JP4355811B2 - Cholesterol binding agent and cholesterol detection kit - Google Patents

Description

  The present invention relates to a cholesterol binding agent and a cholesterol detection kit using the same.

Cholesterol is produced as low density lipoprotein LDL in the liver, transported to peripheral tissues, and taken up. Cholesterol is used in peripheral tissues as a component of biological membranes and as a raw material for steroid hormones. Cholesterol is removed from biological membranes by high-density lipoprotein HDL, transported to the liver, and reused. However, the distribution of cholesterol in cells cannot be easily examined because there is no simple staining method. Actually, the evaluation of intracellular cholesterol dynamics is inferred from the measurement of LDL, VLDL and HDL cholesterol in the bloodstream.
The cholesterol distribution in living tissues can be observed with the fluorescent antibiotic Philippines (Fillipin). However, the fluorescence in the Philippines is weak and will disappear soon. The Philippines reacts with cholesterol in cell membranes, but does not react with intracellular lipid cholesterol, making it unsuitable for observing cholesterol distribution throughout the cell.
DAMP (N- (3-((2,4-dinitrophenyl) amino) propyl) -N- (3-aminopropyl) -methylamine) is taken up into intracellular organelles such as endocrine granules and lysosomes in an acidic pH-dependent manner Since the color develops, it has been used for pH measurement in intracellular organelles due to its strength (Non-Patent Documents 1 to 3). However, it has never been known that DAMP binds to cholesterol and can be used to measure cholesterol.
Orci, L., et al. Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles. J. Cell Biol. 103, 2273-2281 (1986). Anderson, RGW & Orci, L. View of acidic intracellular compartments.J. Cell Biol. 106, 539-543 (1988). Hayashi, M., Yamamoto, A. & Moriyama, Y. The internal pH of synaptic-like microvesicles in rat pinealocytes in culture.J. Neurochem. 82, 698-704 (2002).

  An object of the present invention is to provide a reagent that can specifically detect cholesterol by binding specifically to cholesterol.

  The present inventor has intensively studied to solve the above problems. As a result, it was found that DAMP specifically binds to cholesterol. Thus, the inventors have found that cholesterol can be efficiently detected by utilizing the binding of DAMP and cholesterol, and have completed the present invention.

That is, the present invention is as follows.
(1) A cholesterol binder comprising DAMP.
(2) A cholesterol detection kit comprising the cholesterol binder of (1).
(3) The cholesterol detection kit according to (2), further comprising an anti-DAMP antibody.
(4) A method for detecting cholesterol, comprising adding DAMP to a sample to form a complex of cholesterol and DAMP contained in the sample, and detecting cholesterol via DAMP in the complex.
(5) The method for detecting cholesterol according to (4), wherein detection is performed using an anti-DAMP antibody.

According to the present invention, cholesterol can be detected easily and specifically. In particular, intracellular cholesterol can be easily detected, and the distribution of intracellular cholesterol can be determined using leukocytes and the like, which is suitable for testing for hypercholesterolemia and the like.

The present invention is described in detail below.
DAMP is an abbreviation for N- (3-((2,4-dinitrophenyl) amino) propyl) -N- (3-aminopropyl) -methylamine and is a compound having the structure of formula (I). DAMP may be used by chemical synthesis, or a commercially available product (Molecular Probes) may be used.

Since the present inventors have clarified the property that DAMP specifically binds to cholesterol, DAMP can be used as a cholesterol binder.
Examples of uses of the cholesterol binder include recovery and detection of cholesterol. Examples of a method for recovering cholesterol include a method in which DAMP is bound to an appropriate carrier and brought into contact with a target sample, and cholesterol bound to the carrier via DAMP is recovered. In addition, as a method for detecting cholesterol, there is a method in which DAMP is added to a sample containing cholesterol to form a complex of DAMP and cholesterol in the sample, and cholesterol in the complex is detected via DAMP. It is done. In this case, a detectable substituent (for example, a fluorescent group) may be bound to DAMP and detected using such a modified DAMP. However, detection may be performed using an anti-DAMP antibody. Is preferred.
The detection target is not particularly limited as long as it is a sample that can contain cholesterol, but a biological sample that can contain cholesterol is preferable, and cells and tissue sections that can contain cholesterol are more preferable.
Examples of methods for detecting the amount and distribution of cholesterol in cells and tissue sections that can contain cholesterol include the following methods.
A DAMP solution is added to cells or tissue sections fixed with formalin or the like, reacted for a predetermined time, and then washed. Thereafter, an anti-DAMP antibody is reacted. After washing, the labeled secondary antibody is reacted and detected based on the label.
The anti-DAMP antibody is not particularly limited as long as it specifically binds to DAMP, and a commercially available antibody or an antibody prepared by immunizing an animal with DAMP can be used. It is preferable to use an antibody against the (DNP) group. As a commercially available anti-DAMP antibody, an anti-DAMP polyclonal antibody (Sigma-Aldrich) can be used.
As the labeled secondary antibody, a fluorescent labeled antibody or an enzyme labeled antibody can be used. When a fluorescently labeled antibody is used, it can be detected using a fluorescent microscope or the like. On the other hand, when an enzyme-labeled antibody is used, it can be detected based on light emission or color development caused by a substrate decomposition reaction by adding an enzyme substrate.

