KR20190022762A - Modified cardiolipin-coated magnetic nadobeads and methods for making same - Google Patents

Abstract

The present invention discloses a modified cardiolipin-coated magnetic nadobide and a method of making the same, wherein the modified cardiolipin-coated magnetic nadobide comprises modified cardiolipin and magnetic nadobide; The modified cardiolipin is obtained by oxidation and amination of a hydrophobic fatty acid side chain of cardiolipin, wherein the modified cardiolipin comprises an amino group and the modified cardiolipin is bound to a magnetic nadobide via an amino group.

Description

Modified cardiolipin-coated magnetic nadobeads and methods for making same

The present invention relates to the field of in vitro diagnostics, and more particularly to modified cardiolipin-coated magnetic nadobeads and methods of making the same.

Anticardiolipin antibodies are antibodies capable of reacting with antigenic substances containing various phospholipid structures. Antigens are negatively charged phospholipid components involved in various cell membrane constitutions.

Antiphospholipid antibody syndrome (APS) involves a variety of autoimmune diseases, more commonly occurring in young people, with a male to female ratio of about 2: 8. The patient may exhibit one or more symptoms that may affect various systems and organs including vein and arterial thrombosis, thrombocytopenia, habitual abortion, cardiomyopathy, heart disease, brain and kidney infarction, and pulmonary hypertension. Malignant APS can develop in a short-term, progressive, coherent thrombus formation, resulting in multiple organ failure, even death. May develop in systemic lupus erythematosus and other autoimmune diseases, or may occur alone (primary antiphospholipid antibody syndrome).

It can also produce antiphospholipid antibodies in the body of syphilis, and syphilis is a sexually transmitted disease caused by the spiraling bacterium Treponema pallidum. According to reports, more than 5 million people are infected with syphilis every year, including 30,000 infants due to congenital infection. Syphilis can lurk or hide in the patient's body for years, and can cause a variety of clinical symptoms. Here, the patients in the incubation period of syphilis have no clinical symptoms, and the next step lasts for a lifetime among about two-thirds of the untreated patients. The infected person is not contagious during the incubation period, but the newborns born by the latent mother may be infected with congenital syphilis.

Antiphospholipid antibody detection is widely used for the diagnosis of anti-phospholipid antibody syndrome and the test for treponemapridimom of syphilis. This detection is inexpensive and quick, and has advantages such as convenient handling of a plurality of samples. Alternatively, the more successful the syphilis treatment is, the more gradually the concentration of the anti-phospholipid antibody decreases, but the concentration of the specific anti-syphilis antibody, treponemapalidum antibody, may not only last for years, but may also be high during menstruation. Therefore, the anti-phospholipid antibody detection test is a better option to monitor syphilis treatment.

A method for detecting a conventional anti-phospholipid antibody is to apply cardiolipin to a specific solid phase, such as an ELISA plate, primarily by the method of physical adsorption, and then to remove the cardiolipin attached to the ELISA plate Binds to the anti-phospholipid antibody, thereby capturing the anti-phospholipid antibody in the sample. The concentration of the antiphospholipid antibody can be read indirectly in the sample by detecting the luminescent concentration after coloring the captured antiphospholipid antibody.

Since the cardiolipin immobilized by the physical adsorption method is easily dissociated and separated from the solid plate by the influence of external conditions such as solvent and heating, the stability of the detection product produced by this method is greatly reduced.

Accordingly, there is a need to provide a magnetic nadobide coated with a modified cardiolipin having excellent stability for detecting an anti-cardiopin antibody and a method for producing the same.

The modified cardiolipin-coated magnetic nadobide includes modified cardiolipin and magnetic nadobide,

Wherein the modified cardiolipin is obtained through oxidation and amination of a hydrophobic fatty acid of the cardiolipin, wherein the modified cardiolipin comprises an amino group, wherein the modified cardiolipin is bound to a magnetic nadobide via an amino group .

In one embodiment, the surface of the magnetic nadobide comprises a carboxyl group, and the amino group forms an amide ester structure with the carboxyl group, thereby binding the cardiolipin to the nanobead.

