JP2005221293A - Microplate and measuring method of glycolipid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a production method of a carrier having repeatability and stability of measurement and suitable for high-sensitivity measurement of trace glycolipid, and a measuring method using it. <P>SOLUTION: The glycolipid is measured by using this microplate formed as a solid layer by a covalent bond from a compound including a long hydrocarbon chain. Hereby this method is suitable for micro-measurement of the glycolipid, and is useful for a research field of medical science or biochemistry, a diagnosis field or the like. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a microplate suitable for micro-measurement of glycolipid. The present invention also relates to a method for measuring a trace amount of glycolipid using the microplate.

  A glycolipid is a substance containing a water-soluble sugar chain and a fat-soluble group in the molecule, and includes a sphingoglycolipid and a glyceroglycolipid. Recent studies have revealed that these glycolipids play important roles in vivo such as signal transduction and antigen reaction, and establishment of a method for accurately measuring glycolipids is required.

  In order to measure molecules such as glycolipids that have a hydrophobic group in the molecule, it is essential to bind them to a solid layer first. Is widely used, and a method of binding glycolipids via a hydrophobic bond with polystyrene is used. Although the polystyrene carrier itself exposes the benzene ring, which is a hydrophobic group, it binds to lipids, etc. via a hydrophobic bond with the hydrophobic group, because of the stereochemical limitations of the benzene ring, The coupling is inadequate, resulting in doubts about the reproducibility and authenticity of the measurements. In addition, since the benzene ring has a small stereochemical size, its properties as a hydrophobic group are weak. Therefore, a considerable amount of the immobilized lipids deviates from the solid phase at the stage of washing with a buffer or blocking with a protein, which is an essential operation for the ELISA method or the like. As described above, the amount of glycolipid antigen immobilized on the carrier is unstable in the conventional microplate, and the amount of immobilization varies depending on the glycolipid sugar chain, so there is a limit to the measurement of the amount of glycolipid. there were.

  It has been reported that glycolipids and phospholipids having a hydrophobic group in the molecule bind well to an agarose gel having 8 carbon atoms (octyl) or a silica gel carrier having 18 carbon atoms (stearyl group). It is mainly used for separation of molecular species by the difference in fatty acid chain of these lipids and desalting from lipid solutions (for example, ODS column manufactured by Shimadzu Corporation). However, these agarose gels and silica gel carriers cannot be molded to measure a large number of lipids such as microplates at a high speed, and carriers having long-chain hydrocarbons are not suitable for quantification of lipids. There were no examples used for measurement.

  An object of the present invention is to provide a method for producing a carrier having reproducibility and stability of measurement and suitable for highly sensitive measurement of a minute amount of glycolipid, and a measurement method using the same.

Specifically, the following are the issues.
(1) In order to complete the lipid measurement technique, a carrier having a strong hydrophobic binding ability is provided.
(2) To provide a method for increasing the amount of lipids including glycolipids to be immobilized and preventing deviation due to washing of the immobilized lipids.
(3) Introduce a new method for the synthesis of hydrophobic carriers.

  The inventors of the present invention have made extensive studies in order to solve the above problems. As a result, it has been found that glycolipids can be measured accurately and with good reproducibility by using a plate in which long-chain hydrocarbon chains are solidified, and the present invention has been completed.

That is, the present invention is as follows.
(1) A microplate in which a compound containing a long hydrocarbon chain is solidified by a covalent bond.
(2) The microplate of (1), wherein the long hydrocarbon chain has 8 to 18 carbon atoms.
(3) The microplate of (1) or (2), wherein the covalent bond is an amide bond.
(4) The microplate according to any one of (1) to (3), which is used for glycolipid measurement.
(5) A kit for measuring glycolipid, comprising the microplate of (4).
(6) A method for measuring glycolipid, comprising adding a sample containing glycolipid to the microplate of (4) and measuring the amount of glycolipid bound to the microplate.

