WO2001044814A1

WO2001044814A1 - Support having space-forming member

Info

Publication number: WO2001044814A1
Application number: PCT/JP2000/008766
Authority: WO
Inventors: Hiroaki Sagawa; Haruhisa Sawaragi; Masanori Takayama; Junichi Mineno; Kiyozo Asada; Ikunoshin Kato
Original assignee: Takara Bio Inc.
Priority date: 1999-12-14
Filing date: 2000-12-12
Publication date: 2001-06-21
Also published as: JPWO2001044814A1; AU1736601A

Abstract

A method of measuring the binding property of a ligand to a receptor characterized in that the process for binding the ligand to the receptor comprises using a plate-type support having a plural number of different ligands or receptors immobilized on a preliminarily determined domain on the surface thereof and a space-forming member and injecting a solution containing a substance capable of binding to the substances immobilized on the plate-type support into the space, which is formed between the plate-type support and a coating support covering the same via the space-forming member, by the utilization of capillary action.

Description

Description Technical support

The present invention relates to a measurement method for obtaining information on the binding between a ligand and its receptor useful in the field of biochemistry in a stable state, and a carrier used in the measurement method.

In recent years, experiments using microarrays, in situ hybridization, etc. to test the target immobilized on a solid support and a sample in a liquid, for example, the binding or affinity between a ligand and a receptor, are often performed. It has come to be done. In such a situation, to prevent the sample solution from drying during the reaction, a coated carrier such as a cover glass is applied so that the sample solution is uniformly contacted with the surface of the carrier on which the target is immobilized. In the step of applying the cover glass, it is usual to drop the sample solution on the solid support first and place the cover gently. In some areas, the sample and target are not in contact, and accurate results cannot be obtained. It is very difficult to apply a cover without air bubbles, and once air bubbles enter, if the cover is removed and reapplied, the solid support surface will dry, Scratched or the fixed target may shift. In other words, once bubbles enter, even if corrections are made later, accurate results are often not obtained. For example, as the above cover glass, there is a self-seal hybridization chamber manufactured by MERGEN Co., Ltd. This product has a packing shape with a thickness of about 0.5 mm around the square power bar to form a gap. The cover has two holes diagonally about the corner of the cover for the purpose of injecting the sample solution. When this cover glass is used, it is necessary to cover the cover glass with great care since the four sides of the cover glass are surrounded by the void forming members, and it is necessary to remove the cover glass similarly after the hybridizing process. Also, when the sample solution is injected, the sample solution added from one of the holes is no longer used. When the solution arrives at one of the holes, the solution stops moving, and it may be difficult to remove the bubble in question. Further, the cover glass has a large void volume and requires about 200 μl of a sample solution.

Alternatively, a cover glass can be applied to the surface of the solid support, and then the sample solution can be injected from the side by capillary action. Due to the material of the carrier, the condition of the surface of the solid phase carrier, or dirt or debris attached to the surface, air bubbles are likely to be mixed in, and it is difficult to inject the sample solution without easily mixing air bubbles. is there.

As described above, the conventional cover glass has various problems, and there has been a demand for an air gap forming member holding member that can easily remove bubbles and minimize the amount of a sample solution. Purpose of the invention

It is an object of the present invention to provide a method for injecting a sample solution between a solid support and a coated support in the above-described working procedure, in which a simple operation does not introduce bubbles and the binding between a ligand and a receptor under uniform conditions. An object of the present invention is to develop and provide a method capable of performing an operation of measuring the temperature and obtaining an accurate result, and an instrument used for the measuring method. Summary of the Invention

According to an outline of the present invention, a first invention of the present invention relates to a method for measuring the binding property between a ligand and a receptor, wherein a step of binding the ligand and the receptor includes a plurality of steps in a predetermined region on the surface thereof. A plate formed by immobilizing a different ligand or receptor, and a gap formed between the plate-shaped carrier and the coated carrier covering the upper portion of the carrier via a gap-forming member; The present invention relates to a method for measuring the binding property between a ligand and a receptor, which is performed by injecting a solution containing a substance having a binding property to a substance immobilized on a carrier in a capillary state.