The method of manufacturing the modified cardiolipin-coated magnetic nadobide comprises:

Reacting the cardiolipin and the peroxide sufficiently under the condition that the first solvent is present to obtain an intermediate product;

Adding an excess amount of ammonia water to the intermediate product followed by sufficient reaction to obtain a modified cardiolipin wherein the hydrophobic fatty acid side chain of the modified cardiolipin is oxidized and aminated and the modified cardiolipin comprises an amino group step; And

Mixing the modified cardiolipin and the magnetic nadobide and reacting them sufficiently to obtain a modified cardiolipin-coated magnetic nadobide.

These modified cardiolipin-coated magnetic nadobeads have a characteristic of being excellent in stability and controlling the amount of connection by directly bonding magnetic nadobide and modified cardiolipin through chemical bonding. These modified cardiolipin-coated magnetic nadobeads can be used directly for the detection of anti-phospholipid antibodies and have a higher stability when compared to the detection products prepared by way of hippurization of cardiolipin through conventional physical adsorption .

FIG. 1 is a flow chart illustrating a method of manufacturing a modified cardiolipin-coated magnetic nadobead according to one embodiment.

The above objects, features and advantages of the present invention will become more apparent by describing embodiments of the present invention with reference to the accompanying drawings. For a complete understanding of the present invention, numerous specific details are set forth in the description. However, the present invention may be embodied in many other forms other than those described herein, and those skilled in the art will be able to make similar modifications without departing from the scope of the present invention, It is not limited.

Modified cardiolipin coated magnetic nadobeads according to one embodiment include: modified cardiolipin and magnetic nadobide.

Cardiolipin is an ester material composed of three glycerol, two phosphoric acids and four long chain unsaturated alkyl groups, which contains two hydrophilic centers and four hydrophobic side chains.

The structure of cardiolipin is as follows.

The modified cardiolipin is obtained by oxidizing and aminating the hydrophobic fatty acid side chain of the cardiolipin, wherein the modified cardiolipin comprises an amino group, wherein the modified cardiolipin is bound to the magnetic nadobide via an amino group.

Because the cardiolipin has four side chains, the cardiolipin can contain multiple amino groups since the four side chains can be oxidized at the same time as the cardiolipin is modified. Specifically, the modified cardiolipin may contain 1 to 8 amino groups (-NH ₂ ).

Preferably, the modified cardiolipin comprises one -NH _2.

The surface of the magnetic nad bead may comprise at least one of a carboxyl group (-COOH) and a terminal epoxy group (-CH (O) CH ₂ ).

In one embodiment, the surface of the magnetic nadobide comprises a carboxyl group, and the amino and carboxyl groups form an amide ester structure (-CO-NH-) to link the modified cardiolipin to the magnetic nadobide.

In one particular embodiment, the structural formula of the modified cardiolipin-coated magnetic nadobide is as follows.

Preferably, the surface of the magnetic nadobide comprises a terminal epoxy group, and the amino group and the terminal epoxy group form a secondary amine structure (-NH-) to link the modified cardiolipin to the magnetic nadobide.

In one particular embodiment, the structural formula of the modified cardiolipin-coated magnetic nadobide is as follows.

Due to the very small size of the cardiolipin molecule, there is very little space available for modification, and deformation on the hydrophilic phosphate center reduces the affinity of the cardiolipin and anti-phospholipid antibodies, and even destroys the antigenic activity.

The modified cardiolipin-coated magnetic nadobide maintains a modification to the cardiolipin center of the cardiolipin hydrophilic phosphate center through modification to the hydrophobic side chain of the cardiolipin, while the amino group of the modified cardiolipin acts as a magnetic nanobead By combining, the modified cardiolipin is firmly attached to the surface of the magnetic nanobead.

The modified cardiolipin-coated magnetic nadobide has a feature that the cardiolipin modified to the magnetic nadobide is directly and firmly connected to the magnetic nadobide through chemical bonding, and the stability is excellent and the amount of connection can be controlled. These modified cardiolipin-coated magnetic nadobeads can be used directly for the detection of anti-phospholipid antibodies and have higher stability when compared to the detection products prepared by the method of hopping the cardiolipin through conventional physical adsorption .

As shown in FIG. 1, the method of manufacturing the modified cardiolipin-coated magnetic nadobead includes the following steps.

Step S10: The cardiolipin and the peroxide are sufficiently reacted under the condition that the first solvent is present to obtain an intermediate product.

The fatty acid side chain of the cardiolipin is oxidized by the reaction of cardiolipin with peroxide.