  The plate of the present invention has a strong hydrophobic binding ability to glycolipids, and the glycolipids are hardly deviated by washing or the like. Therefore, by using the plate of the present invention, glycolipid can be accurately measured with good reproducibility.

  The microplate of the present invention is a microplate in which a compound containing a long hydrocarbon chain is solidified by a covalent bond.

  The microplate refers to a container to which a small amount of liquid sample can be added, but may have a plurality of wells (holes) so that various types of samples can be added. For example, 6, 12, 24, 48, 96-well multiwell plates can be mentioned.

  On the microplate of the present invention, a compound containing a long hydrocarbon chain is solidified by a covalent bond. The length of the long-chain hydrocarbon chain contained in the solidified compound is not limited as long as the glycolipid can be sufficiently hydrophobically bonded, but preferably has 8 to 24 carbon atoms, and 8 to 18 It is more preferable that In addition, the long-chain hydrocarbon chain in the compound may be branched. In this case, the above carbon number refers to the carbon number of the longest chain. Further, the long hydrocarbon chain may contain unsaturated carbon.

  The type of the covalent bond is not particularly limited, and examples thereof include an amide bond, an ester bond, a thioester bond, and an imino bond, and among them, the amide bond is particularly preferable. Examples of the plate in which a compound containing a long hydrocarbon chain is solidified by an amide bond include a plate in which an octyl group is bonded through an amide bond, a plate in which an octadecyl group is bonded through an amide bond, and the like.

  The microplate of the present invention can be produced by solidifying a compound containing a long hydrocarbon chain on a microplate having a functional group to which the compound can be covalently bonded via a covalent bond. Examples of the compound containing a long hydrocarbon chain used for the production include compounds in which a reactive group such as a carboxyl group or an amino group is bonded to the long chain hydrocarbon chain. These reactive groups are preferably bonded to the end of a long-chain hydrocarbon chain or a hydrophobic functional group. Examples of such compounds include long chain fatty acids and long chain alkylamines.

On the other hand, as a microplate having a functional group capable of covalently bonding with a reactive group of a compound, for example, when the compound is a compound containing a carboxyl group, a plate having an amino group is used, and the compound contains an amino group. In the case of a compound, a plate having a carboxyl group, N-hydroxysuccinimide activated ester (NHS ester), maleimide group or the like can be used. Such a plate can be obtained by introducing a functional group into an ordinary plastic plate or the like. Examples of the plate having an amino group include A plate of Sumitomo Bakelite Co., Ltd., and examples of the plate having a carboxyl group include C plate of the same company.

  Specifically, for example, a long-chain carboxylic acid is covalently bonded onto a plate having an amino group using a condensing agent such as carbodiimide, thereby producing a plate in which a long-chain alkyl compound is solidified by an amide bond. can do. Such a plate can also be obtained by covalently bonding a long chain alkylamine onto a plate having a carboxyl group. The combination of the functional group solidified on the plate and the functional group contained in the compound is not particularly limited as long as it is solidified by a covalent bond.

  Since the long hydrocarbon chain solidified on the microplate of the present invention interacts with the hydrophobic chain contained in the glycolipid, the microplate of the present invention can be suitably used for the measurement of glycolipid.

  In the present invention, the glycolipid refers to a substance containing a water-soluble sugar chain and a fat-soluble group in the molecule. The glycolipid in the present invention includes a sphingoglycolipid, a glyceroglycolipid, and the like. In addition, the glycolipid in the present invention includes acidic sugars such as ganglioside which is a glycolipid containing sialic acid, sulfatide (sulfolipid) which is a glycolipid containing sulfuric acid, a glycolipid containing uronic acid, or a glycolipid containing phosphoric acid. Lipids and neutral glycolipids are included. Ganglioside (sialoglycolipid) is a general term for glycosphingolipids containing sialic acid. Examples of the ganglioside include GM1a, GM1b, GM1-Fuc, GM2, GM3, GM3 (NeuGc), GM4, GD1a, GD1b, GD2, GD3, GD3 (NeuAc / NeuGc), GD3 (NeuTc, 1G , GT1c, GQ1a, GQ1b, GQ1c, GP1a, GP1b, GP1c, or LM1.

