FIG. 10A) in which glycolipids separated and developed secondarily on an HPTLC-plate by the method shown in Example 9 and HPTLC-
It is a photograph substituted for a drawing which shows the result (b of FIG. 10) in which the glycolipid secondary-developed and separated on the plate was transferred to a PVDF membrane, extracted from the PVDF membrane, and the degree of purification was confirmed by HPTLC-plate.

FIG. 11 is a graph showing the yield of glycolipid purified and separated from the PVDF membrane by transferring the glycolipid separated and developed on the HPTLC-plate by the method described in Example 9.

FIG. 12 shows a PVD obtained by the method of the present invention shown in Example 11.
The antibody MSG-15 was allowed to act on the glycolipid transcribed on the F membrane, and the result was detected with a peroxidase-labeled second antibody, and the antibody was allowed to act as it was on an HPTLC-plate, and was detected with the labeled second antibody. It is a photograph substituted for a drawing which shows and compared the result.

Claims (8)

(57) [Claims] 1. A After impregnating the transfer organic solvents lipophilic compound to the fixed layer of the supported analytical element, is contacted under heating synthetic resin film on the fixed layer surface carrying the lipophilic compound, the A method for transferring a fat-soluble compound from an analytical element, comprising transferring a fat-soluble compound from the fixed layer to the synthetic resin film. 2. The method according to claim 1, wherein an organic solvent for transfer containing a cation is used. 3. The process as claimed in claim 1 or 2 contacting the synthetic resin film on the fixed layer surface under pressure. 4. A method for detecting a fat-soluble compound transferred to a synthetic resin film by the method according to claim 1 on the synthetic resin film. 5. A method for purifying a fat-soluble compound transferred to a synthetic resin film by the method according to claim 1 from the synthetic resin film. 6. (1) The pressing plate portion having a smooth flat surface
Upper lid part provided in the center of the side, (2) Make sure that the fixed layer of the analysis element and the synthetic resin film are in contact with each other.
The analysis element and the synthetic resin film are placed in an overlapping state.
A mounting plate that can be placed at the center of the upper surface
Table, (3) connecting means for connecting the upper lid part and the mounting table,
as well as (4) From the fixed layer impregnated with the organic solvent for transfer,
The fat-soluble compound supported on the fixed layer to the synthetic resin film
Temperature setting means that can control the heating temperature for transfer The pressing plate portion of the upper lid portion or the mounting plate of the mounting table.
At least one of the parts is connected to a heating source to form a heating plate
A transfer device, characterized in that: 7. A contact between the analysis element and the synthetic resin film.
The transfer device according to claim 6, further comprising a means for adjusting the pressure.
Place . Claim 8. The pressing plate portion and the placing plate portion are heat-resistant.
And a material having organic solvent resistance, and
8. The transfer device according to 7.