Peroxides are peroxybenzoic acid, M-Chloroperoxybenzoic acid, peracetic acid, or peroxypropionic acid.

The molecular ratio of cardiolipin and peroxide is 1: 1 ~ 8.

The first solvent is dichloromethane, trichloromethane, chloroform, benzene, or toluene.

Step S10 further comprises a step of purifying the intermediate product which is capable of purifying by liquid chromatography. After purification, a modified cardiolipin having a purity of about 80% can be obtained.

In the step S10, the reaction temperature is 60 ° C to 100 ° C

Step S20: An excessive amount of ammonia water is added to the intermediate product obtained in step S10 and reacted sufficiently to obtain a modified cardiolipin.

The hydrophobic fatty acid side chain of the cardiolipin is oxidized and aminated with peroxides and ammonia water, and the resulting modified cardiolipin contains an amino group.

Because cardiolipin has four side chains, the cardiolipin can contain multiple amino groups since the four side chains can be oxidized simultaneously when the cardiolipin is modified. Specifically, the modified cardiolipin may contain 1 to 8 amino groups (-NH ₂ ).

Preferably, the modified cardiolipin comprises one -NH _2.

The mass concentration of ammonia water is 10% to 30%.

In the step S20, the reaction temperature is 60 ° C to 100 ° C.

Step S30: The modified cardiolipin and the magnetic nadobide obtained in the step S20 are mixed and reacted sufficiently to obtain a modified cardiolipin-coated magnetic nadobide.

In the obtained modified cardiolipin-coated magnetic nano-beads, the modified cardiolipin is linked to the magnetic nano-beads through the amino group.

The surface of the magnetic nad beads may comprise at least one of a carboxyl group (-COOH) and a terminal epoxy group (-CH (O) CH ₂ ).

Magnetic nad beads are available on the market. For example, MagnaBind Cat. No. 21353 carboxylated magnetic beads and MagnaMedics Cat. No. MD01010, or a plurality of MD01010 can be selected and mixed at a predetermined ratio.

Ethyl-3- (3-dimethylaminopropyl) carbodiimide in the step S30 when the magnetic beads contain a carboxyl group on the surface of the beads, (EDCi) and 3-sulfonate-N-hydroxysuccinimide (Sulfo-NHS) in the presence of a second solvent to produce an active intermediate, Lipid is added and reacted sufficiently to obtain a modified cardiolipin-coated magnetic nadobide. Wherein the second solvent is a phosphate buffer solution of DMSO, DMF, tetrahydrofuran or a pH of 6.5 to 8.5, the concentration of the magnetic nadobide is 5 mg / mL to 10 mg / mL, 1-ethyl-3- Dimethylpropyl) carbodiimide is 0.5 mg / mL to 1.5 mg / mL, and the concentration of 3-sulfonic acid-N-enhydroxy succinimide is 0.5 mg / mL to 1.5 mg / mL.

When an activated cardiolipin containing one amino group is taken as an example and a carboxyl group is contained on the surface of the nano bead, the structural formula of the modified cardiolipin-coated magnetic nano beads obtained by the reaction between the modified cardiolipin and the magnetic nano- As shown below.

If the magnetic nanoparticle comprises a terminal epoxy group on the surface of the bead, then in step S30, the magnetic nadobide and the modified cardiolipin are sufficiently reacted in a phosphate buffer solution of pH 6.5 to 8.5 to obtain a modified cardiolipin-coated magnetic napid bead. Here, the concentration of the magnetic nanobead is 5 mg / mL to 10 mg / mL.

The active cardiolipin containing one amino group is exemplified by a structural formula of a modified cardiolipin-coated nano-bead obtained by reacting a modified cardiolipin with a magnetic nano-bead when a terminal epoxy group is contained on the surface of the magnetic nano- Is as follows.

This modified method of producing cardiolipin-coated magnetic nadobeads maintains the modified cardiolipin at the hydrophilic phosphate center of the cardiolipin through modification to the hydrophobic side chain of the cardiolipin, and the amino group of the modified cardiolipin acts as a magnetic It binds to the nanobeads and binds the modified cardiolipin tightly to the surface of the magnetic nanobeads.

This modified method of manufacturing magnetic nadobead coated with cardiolipin has a characteristic that it directly binds the cardiolipin modified to the magnetic nadobide through a chemical bond and is stable and can control the amount of connection. These modified cardiolipin-coated magnetic nadobeads can be used directly for the detection of anti-phospholipid antibodies and have a higher stability when compared to the detection products prepared by the method of hopping the cardiolipin through conventional physical adsorption .