In the first invention of the present invention, air bubbles generated when a solution containing a substance having a binding property to a substance immobilized on a plate-shaped carrier is injected into the plate-shaped carrier and the carrier via a void forming member From the side of the void formed between the carrier and the top of the carrier Is preferably performed. Further, the capacity of the void is preferably 200 / X1 or less, and the void formed between the plate-shaped carrier and the coating carrier covering the upper part of the carrier via the void forming member. Is preferably equal to or less than 300 μιη. Further, the contact area of the space forming member with the carrier is preferably 2.0 cm ² or less. In the first invention of the present invention, as the ligand, a nucleic acid, a peptide, a protein, an antibody, a saccharide, a sugar chain, or a cell can be suitably used. As the receptor, nucleic acids, peptides, proteins, antibodies, carbohydrates, sugar chains, or cells can be suitably used.

In the first aspect of the present invention, the coated carrier may be a coated carrier holding a space forming member. Alternatively, the plate-shaped carrier may be a plate-shaped carrier holding a gap forming member.

A second invention of the present invention provides a plate-like carrier having a plurality of different ligands or receptors immobilized in a predetermined region on the surface of the carrier, and the plate-like carrier and the carrier being interposed via a gap forming member. The present invention relates to a coated carrier characterized by holding a gap forming member for forming a gap between the coated carrier and an upper coated carrier.

The third invention of the present invention provides a plate-like carrier having a plurality of different ligands or receptors immobilized in a predetermined region on the surface of the carrier, and the plate-like carrier and the carrier being interposed via a gap forming member. The present invention relates to a plate-shaped carrier which holds a member for forming a space for forming a space between the cover-shaped carrier and an upper cover.

In the second invention or the third invention of the present invention, bubbles generated when injecting a solution containing a substance having a binding property to a substance immobilized on a plate-shaped carrier are generated via a void forming member. A combination of a coated carrier and a plate-shaped carrier that can be removed from the side surface of a void formed between the plate-shaped carrier and the coated carrier covering the upper portion of the carrier can be suitably used. Further, the volume of the void is preferably 200 μl or less, and the void formed between the plate-shaped carrier and the covered carrier covering the upper portion of the carrier via a void forming member. The thickness of the layer is preferably less than 300 μππι. Further, it is preferable that the contact area of the space forming member with the carrier is 2.0 cm ² or less. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a plan view showing a coated carrier holding a space forming member of the present invention.

FIG. 2 is a side view showing a coated carrier holding the void forming member of the present invention. Detailed description of the invention

In the present invention, the ligand is not particularly limited as long as it is a molecule recognized and bound by a specific receptor, and examples thereof include a nucleic acid, a peptide, a protein, an antibody, a carbohydrate, a sugar chain, and a cell.

Examples of ligands include agonists and antagonists for cell membrane receptors, toxins (to Xin and Venom), virus epitopes, hormones (eg, peptide hormones, steroid hormones, etc.), hormone receptors, peptides , Enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, polynucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies. In the present invention, the receptor is not particularly limited as long as it shows a binding property to the ligand, and may be a natural molecule or an artificial molecule. For example, nucleic acids, peptides, proteins, antibodies, carbohydrates, sugar chains, cells, proteins, such as enzymes, cell surface proteins (receptors), glycoproteins, and monoclonal antibodies.

That is, in the present invention, the contact surface characteristics of the ligand and the receptor having the binding property to the ligand are mutually complementary.

In the present invention, the carrier is not particularly limited as long as it is a non-porous material having a structure with a smooth surface, and for example, glass such as a cover glass or a slide glass or a polymer such as a transparent plastic is preferable. Can be used for That is, there is no particular limitation as long as the sample solution can be held.

In the present invention, the gap forming member for forming a gap for injecting the sample solution by capillary action between the plate-shaped carrier and the coated carrier is at least one of a plate-shaped carrier and a coated carrier. It is sufficient that the gap is held on one side, and at least one end of the gap to be formed is open. The material of the void forming member and the holding method are not particularly limited, as long as they do not elute or emit fluorescence during the binding reaction between the ligand and the receptor. Although not particularly limited, Teflon or the like can be suitably used. In the present invention, the capacity of the void is not particularly limited, but refers to, for example, the capacity of the void formed between the plate-shaped carrier and the coated carrier. The void preferably includes a predetermined region where the ligand or receptor is immobilized. The space forming member may be added to the plate-shaped carrier or the coated carrier, or may be formed integrally.

