阅读说明：本技术 用于识别低密度脂蛋白的酸性状态及糖化状态的检测试剂及试剂盒 (Detection reagent and kit for identifying acidic state and glycated state of low-density lipoprotein ) 是由 山田健一 井手友美 石田悠马 一圆刚 山本启一 于 2018-04-27 设计创作，主要内容包括：本发明提供一种用于全面地检测低密度脂蛋白的氧化状态及糖化状态的检测试剂。更详细的,根据本发明,在使用发色团标记抗体来检测氧化低密度脂蛋白及糖化脂蛋白的同时,使用荧光氮氧自由基2,2,6-三甲基-4-(硝基苯并[1,2,5]噁二唑-7-基氨基)-6-戊基哌啶-1-氧基(NBD-Pen)来检测脂质自由基。(The present invention provides a detection reagent for comprehensively detecting the oxidation state and the glycation state of low-density lipoprotein. More specifically, according to the present invention, while oxidized low density lipoproteins and glycated lipoproteins are detected using a chromophore-labeled antibody, lipid radicals are detected using fluorescent nitroxide radical 2,2, 6-trimethyl-4- (nitrobenzo [1,2,5] oxadiazol-7-ylamino) -6-pentylpiperidin-1-yloxy (NBD-Pen).)

1. A detection reagent, comprising:

a primary detection antibody that recognizes oxidized low density lipoprotein,

a primary detection antibody recognizing glycated low-density lipoprotein, and

as shown in structural formula (1):

[ chemical formula 1]

Fluorescent nitroxyl radicals are shown.

2. The detection reagent according to claim 1, wherein,

the primary detection antibody recognizing oxidized low-density lipoprotein is a mixture containing at least an antibody recognizing malondialdehyde-lysine, an antibody recognizing 4-hydroxy-2-nonenal, and an antibody recognizing acrolein-lysine.

3. The detection reagent according to claim 1 or 2, wherein,

the primary detection antibody that recognizes glycated low-density lipoproteins is a mixture containing at least an antibody that recognizes pentosan, an antibody that recognizes Croelin, an antibody that recognizes carboxymethyllysine, an antibody that recognizes carboxyethyllysine, and an antibody that recognizes pyrroline.

4. A kit for discriminating between an acidic state and a glycated state of a low-density lipoprotein, comprising:

a microplate immobilized with an antibody for recognizing low-density lipoprotein,

the detection reagent according to any one of claims 1 to 3,

and a fluorescence-labeled secondary detection antibody for recognizing the primary detection antibody for oxidized low-density lipoprotein and for recognizing the primary detection antibody for glycated low-density lipoprotein.

5. The kit according to claim 4, wherein,

the fluorescence chromophore of the labeled secondary detection antibody is a 7-nitrobenzofurazan inducer.

Technical Field

The present invention relates to a technique for visually detecting oxidized and glycated lipoproteins. More specifically, the present invention provides a detection reagent and a kit for detecting the oxidation state of lipids and the glycation state of proteins in low-density lipoproteins by fluorescence observation.

Background

In recent years, it has been found that oxidation and glycation of lipids promote aging. It is known that lipids that are deteriorated by oxidation, glycation, and the like are associated with various diseases, and studies have been increasingly conducted to find the cause of such deterioration (oxidation and glycation) of lipids, thereby making them useful not only in the fields of aging prevention and beauty but also in the prevention and treatment of diseases.

Superoxide anion radicals, hydroxyl radicals, hydrogen peroxide, singlet oxygen, and other Reactive Oxygen Species (ROS) affect various phenomena in the living body, and among them, hydroxyl radicals are known to have extremely high reactivity and cause various diseases, and thus, research on these is being actively conducted. It has been found that this hydroxyl radical acts on lipids to generate lipid radicals (lipid radics).

Since lipid radicals are highly reactive and unstable, lipid peroxidation occurs in a chain to generate lipid radicals, which in turn generate electrophilic compounds as metabolites. Since lipids contain many unsaturated fatty acids and hydrogen atoms of active methylene moieties thereof are abstracted, they are easily attacked by radicals, and lipid peroxidation chain reactions composed of the processes shown in reaction formulas (a) to (c) are induced (fig. 1).

(formula 1)

LH+R·→L·+RH (a)

LOO·+LH→LOOH+L· (c)

The free radical (R.cndot.) abstracts hydrogen from unsaturated fatty acid (LH) to start chain reaction (a); generating a Lipid peroxy radical (LOO.) (b) by reacting the generated Lipid radical (L.) with an oxygen molecule; then, the lipid peroxy radicals abstract hydrogen atoms from the surrounding unsaturated fatty acids, generating lipid peroxides (LOOH) and lipid radicals (L.) (c). Under the action of the regenerated lipid radicals (L.), the next chain reaction cycle is started.

Lipid peroxides (LOOH) can be converted into hundreds or more of electrophilic compounds including malondialdehyde, 4-hydroxy-2-nonenal, acrolein, propionaldehyde, and glyoxal as metabolites thereof.