The present invention also provides a cholesterol detection kit containing DAMP. The kit is preferably a kit that contains instructions for use that describe its use for cholesterol detection and a protocol for its detection method. The kit of the present invention may contain other reagents such as an anti-DAMP antibody and a secondary antibody.
The kit of the present invention can be used not only for research purposes but also for diagnostic purposes such as hypercholesterolemia.

  The following examples illustrate the present invention more specifically. However, the present invention is not limited to the following examples.

[Example 1]
Solutions of 0, 0.02, 0.2, 2.0, and 20 μg of cholesterol were dot blotted onto membranes (Protoran: Schleicher & Schuell BioScience Inc), respectively. As comparative controls, 20 μg of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin were blotted on the membrane, respectively. 1% BSA (bovine serum albumin) / PBS was added to the obtained membrane and blocked for 30 minutes. Next, 10 μg / ml DAMP diluted in 1% BSA / PBS was added and incubated at room temperature for 4 hours. Next, it was washed with PBS for 5 minutes, and this was repeated 3 times. Next, an anti-DAMP polyclonal antibody (Sigma-Aldrich) diluted 1: 100 with 1% BSA / PBS was added and incubated overnight at 4 ° C. The operation of washing with PBS for 5 minutes was repeated 3 times, and then a secondary antibody (anti-rabbit secondary antibody: Jackson) diluted 1: 500 with 1% BSA / PBS was added and incubated at room temperature for 2 hours. The operation of washing with PBS for 5 minutes was repeated three times, and then detection was performed by adding a luminescent substrate (ECL Western Blotting Detection kit; Amersham Biosciences). The results are shown in FIG. It was found that DAMP binds to cholesterol in a concentration-dependent manner. DAMP did not react with any of the control lipids, indicating that DAMP specifically binds to cholesterol.

[Example 2]
The pituitary gland was removed from a rat (Wister rat, male, 10 weeks old) and frozen. From this, a frozen section having a thickness of 4 μm was prepared on a slide glass. Sections were washed with PBS for 5 minutes and this was repeated 3 times. Next, 1% BSA / PBS was added to perform blocking for 30 minutes. Next, 10 μg / ml DAMP diluted in 1% BSA / PBS was added and incubated at room temperature for 4 hours. Next, it was washed with PBS for 5 minutes, and this was repeated 3 times. Next, an anti-DAMP polyclonal antibody (Sigma-Aldrich) diluted 1: 100 with 1% BSA / PBS was added and incubated overnight at 4 ° C. After washing 3 times with PBS for 3 minutes, the secondary antibody diluted 1: 500 with 1% BSA / PBS (Redx anti-rabbit
IgG: Jackson) was added and incubated at room temperature for 2 hours. The operation of washing with PBS for 5 minutes was repeated 3 times, and then a cover glass was placed and observed with a fluorescence microscope. In addition, fluorescence was detected from successive sections using an anti-cholesterol antibody (Rabbit Anti-Cholesterol 3 Polyclonal Antibody; Biogenesis) as a primary antibody in the same procedure as described above. Furthermore, detection using the fluorescent substance Philippines (Sigma-Aldrich) was also performed.
The results are shown in FIG. According to this, the fluorescence pattern obtained using DAMP and the fluorescence pattern obtained using anti-cholesterol antibody coincided, and it was found that intracellular cholesterol distribution can be measured by using DAMP. Cholesterol could also be detected using Philippine autofluorescence, but the fluorescence was weak and the signal disappeared in a short time.
The Philippines reacts with cholesterol in the cell membrane, but does not react with intracellular lipid cholesterol, making it unsuitable for observing the distribution of cholesterol throughout the cell. On the other hand, if DAMP is used, it is possible to know the distribution of cholesterol in tissues and cells only by incubating with tissue pieces and cell clusters fixed on a slide glass for several minutes. Since DAMP binds to plasma membranes, endocrine granule membranes, and lipid droplets with high concentrations of cholesterol, DAMP can observe cholesterol localization and accumulation in greater detail and accuracy than existing Philippines.

[Example 3]
Next, fluorescent tissue staining was performed using a growth hormone antibody diluted 1:50 (anti-Growth Hormone antibody: Biogenesis) and compared with the fluorescence pattern of DAMP. As a result, it was found that cholesterol is present not only in growth hormone-secreting cells but also in many endocrine cells.

The figure (photograph) which shows the coupling | bonding of DAMP with respect to each concentration cholesterol (upper stage) and each lipid (lower stage). Abbreviations are as follows: PC: phosphatidylcholine, PE: phosphatidylethanolamine, PI: phosphatidylinositol, PS: phosphatidylserine, SM: sphingomyelin, Chol: cholesterol. The figure (photograph) which shows the detection result of the cholesterol of a pituitary tissue section. From the left, the detection pattern by the Philippines, DAMP, and anti-cholesterol antibody is shown. The figure (photograph) which shows the fluorescence dyeing | staining result of a pituitary tissue section. From the left, an image using DAMP, an image using anti-growth hormone antibody, and an image in which both are superimposed are shown.

Claims (5)

A cholesterol binder comprising DAMP (N- (3-((2,4-dinitrophenyl) amino) propyl) -N- (3-aminopropyl) -methylamine). A cholesterol detection kit comprising the cholesterol binding agent according to claim 1. The cholesterol detection kit according to claim 2, further comprising an anti-DAMP antibody. A method for detecting cholesterol, comprising adding DAMP to a sample to form a complex of cholesterol and DAMP contained in the sample, and detecting cholesterol via DAMP in the complex. The method for detecting cholesterol according to claim 4, wherein the detection is performed using an anti-DAMP antibody.

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