That is, air bubbles generated when a solution containing a substance having a binding property to the substance immobilized on the plate-shaped carrier is injected into the plate-shaped carrier and the coated carrier that covers the upper portion of the carrier via the space forming member. The shape of the member for forming the gap and the shape of the member for forming the gap and the member may be a combination of the coated carrier and the plate-shaped carrier so as to be removed from the side surface of the gap formed between them. However, it may be held out of alignment. The volume of the void formed by the void forming member of the present invention is preferably not more than 200 μ1 per 4.84 cm ² of the area on the carrier to be brought into contact with the solution. It is more preferably at most 100, particularly preferably at most 50 μl. Further, the thickness of the void formed between the plate-shaped carrier and the coated carrier covering the upper portion of the carrier via the void forming member is preferably in the range of 1 to 300, more preferably 3 to 300. 1100 / xm, particularly preferably 5 to 60 μπι. Further, the contact area of the void forming member with the carrier is preferably 4.84 cm ² or less, more preferably 2.0 cm ² or less, more preferably, per area of 4.84 cm ² on the carrier to be brought into contact with the solution. It is 1.5 cm ² or less, particularly preferably 1.3 cm ² or less.

The plate-like carrier used in the present invention is not limited as long as it is used for measuring the binding property between the ligand and the receptor.For example, a slide glass on which the ligand is immobilized is preferably used. can do.

The coated carrier is not particularly limited as long as it is used for performing a binding reaction between a ligand and a receptor on a solid phase carrier.For example, the coated carrier is used for coating slide glass on which the above ligand is immobilized. Can be suitably used. The space formed via the space forming member is not particularly limited as long as the sample solution can be injected by capillarity from at least one end of the space while the coated carrier is placed on the plate-shaped carrier.

The material, size, etc. of the plate-shaped carrier and the coated carrier are not particularly limited, and are not limited to ligands. In the step of measuring the binding of the receptor, whatever is suitable for the purpose of the present invention.

A preferred example of the present invention is a coated carrier obtained by adding Teflon to a cover glass as a member for forming voids by Teflon printing. The teflon printing may be performed by a method usually performed on the glass surface, and the method is not particularly limited as long as the space forming member does not elute or the eluted member does not emit fluorescence during the binding reaction between the ligand and the receptor. Absent. In addition, a plate-shaped carrier obtained by adding Teflon as a member for forming a gap to a slide glass by Teflon printing is also provided. The present invention also encompasses a case where a teflon film is sandwiched between a plate-shaped carrier and a coated carrier and used as a member for forming voids.

Figure 1 shows an example of a Teflon print pattern on a cover glass. That is, FIG. 1 is a diagram showing a coated carrier holding the void forming member of the present invention. The black painted area in the figure is the Teflon printed area. FIG. 2 is a schematic view showing the thickness of the gap forming member used in the present invention. That is, FIG. 2 is a view of the coated carrier and the plate-shaped carrier as viewed from the side, and the black-painted portions in the figure are the void forming members. A shows a case without a gap forming member, and B shows a case with a gap forming member.

According to the present invention, a gap is formed between the plate-shaped carrier and the coated carrier via a gap-forming member, and contains a substance having a binding property to a substance fixed to the plate-shaped carrier from an end of the gap. It became possible to inject the sample solution by capillary action, and it was possible to provide an appropriate space for efficiently performing the binding reaction.

Further, by using a container for binding reaction between a ligand and a receptor comprising the plate-shaped carrier, the coated carrier, and the void-forming member of the present invention, it becomes possible to inject the sample solution into the voids formed by capillary action. This has made it possible to easily and reliably prevent the incorporation of air bubbles, which has been a problem with the conventional method of applying the cover after dropping the liquid.

In addition, normal coated carriers without voids, for example, the surface condition of coated carriers or plate-shaped carriers that pose a problem in the method of injecting a sample solution from the side with a cover glass placed first, or adhered to the surface The sample solution can be injected by capillary action into the space formed between the plate-shaped carrier and the coated carrier without being affected by dirt, dust, and the like.