These metabolites have been found to be cytotoxic, inflammatory, mutagenic, either alone or after forming complexes with proteins.

In the living body, a lipid insoluble in water is combined with an apolipoprotein to form a lipoprotein. In addition, cholesterol, which is essential for the formation of cell membranes, is also insoluble in water and binds to apolipoproteins as well. Lipoproteins are classified into High Density Lipoproteins (HDL), Low Density Lipoproteins (LDL), and the like according to specific gravity.

In particular, the lipid in LDL (fig. 2a) generates lipid radicals by the action of ROS and the like, and generates a metabolite through peroxidation of the lipid. Thus, LDL (oxidized-state LDL) in which only lipids are oxidized and deteriorated is referred to as lightly modified LDL (MM-LDL) (FIG. 2 b). The produced metabolites are bound to each other by lysine residues and arginine residues of the proteins in LDL, and become so-called oxidized LDL (oxidized LDL) (FIG. 2 c).

As shown in FIG. 3, it has been found through extensive studies that such oxidatively modified LDL causes various diseases including age-related macular degeneration (AMD) and arterial sclerosis (for example, non-patent documents 1 and 2).

In addition, it has been found that when the blood glucose level rises, saccharides are bonded to proteins to form carbonyl compounds such as 3-deoxyglucosone (3 DG), Glyoxal (GO), Methylglyoxal (MGO), glyceraldehyde (glyceraldehyde), glycolaldehyde (glycolaldehyde), and the like, and finally, lysine residues (Lys) and arginine residues (Arg) of the proteins are bonded to form pentostatin (pentostatin), crospovidone (crosline), carboxymethyllysine (N.epsilon. - (carboxmethyl) lysine; CML), carboxyethyllysine (N.epsilon. - (carboxyethyl) lysine; CEL), and glycosylation end products (AGE) represented by pyrroline. It has been found that such AGEs accumulate in the body, and cause various diseases. For example, it has been reported that AGE accumulates in blood vessels to cause arteriosclerosis (non-patent document 3), bone to cause osteoporosis (non-patent document 4), and brain to cause alzheimer's disease (non-patent document 5).

The proteins on LDL are glycated to become glycated LDL.

As described above, it has been found that when lipids and proteins constituting LDL are oxidized or glycated, various diseases are brought to the whole body of a living body, and various countermeasures have been studied, and technologies for detecting oxidized LDL (MM-LDL, OxLDL) and glycated LDL have been developed.

(Prior art document)

(non-patent document)

Non-patent document 1: javadzadeh, a.et al.retina.2012,32(4),658

Non-patent document 2: holvoet, p.et al.arterioscler.thromb.vasc.biol.2003,23(8),1444

Non-patent document 3: brown M., et al: science.1986; 232:1629-1632

Non-patent document 4: saito m., et al: Osteoporos int.2006; 17:986-995

Non-patent document 5: reddy VP, et al: Neurotox Res.2002; 4:191-209

Non-patent document 6: itabe H.et al.J.Atheroscher, Thromb.2007,14(1),1-11

Non-patent document 7: cerami a., et al., sci.am.256; 90-96:1987

Non-patent document 8: kotani K et al, Biochim Biophys acta.1215: 121-Achilles 5,1994

Non-patent document 9: miyata T, et al, FEBS Lett 445:202-206,1999

Disclosure of Invention

(problems to be solved by the invention)

The results obtained by various immunological detection methods for oxidized modified LDL were comprehensively considered by plate et al, and differences between the various detection methods were confirmed (non-patent document 6). More specifically, the method of plate et al and the MX kit from medex corporation were used to measure the concentration of oxidatively modified LDL by a capture ELISA method using DLH3 antibody, but the correlation was weak (fig. 4).

Although the DLH3 antibody is used for recognizing oxidized phosphatidylcholine (Oxidizedphosphatidolcholesine) formed on oxidatively modified LDL, as described above, various protein-associated complexes such as malondialdehyde (MDM), 4-hydroxy-2-nonenal (HEN), Acrolein (ACR), propionaldehyde (propanal), and glyoxal (glyoxal) exist as metabolites of lipid peroxide (LOOH) on oxidatively modified LDL. That is, only a part of the oxidation marker for oxidatively modified LDL can be observed in the conventional method.

AGEs on glycated LDL are often fluorescent and emit fluorescence having an emission wavelength of 440nm at an excitation wavelength of 370nm (for example, non-patent document 7). Thus, a method of directly performing fluorescence measurement from human skin has been studied. Although it has been confirmed that the fluorescent AGE present in the skin is pentostatin, the detection has not been successful.

On the other hand, ELISA kits for recognizing antibodies such as pentostatin, CML, CEL, and pyrrolate and detecting ACE are commercially available.

Thus, although the respective sites of oxidation and glycation were measured by fluorescence, no method for comprehensively observing the state of change of LDL (oxidation state and glycation state) has been developed. Accordingly, the present inventors have made an object to provide a method for observing the state of LDL deterioration in a comprehensive manner.