  As a method for measuring the glycolipid as described above using the microplate of the present invention, for example, a glycolipid-containing sample extracted from a living tissue or a cell such as an animal or a plant by a conventional method is used. Examples include a method of detecting glycolipid bound to the microplate via a long-chain hydrocarbon chain after adding to the microplate and washing. Glycolipids may be measured directly, but are preferably measured using antibodies or the like. It is also possible to measure multiple samples simultaneously using a microplate reader.

  Examples of detection methods using antibodies include ELISA (Enzyme-Linked Immunosorbent Assay). Specifically, for example, after adding a glycolipid-containing sample to the microplate of the present invention, washing and the like, the glycolipid is specifically identified to the glycolipid bound to the microplate via a long hydrocarbon chain. The antibody that recognizes the target can be reacted, and then detected using a secondary antibody that recognizes the Fc site of the antibody. The secondary antibody is preferably labeled with a fluorescent substance or an enzyme for detection. Alternatively, a secondary antibody having biotin bound thereto and an avidin bound enzyme can be used for detection. Examples of the enzyme include peroxidase and alkaline phosphatase. In addition, the antibody with respect to glycolipid can use what is marketed.

  The present invention also provides a kit for measuring a glycolipid comprising the microplate of the present invention. Such a kit may further contain a detection antibody or the like. For example, in the case of an ELISA kit, a kit containing an anti-glycolipid antibody, a labeled secondary antibody, a detection substrate and the like can be mentioned.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(1) Preparation of reagents Glycolipid blocking proteins include 1% bovine serum albumin (BSA), 1% human serum albumin (HSA), 1% chicken albumin (OVA), 0.3% gelatin, 5% Phosphate buffer (PBS) (pH 7.4) containing skim milk (SM), Block Ace (BA; Snow Brand) or Super Block (SB; Pearce) was used. These proteins are conventionally used for blocking after glycolipid immobilization. As a washing solution, a PBS (pH 7.4) buffer solution containing 0.02% of Tween (Tw20; Poly (Oxyethylene) sorbitan monolaulate) was used.

(2) Preparation of plate A-plate (0.17 nmol exposure) from which Sumitomo Bakelite exposed amino groups and C-plate (0.30 nmol exposure) from which carboxyl groups were exposed were used as the long-chain hydrocarbon chain introduction plate.

  Add 5 nmol octanoic acid (8 carbon atoms) per well of A-plate or 5 nmol octylamine per well of C-plate, the following is commonly 5 μl DMSO solution, 1 mg water-soluble carbodiimide (WSC) 95 μl of PBS (pH 5.8) was added and reacted at 37 ° C. for 1 to 5 hours. After the reaction, the reaction solution was removed, washed 5 times with 0.3 ml of ethanol, then washed 3 times with 0.3 ml of PBS (pH 7.4), and the same buffer was added and stored in a cold place until use. A plate in which octanoic acid was bound to A plate was referred to as Oct-A plate, and a plate in which octylamine was bound to C plate was referred to as Oct-C plate (see FIG. 1). In these reactions, the same concentration of octadecanoic acid (18 carbon atoms) was used instead of octanoic acid, and octadecylamine (18 carbon atoms) was used instead of octylamine to prepare hydrophobic plates corresponding to each. (OD-A plate, OD-C plate).