In particular, a gap is formed between the plate-shaped carrier and the coated carrier via a gap forming member, When removing air bubbles generated when a sample solution containing a substance having a binding property to the substance immobilized on the plate-shaped carrier from the end of the gap is injected by capillary action, the bubbles are removed from the side surface of the gap forming member. Therefore, there is an advantage that the removal can be performed more easily than the removal from the holes provided in the surface of the conventional coated carrier.

In addition, the carrier for holding a void-forming member of the present invention has an advantage that the surface of the DNA array is less likely to be damaged when voids are formed since the contact area with the carrier is minimal. In particular, since the contact area is small, there is an advantage that the diffusion of the sample solution into the voids is efficient with respect to an object in which the entire area of the force bar glass is surrounded by a spacer.

Further, since the void-forming member holding carrier of the present invention does not have an adhesive substance, the installation position of the coated carrier can be easily corrected at the time of forming the void.

In the method for measuring the binding property between a ligand and a receptor according to the present invention, the step of binding the ligand and the receptor by using the above-mentioned void-forming member-holding carrier may include the steps of: Alternatively, a plate-like carrier having a receptor immobilized thereon, and a gap formed between the plate-like carrier and a covering carrier covering the upper portion of the carrier via a gap-forming member, the plate-like carrier from the end of the gap. It can be carried out by injecting a solution containing a substance having a binding property to the substance immobilized on the capillary by capillary action. Further, bubbles generated when a solution containing a substance having a binding property to the substance immobilized on the plate-shaped carrier are injected into the plate-shaped carrier and a coated carrier that covers an upper portion of the carrier through a gap forming member. The sample solution can be easily removed from the side surface of the void formed between the DNA chip and the DNA chip, even for beginners handling the DNA chip. Further, by using the carrier for holding a void-forming member of the present invention, the amount of the sample solution can be reduced in the measurement of the binding property between the ligand and the receptor, so that a strong signal is obtained after the hybridization. Can be.

From the above, it has become possible to provide a method and a container that allow anyone to obtain a stable and reliable result in a binding affinity experiment between a ligand and a receptor. Example Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to the scope of the examples.

Example 1

As in the pattern shown in Fig. 1, Teflon printing is performed on cover glass (Matsunami Glass Industry Co., Ltd.) using the screen printing method described in Screen Printing Handbook, Second Edition, 1990 (Japan Screen Printing Technology Association). As a result, cover glasses (22 mm × 22 mm and 22 mm × 43 mm) to which a member for forming a gap was added were produced. In addition, Teflon printing was performed on the slide glass (Matsunami Glass Industry Co., Ltd.) at the position corresponding to Fig. 1 to create a slide glass with a gap forming member added. Example 2

The cover glass with the void-forming member prepared in Example 1 (indicated by B in FIG. 1), the cover glass with no added member (Matsunami Glass Co., Ltd., and a commercially available DNA microarray (Cyano CH IP Version 1) 0: manufactured by Takara Shuzo Co., Ltd.), and the size of the cover glass was 22 x 43 mm.

The test solution was a culture of the cyanobacterium PCC 683 under light conditions, the total RNA was extracted by a conventional method, and the resulting RNA was used as a template to perform a reverse transcription reaction in the presence of a fluorescently labeled substrate nucleotide. Then, a fluorescently labeled probe was prepared. The fluorescently labeled probe was dissolved in water and then subjected to hybridization.

In the experiment, in the hybridization step, about 201 drops of the hybridization solution was added to the above-mentioned DNA microarray, and then a normal cover glass was placed on the DNA microarray, and a gap-forming member with a thickness of 20 m and 70 / zm was attached first. After the cover glass was grounded to the DNA microarray, a comparison was made by injecting a hybridizing solution of 30 μl for a thickness of 20 μm and 70 μl for a thickness of 70 μm.

As a result, in the method using the cover glass with the member for forming the voids, no bubbles were mixed in, the operation was simple, the background was very low, and a highly sensitive result was obtained.

On the other hand, in a normal method using a normal cover glass, bubbles are mixed in, The operation has to be redone. In addition, the results obtained were often impossible to analyze.

In particular, a comparison of the above methods was performed for 10 novice users of the DNA microarray, and 9 out of 10 people were mixed with air bubbles in the usual way.

In contrast, with the method using a cover glass with spacer, 8 out of 10 persons could inject the hybridizing solution without introducing air bubbles.