(measures taken to solve the problems)

The inventors of the present invention developed the following method of fluorescence detection: lipids were extracted from organisms that were subjected to oxidative stress in another study by reacting a compound of formula (1):

(chemical formula 1)

The fluorescent nitroxide radical 2,2, 6-trimethyl-4- (nitrobenzo [1,2,5] oxadiazol-7-ylamino) -6-pentylpiperidin-1-yloxy (NBD-Pen) is shown to act on this lipid extract to capture lipid radicals or their radical fragments.

Therefore, the present inventors have conducted simultaneous or stepwise detection of lightly modified LDL by fluorescence detection using the above-mentioned fluorescent nitroxide radicals, detection of oxidatively modified LDL by ELISA, and detection of glycated LDL by ELISA, and visualized the oxidation state and the glycation state of LDL all by fluorescence, thereby realizing individual or comprehensive detection of the deterioration state of LDL.

When the wavelength of fluorescence emission of the slightly modified LDL, the wavelength of fluorescence emission of the oxidized modified LDL and the wavelength of emission of the glycated LDL are different from each other, three kinds of deterioration states can be identified. Furthermore, if all the fluorescence wavelengths are the same, the state of LDL deterioration can be confirmed in a comprehensive manner.

(Effect of the invention)

By visualizing the state of alteration (oxidation and glycation) of LDL by fluorescence, information useful for early detection of diseases caused by altered LDL, improvement of diagnosis and treatment can be obtained.

Drawings

FIG. 1 is a schematic diagram illustrating lipid peroxidation.

Fig. 2 is a schematic diagram showing the oxidation state of low-density lipoprotein (LDL).

FIG. 3 is a disease associated with oxidatively modified LDL.

FIG. 4 is a graph showing the correlation between two conventional methods for detecting oxidized modified LDL (see non-patent document 6).

FIG. 5 is a graph showing the difference in detection sensitivity for detecting lipid radicals in LDL by NBD-Pen, due to the radical generator.

FIG. 6 is a graph showing a comparison of the sensitivity of detecting oxidized modified LDL using the detection method using NBD-Pen of the present invention and a conventional detection method.

FIG. 7 shows a fluorescence microscope image (a) of an aortic sample of an arteriosclerosis model mouse to which NBD-Pen was applied and an electronic image (b) of a plaque after oil red staining.

Detailed Description

Reference example one: development of fluorescent nitroxide radicals

The inventors of the present invention have developed a compound of formula (2):

(chemical formula 2)

A novel method for synthesizing a 2, 6-substituted product (stabilization product) of a TEMPO nitroxide radical 2,2,6, 6-tetramethylpiperidine-N-oxyl is disclosed, and it has been found that lipophilicity and lipid peroxidation inhibiting ability are improved and a lipid radical can be efficiently trapped by introducing an alkyl chain around the radical.

Further, since the nitroxide (No.) is a stable radical having paramagnetism and has a property of reducing fluorescence by intersystem crossing due to photoinduced electron transfer and electron spin exchange in a charge separation state, fluorescence of a fluorescent nitroxide to which a fluorescent chromophore is covalently bonded to the nitroxide is in a quenched state by intramolecular electron transfer. However, it was confirmed that when the nitroxide radical reacts with the radical to lose paramagnetism, electron transfer does not occur, and thus a fluorescence emission state is obtained. That is, by fluorescence observation, a fluorescent nitroxide radical can be used as a probe for detecting the capture of a radical.

The inventors of the present invention converted the 4-carbonyl group of nitroxide radical TEMPO to an amino group, resulting in the following structural formula (3):

(chemical formula 3)

The fluorescent chromophore shown, 7-Nitrobenzofurazan (NBD), is covalently bound to have access to the radical site of nitroxide radical TEMPO.

Most of the lipid molecules to be detected are present in the biological membrane, and form a hydrophobic environment. Thus, while fluorescence decays in a hydrophilic environment, environmentally responsive fluorescent chromophores that selectively emit highly fluorescent light in a hydrophobic environment are preferred. Therefore, the inventors of the present invention selected NBD, which is widely used in lipid fields such as phase transition of biological membrane, membrane fusion, or intracellular lipid metabolism, as a fluorescent chromophore.

The NBD inducer has an excitation wavelength of about 470nm, is suitable for argon laser excitation (488nm), and is therefore suitable for imaging using a fluorescence microscope, and is therefore very advantageous. Further, the maximum luminescence wavelength is 530nm, and the use of the NBD inducer is also advantageous from the viewpoint that autofluorescence due to substances in the living body can be reduced.

The present inventors have found that introduction of a chain alkyl group into the vicinity of the radical site of a TEMPO-based nitroxide radical changes the lipophilicity and steric hindrance of the compound, and as a result, lipid radicals can be efficiently trapped.

The present inventors synthesized compound a (NBD-Pen) in which the 2-position was replaced by two methyl groups and the 6-position was replaced by a methyl group and a pentyl group, and used it as a highly lipid-reactive NBD-nitroxide radical.

(chemical formula 4)

(A)