Specific examples are as follows.

Example 1: Preparation of Modified Cardiolipins

Under the protection of argon gas, 1 mmol of cardiolipin is added to a 20 mL glass flask containing 5 mL of dichloromethane and dissolved sufficiently by magnetic stirring at room temperature. 3.5 mmol of m-chloroperoxybenzoic acid was added, and the mixture was allowed to react at room temperature for 36 hours. The reaction mixture is poured into an ice-water bath and extracted with petroleum ether. Petroleum ether was evaporated under reduced pressure to dryness, then ammonia water with a mass fraction of 15% of 5 mL was added, and the mixture was heated to reflux for 8 hours. After cooling, it is poured into ethyl acetate and washed three times with water. After drying, a colorless oil-like material is obtained, which is separated and purified by liquid chromatography to give a modified cardiolipin having an amino group in its side chain. MS (ESI ^& lt ^{; +} & gt ^; , m / z): 1481.99142.

Example 2: Preparation of modified cardiolipin-coated magnetic nadobeads

8 μl of a suspension containing magnetic beads having terminal epoxy groups on its surface was taken and 850 μl of 30 mmol / L phosphate buffer was added. 100 μl of cardiolipin modified with 1 mg / ml of amino group was added And reacted at 37 캜 for 4 hours. The unbound modified cardiolipin is magnetically separated and the remaining magnetic beads are dispersed in 2 mL of 50 mmol / L Tris buffer.

Example 3: Preparation of magnetic nadobide coated with modified cardiolipin

200 μL of a suspension containing 10 mg of magnetic beads having a terminal epoxy group on the surface was added thereto, and 400 μL of 20 mg / mL of EDCi phosphate buffer and 400 μL of 20 mg / mL of NHS phosphate buffer were sequentially added, followed by shaking at 37 ° C. for 30 minutes . After magnetic separation, the magnetic beads were redissolved in 900 mmol of phosphate buffer at 20 mmol / l. In the course of shaking and mixing uniformly, 100 μL of cardiolipin modified with 1 mg / mL aminomycin was added and incubated at 37 ° C For 2 hours. The unbound modified cardiolipin is magnetically separated and the remaining magnetic beads are dispersed in 2 mL of 50 mmol / L Tris buffer.

Example 4 Reaction of Modified Cardiolipin-Coated Magnetic Nado Beads and Anti-Phospholipid Samples

200 μl of the modified cardiolipin-coated magnetic nadobide solution prepared in Examples 1 to 3 is taken respectively, 5 μl of anti-phospholipid antibody serum sample is added, and the mixture is incubated at 37 ° C. for 30 minutes. After magnetic separation, the modified cardiolipin-coated magnetic nadose beads were redissolved to 200 μL, followed by addition of a secondary antibody labeled with Horseradish peroxidase, incubation at 37 ° C. for 30 minutes , Followed by incubation for 10 minutes with addition of TMB substrate solution. 100 μl of a stop solution was added, and the fluorescence signal value of the sample was measured with an OD value in a microplate reader within 10 minutes .

Three cardiolipin positive serum samples and three cardiolipin negative serum samples were taken respectively and the OD values detected in comparison with the conventional physisorption method are shown in Table 1.

Table 1: OD values detected in different samples of Examples 1 to 3 and control (physisorption method)

Referring to Table 1, the emission signal when the anti-phospholipid antibody samples were detected in the magnetic beads coated with the modified cardiolipin-coated magnetic beads prepared in Examples 1 to 3 and the magnetic beads adsorbed by the physical adsorption method (control group) In contrast, the negative samples detected by the modified cardiolipin-coated magnetic nanobeads prepared according to Examples 1 to 3 showed significantly lower emission values (4 to 8-fold reduction) than the control, The sample significantly improves the emission signal value (4 ~ 8 times better) than the control.

Therefore, the anti-phospholipid samples detected by the modified cardiolipin-coated nanomagnetic beads prepared in Examples 1 to 3 significantly improve the detection sensitivity when magnetic beads are hindered by the conventional physical adsorption method.