From the above results, it was confirmed that the binding reaction between the ligand and the receptor, which was conventionally difficult, can be efficiently performed in a uniform system by using the coated carrier to which the member for forming the voids is added. In addition, a DNA microarray was prepared using a slide glass to which a gap-forming member was added, and a hybridization solution was injected into the gap formed between the cover glass and the DNA microarray. We were able to set good high-prid soybean conditions. Example 3

(1) Using a commercially available DNA microarray (Human Cancer CH IPVer. 2.0: manufactured by Takara Shuzo Co., Ltd.) and the slide glass with a holding member for forming a gap prepared in Example 1, the gap formed in Example 1 Hybridization experiments were performed on cover glass with forming members, cover glass without components (Matsunami Glass Co., Ltd.), and commercially available Self-Seal Hybridization Chamber & Cover Dots (Mergen). .

As a test solution, HL60 cells (Dainippon Pharmaceutical Co., Ltd. ± M) were cultured. After recovering the cells from the culture solution, total RNA was extracted using a Trizol reagent (Gibco BRL † ± M). Using the obtained RNA as a template, a reverse transcription reaction was carried out in the presence of a fluorescently-labeled substrate nucleotide using an RNA Fluorescent LaCeLiteCoreKit to prepare a fluorescent-labeled probe. The fluorescent labeled probe was subjected to hybridization after dissolving in water.

The same method as in Example 2 was used for evaluating the cover glass.

As a result, in the method using the cover glass with the void forming member prepared in Example 1, no air bubbles were mixed, the operation was simple, and the background was very low. Highly sensitive results were obtained. On the other hand, when a MERGEN cover glass was used, air bubbles could not be removed from the cover glass in some cases. From this, it was confirmed that when removing bubbles, it is better to remove from the side surface of the void formed by the void forming member than to remove from the upper surface of the coated carrier. In addition, when using normal cover glass, air bubbles were mixed in and the operation had to be repeated. In addition, the results obtained were often impossible to analyze.

For the amount of sample solution used for hybridization, if the area of the carrier that comes into contact with the solution is 4.84 cm ² , when using a cover glass without a void forming member, 101, MERGEN The value was 200 μ1 when using the company cover glass, and 15/1 when using the cover glass with a 20 m-thick void forming member prepared in Example 1.

Also, in a comparative experiment in which 10 beginners handling DNA microarrays as described in Example 2 were used as panels, when MERGEN Neh slide glass was used, air bubbles were mixed in 6 out of 10 persons.

On the other hand, in the case of using the carrier for holding a void-forming member of the present invention, eight out of ten persons could inject the hybridizing solution without mixing air bubbles. From the above results, it was confirmed that the binding reaction between the ligand and the receptor, which had been difficult in the past, could be efficiently performed in a uniform system by using the coated carrier to which the void forming member was added. In addition, a DNA microarray was prepared using a slide glass to which a gap-forming member was added, and a hybridization solution was injected into the gap formed between the slide glass and the cover glass. It was possible to set good hybridization conditions. Example 4

A member holding carrier for forming a gap was examined. The carrier, as shown in FIG. 2 B, in the case the contact area of the hybridization solution of 4. 84 cm ² and 9. 46 cm ^2, the thickness of the air gap forming member in the range of 0 to 500 m The adjusted one was used. Experimental conditions other than the height of the void forming member were the same as in Example 3.

As a result, the thickness of the gap forming member is 300 m or less, especially 100 μm or less. Within the range, bubbles could be easily removed, and the hybridization with the sample specimen was good.

In addition, when the volume of the voids formed by the void forming member was analyzed from the amount of the Hypuri solution, the signal intensity obtained was higher as the Hypuri solution concentration (sample solution concentration) was increased and the No. and Iburi solutions were smaller. Was confirmed. High Priestess in contact area 4. 84 cm ² of the solution (22 X 22 mm), using liquid amount. 10 to 50 mu 1 of the range, or the contact area is used solution at ^{9. 46 cm 2 (22 X 43} mm) It was confirmed that the range of the amount of 20 to: L00 μl was effective from the viewpoint of the obtained signal intensity, and that it was good to adjust the gap so that the above volume was obtained.