In order to investigate the amount of long-chain hydrocarbon bound to the plate, [ ¹⁴ C] -labeled octanoic acid or [ ¹⁴ C] -labeled octadecanoic acid was added to the amino group of the A-plate as a carboxylic acid binding reaction solution. Thereafter, a part of the reaction supernatant was collected, the radioactivity of unreacted carboxylic acid was measured, and the amount of carboxylic acid bound to the well was determined (FIG. 3). As a control, a reaction system without adding WSC was used. On the other hand, for determination of the amount of aliphatic amine bound to the C-plate, labeled aliphatic amine was added, and the unreacted aliphatic amine in the reaction supernatant was analyzed by a fluorescence analysis method (Florescamine method).

(3) Glycolipid antigen LactosylCeramide (LC) purified from equine kidney as neutral glycolipid and GM1 or GD1a purified from bovine brain as acidic glycolipid were used. The molecular images of these glycolipids are relatively hydrophobic because LC has short sugar chains, and GM1 and GD1a are relatively hydrophilic because of long sugar chains (see FIG. 2). In order to measure the amount of glycolipid bound to the plate and the amount of deviation due to washing, a part of each glycolipid was labeled with tritium by a known method.

(4) Glycolipid binding For measurement of the amount of glycolipid bound to the plate and the amount of deviation due to washing, 5 μl alcohol solution containing 0.1 μg of radiolabeled glycolipid (GM1 or LC) per well was added, washed and dried. To bind glycolipids. The binding amount was measured from the recovery rate of the labeled glycolipid in the washing solution (FIGS. 4 and 5). As a plate for comparison, Immulon 1B (Dynatech, made of polystyrene) and MaxiSorp plate (Nunc, made of polypropylene) were used.

(5) Examination of binding force and blocking agent of plate After binding glycolipid to each plate, it was washed with PBS + 0.3% gelatin containing surfactant Tw at a concentration of 0-0.06%, and then ELISA method ( The degree of liberation (ease of liberation) of the glycolipid bound to each plate was measured (FIG. 6).

  Next, as a blocking agent to reduce the background when performing ELISA using each plate, 0.3% gelatin in PBS, 1% HSA, 1% BSA, 4 times diluted block ace, Super Block, 1% OVA, Their effects were examined using 1% skim milk (FIG. 7). A PBS buffer containing each protein and Tween 0.02% was used as the washing solution.

(6) Measurement by ELISA
For the measurement of glycolipids by ELISA, 5 μl alcohol solution containing 0 to 100 ng of unlabeled glycolipid per well was added. For comparison purposes, an equal volume of alcohol without glycolipid was added to another well.

  The ELISA method was based on a known method. Among antibodies against glycolipids, monoclonal antibodies against GM1 (IgG) and GD1a (IgM) are commercially available, so these are used as primary antibodies, secondary antibodies as peroxidase-labeled anti-mouse IgG sheep antibodies (Jackson ImmunoResearch) The latter used peroxidase-labeled anti-mouse IgM antibody (Jackson ImmunoResearch).

  Oct-A, OD-A, Oct-C, OD-C plates, Immullon1B, Nunc plates, untreated A-plates and C-plates with standard GD1a 100ng, 50ng, 25ng, 10ng, 5ng, Add 2.5 ng, 1 ng, and 0 ng, block with 100 μl of 0.3% gelatin-PBS (pH 7.8) for 2 hours at room temperature, discard the gelatin solution, add 500-fold diluted anti-GM1 or GD1a mouse monoclonal antibody, and react for 1 hour at room temperature did. Next, after washing 3 times with 200 μl of 0.3% gelatin-PBS (pH 7.8) containing 0.02% Tw (washing solution), add 50 μl of a 5,000-fold diluted Donkey-derived Peroxidase-labeled anti-IgM antibody, react for 2 hours at room temperature, and 3 times with the above washing solution. After washing, 50 μl of substrate (ABTS) was added and allowed to react for 10 minutes at room temperature. After adding 50 μl of the reaction stop solution, the absorbance at 405 nm was measured. For comparison, a corresponding non-immune immunoglobulin (NonImIgM) was used instead of the antibody, or a system containing no glycolipid was used.