The foregoing embodiments are indicative only of one or several embodiments of the invention and, although the description has been described in more detail and detail, it is not to be construed as limiting the scope of the invention. It will be understood by those skilled in the art that changes and modifications may be made without departing from the spirit and scope of the invention, all of which fall within the scope of protection of the present invention. Accordingly, the scope of protection of the present invention will vary depending on the appended claims.

Claims (10)

Modified cardiolipin and magnetic nadobide,
Wherein the modified cardiolipin is obtained by oxidation and amination of a hydrophobic fatty acid side chain of a cardiolipin, wherein the modified cardiolipin comprises an amino group, wherein the modified cardiolipin is coupled to the magnetic nadobide via the amino group ≪ / RTI > coated with a modified cardiolipin. The method according to claim 1,
Wherein the surface of the magnetic nadobide comprises a carboxyl group and the amino group and the carboxyl group form an amide ester structure to link the modified cardiolipin to the magnetic nadobide. Bead. 3. The method of claim 2,
Wherein the modified cardiolipin-coated magnetic nadobide is characterized in that the structural formula of the modified cardiolipin-coated magnetic nadobide is as follows.

The method according to claim 1,
Wherein the surface of the magnetic nadobide comprises a terminal epoxy group and the amino group and the terminal epoxy group form a secondary amine structure to link the modified cardiolipin to the magnetic nadobide. Magnetic magnetic beads. 5. The method of claim 4,
Wherein the modified cardiolipin-coated magnetic nadobide is characterized in that the structural formula of the modified cardiolipin-coated magnetic nadobide is as follows.

A method for manufacturing magnetic nadobeads coated with a modified cardiolipin of any one of claims 1 to 5,
The cardiolipin and the peroxide are sufficiently reacted under the condition that the first solvent is present to obtain an intermediate product;
Adding an excess amount of ammonia water to the intermediate product and reacting sufficiently to obtain a modified cardiolipin wherein the hydrophobic fatty acid side chain of the modified cardiolipin is oxidized and aminated and the modified cardiolipin comprises an amino group ; And
And mixing and reacting the modified cardiolipin with magnetic nadobide to obtain a modified cardiolipin-coated magnetic nadobide. ≪ Desc / Clms Page number 20 > The method according to claim 6,
Wherein the peroxide is benzoic acid peroxide, meta-chloroperbenzoic acid, peracetic acid or peroxypropionic acid, and wherein the ratio of the cardiolipin to the peroxide is in the range of 1: 1 to 8, and the first solvent is dichloromethane, trichloromethane, chloroform, benzene or toluene. The method according to claim 6,
Wherein the ammonia water has a mass concentration of 10% to 30% in the step of adding an excess amount of ammonia water to the intermediate product and sufficiently reacting the ammonia water. The method according to claim 6,
Wherein the surface of the magnetic nadobide comprises a carboxyl group;
The step of mixing and sufficiently reacting the modified cardiolipin and the magnetic nadobide to obtain the modified cardiolipin-coated magnetic nadobide is characterized in that the magnetic nadobide, 1-ethyl-3- (3-dimethylaminopropyl) Hydroxyimine and 3-sulfonate-N-hydroxysuccinimide are reacted in the presence of a second solvent to produce an active intermediate, the modified cardiolipin is then added and reacted sufficiently to form a coated cardiolipin Wherein the second solvent is DMSO, DMF, tetrahydrofuran or a phosphate buffer solution having a pH of 6.5 to 8.5, the concentration of the magnetic nadobide is 5 mg / mL to 10 mg / mL, and 1 -Ethyl-3- (3-dimethylaminopropyl) carbodiimide is 0.5 mg / mL to 1.5 mg / mL, and the concentration of the 3-sulfonate-N-hydroxysuccinimide is 0.5 mg / 1.5 mg / mL < / RTI > It coated with the modified magnetic cardiolipin I process for producing a bead. The method according to claim 6,
Wherein the surface of the magnetic nadobide comprises a terminal epoxy group;
The step of mixing and sufficiently reacting the modified cardiolipin and magnetic nadobide to obtain a modified cardiolipin-coated magnetic nadobide is characterized in that the magnetic nadobide and the modified cardiolipin are dissolved in a phosphate buffer solution having a pH of 6.5 to 8.5 To obtain the modified cardiolipin-coated magnetic nadose bead. Wherein the concentration of the magnetic nanobeads is 5 mg / mL to 10 mg / mL.

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