Further, the area where the void forming member is in contact with the coated carrier or the plate-shaped carrier may be adjusted so as to have the capacity of the void. If the contact area of the Hypli solution is 4.84 cm ² , 0 . 9 cm ² or less, if the contact area of 9. 46 cm ^2, 1. good results in 3 cm ² or less was obtained.

From the above, it was confirmed that the carrier for forming voids of the present invention was useful in hybridization of a DNA chip. Industrial applicability

According to the present invention, there is provided a container comprising a plate-shaped carrier, a coated carrier, and a member for forming voids, for performing receptor detection with good reproducibility using a high-density ligand array. Thus, a simple and efficient method for measuring the binding property between a ligand and a receptor is provided.

Claims

The scope of the claims

1. In the method for measuring the binding property between a ligand and a receptor, the step of binding the ligand and the receptor is performed by using a plate-like carrier having a plurality of different ligands or receptors immobilized on a predetermined region of its surface. A gap formed between the plate-shaped carrier and the coated carrier covering the upper portion of the carrier via the space-forming member has a property to bind to a substance fixed to the plate-shaped carrier from an end of the gap. A method for measuring the binding property between a ligand and a receptor, which is performed by injecting a solution containing a substance having the above-mentioned substance by capillary action.

2. Bubbles generated when a solution containing a substance having a binding property to the substance immobilized on the plate-shaped carrier is injected into the plate-shaped carrier and the coated carrier covering the upper part of the carrier via a space forming member. 2. The method for measuring the binding property between a ligand and a receptor according to claim 1, wherein the binding between the ligand and the receptor is removed from a side surface of a void formed between the two.

3. The method for measuring the binding property between a ligand and a receptor according to claim 1 or 2, wherein the capacity of the void is 200 μI or less.

4. The thickness of the gap formed between the plate-shaped carrier and the coated carrier covering the upper portion of the carrier via the gap forming member is 300 m or less. The method for measuring the binding property between a ligand and a receptor according to any one of Items 1 to 3.

5. The measurement of the binding property between the ligand and the receptor according to any one of claims 1 to 4, wherein the contact area of the gap forming member with the carrier is 2.0 cm ² or less. Method.

6. The ligand is a nucleic acid, peptide, protein, antibody, carbohydrate, sugar chain, or cell. 6. The method for measuring binding '14 between a ligand and a receptor according to any one of Items 5 to 5.

7. The method according to any one of claims 1 to 6, wherein the receptor is a nucleic acid, a peptide, a protein, an antibody, a carbohydrate, a sugar chain, or a cell.

8. The coated carrier is a coated carrier holding a member for forming voids. The method for measuring the binding property between a ligand and a receptor according to any one of claims 1 to 7.

9. The method for measuring the binding property between a ligand and a receptor according to any one of claims 1 to 7, wherein the plate-shaped carrier is a plate-shaped carrier holding a void forming member.

10. A plate-like carrier in which a plurality of different ligands or receptors are immobilized in a predetermined region on the surface of the carrier, and a coated carrier that covers the upper part of the plate-like carrier and the carrier via a gap forming member. A coated carrier characterized by holding a gap forming member for forming a gap between the coated carriers.

11. A plate-like carrier in which a plurality of different ligands or receptors are immobilized in a predetermined region on the surface of the carrier, and a coated carrier that covers the plate-like carrier and the upper part of the carrier via a gap-forming member. A plate-shaped carrier which holds a member for forming a gap for forming a gap between the plates.

12 2. Bubbles generated when a solution containing a substance having a binding property to the substance immobilized on the plate-like carrier are injected into the plate-like carrier and the upper part of the carrier via the gap forming member. 13. The coated carrier and the plate-shaped carrier according to claim 11 or 12, wherein the shape is such that the shape is removed from a side surface of a void formed between the coated carrier and the covered carrier.

13. The coated carrier and the plate-shaped carrier according to claim 12, wherein the capacity of the void is 20 O / x1 or less.

14. The thickness of the void formed between the plate-shaped carrier and the coated carrier covering the upper portion of the carrier via the void-forming member is 300 / zm or less. Item 14. The coated carrier and the plate-shaped carrier according to Item 12 or 13.

1 5. Contact area to the carrier of the gap forming member, 2. O cm ² coated carrier and the plate-like carrier as claimed in any one of claims 1 2 to 1 4, characterized in that less.