(7) Measurement results Binding long-chain hydrocarbon chains to plates
The amount of octanoic acid (Oct-A) or octadecanoic acid (OD-A) bound to the A-plate was 0.23 and 0.21 nmol / well, respectively. On the other hand, the binding of octylamine (Oct-C) and octadecylamine (OD-C) to the C-plate was 0.13 and 0.11 nmol / well. In FIG. 3, the amount of octanoic acid that did not bind to the A plate was shown on the vertical axis. P1 and P2 are the respective values of two experimental results. Compared with the control (C), the radioactivity in the recovered solution was decreased in P1 and P2, indicating that the radiolabeled octanoic acid was bound to the plate.

2. Glycolipid binding to the plate After adding the radiolabeled LC, GM1 or GD1a to the 4 types of plates prepared above, Immuln1B, Nunc plate, untreated A-plate and untreated C-plate, The binding amount was measured from the recovery rate of the labeled glycolipid. As a result, in GM1, the binding amount to the long-chain hydrocarbon chain plate was significantly superior to that of the polystyrene plate (Immulonn1B, Nunc plate) (FIG. 4). However, LC with strong hydrophobicity also binds in large quantities to Immulon (FIG. 5), and no difference in binding amount was observed between the long-chain hydrocarbon chain plate. This is probably because the amount of long-chain hydrocarbon chains in the Oct-A-plate and Oct-C-plate prepared above was insufficient compared to the amount of benzene ring exposed on the polystyrene plate. This can be solved by increasing the exposure amount of the amino group and carboxyl group of the Oct-A-plate and Oct-C-plate.

3. Effect of the binding force of the plate and the blocking agent Glycolipids were retained in the plate up to 0.02% Tw concentration in the long-chain hydrocarbon chain solidified plate, but the Tw concentration 0.01% in Immulon and Nunc plates About 80% were released, and the strength of the long-chain hydrocarbon chain plate was proved (FIG. 6).

  As a blocking agent for reducing the background, gelatin and skim milk were superior in the long-chain hydrocarbon chain plate (FIG. 7). And the measured value of the comparative control was kept low. This was considered due to the presence of Tw in the washing solution.

4). Measurement of glycolipid antigen by ELISA The glycolipid of 2.5 to 50 ng was able to be measured logarithmically in any long chain plate. On the other hand, Immulon and Nunc plates have low sensitivity because the amount of binding in GD1a is reduced by Tw in the washing solution (FIG. 8). This clearly shows that long-chain hydrocarbon chain plates are superior to conventional microplates. In addition, the vertical axis | shaft of FIG. 8 has shown the light absorbency (OD405nm) of 405nm.

  The microplate of the present invention is suitable for the measurement of a minute amount of glycolipid, and is useful in the medical or biochemical research field or the diagnostic field.

The schematic diagram of the plate of this invention. The figure which shows the structure of a glycolipid. The graph which shows the amount of solidification of the long-chain hydrocarbon chain to a plate. The graph which shows the coupling | bonding amount of GM1 to each plate. The graph which shows the coupling | bonding amount of LC to each plate. The graph which shows the release | release degree of the glycolipid couple | bonded with each plate. The graph which shows the effect of a blocking agent. The graph which shows the sensitivity of ELISA method using each plate.

Claims (6)

A microplate in which a compound containing a long hydrocarbon chain is solidified by a covalent bond. The microplate according to claim 1, wherein the long-chain hydrocarbon chain has 8 to 18 carbon atoms. The microplate according to claim 1 or 2, wherein the covalent bond is an amide bond. The microplate according to any one of claims 1 to 3, which is used for glycolipid measurement. A glycolipid measurement kit comprising the microplate according to claim 4. A method for measuring glycolipid, comprising adding a sample containing glycolipid to the microplate according to claim 4 and measuring the amount of glycolipid bound to the microplate.

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