MYO monoclonal antibody nanoparticle conjugate, preparation method thereof and immunochromatography test strip

Abstract

The invention discloses an MYO monoclonal antibody nanoparticle conjugate, a preparation method thereof and an immunochromatography test strip, relates to the technical field of biological detection, and solves the problems of high cost, poor instantaneity and low sensitivity of existing MYO detection. Mixing and stirring the gold-palladium / iron oxide nanoparticle solution with a sodium polyacrylate aqueous solution, centrifuging, dispersing in a 2-(N-morpholine) ethanesulfonic acid buffer solution, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, fully reacting, adding an MYO monoclonal antibody dAb to continuously react, finally adding BSA (Bovine Serum Albumin) for sealing, centrifuging, washing with water, and drying to obtain the gold-palladium / iron oxide nanoparticle composite material. The gold-palladium / iron oxide nanoparticle conjugate modified by the MYO monoclonal antibody dAb is obtained, and the gold-palladium / iron oxide nanoparticle conjugate modified by the MYO monoclonal antibody dAb is dispersed in a Tris-HCl solution; the kit can be applied to preparation of instant detection tools for acute myocardial infarction patients and in the MYO detection process.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a MYO monoclonal antibody nanoparticle conjugate and a preparation method thereof and an immunochromatographic test strip. Background Art

[0002] MYO (myoglobin) is one of the main markers of myocardial injury. It has the advantages of high sensitivity and high clinical value for the early diagnosis of acute myocardial infarction. Usually, the MYO level in the blood of healthy people is no higher than 15ng / mL. It can rise rapidly within 1-3h after the onset of acute myocardial infarction and reach a peak in about 6-12h. Therefore, detecting the level of MYO in the blood is crucial for the early diagnosis and treatment of acute myocardial infarction. At present, conventional MYO detection mainly uses enzyme-linked immunosorbent assay and chromatography. However, these methods require expensive instruments, cumbersome operations, and rely on professional personnel for detection, which cannot meet the requirements of immediate detection of patients with acute myocardial infarction.

[0003] Immunochromatographic test strips have the advantages of rapid detection, convenient operation, and low cost, and have been widely used in the instant detection of food, environmental and clinical markers. However, the commonly used gold nanoparticles have low detection sensitivity and the detection limit cannot meet the detection requirements.

[0004] Gold palladium / iron oxide nanoparticles have a strong surface plasma effect, and applying them to immunochromatographic test strips can significantly improve the detection sensitivity. However, at present, there is still a gap in the immunochromatographic technology based on gold palladium / iron oxide nanoparticles for the detection of MYO. Summary of the invention

[0005] In order to solve the problems of high cost, poor immediacy and low sensitivity of existing MYO detection, the present invention provides a MYO monoclonal antibody nanoparticle conjugate and a preparation method thereof and an immunochromatographic test strip.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing a MYO monoclonal antibody nanoparticle conjugate comprises the following steps:

[0008] The gold palladium / iron oxide nanoparticle solution and the sodium polyacrylate aqueous solution were mixed and stirred, dispersed in 2-(N-morpholine)ethanesulfonic acid buffer after centrifugation, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added, and after sufficient reaction, MYO monoclonal antibody dAb was added to continue the reaction, and finally BSA was added for blocking and centrifugation to obtain MYO monoclonal antibody dAb-modified gold palladium / iron oxide nanoparticle conjugates, which were dispersed in Tris-HCl solution.

[0009] Preferably, the Tris-HCl solution has a pH of 8.0 and contains 20% sucrose, 5% trehalose, 1% BSA and 1% Tween-20.

[0010] Preferably, the gold palladium / iron oxide nanoparticle solution is prepared by the following steps:

[0011] 1) Add 6 mL of 0.024 mmol / L HAuCl4 solution and 4 mL of 0.024 mmol / L Na2PdCl4 solution to 1 L of deionized water and stir to obtain a mixed solution;

[0012] 2) adding 3.7 mL of 0.75% ammonia solution to the mixed solution of 1) and stirring to obtain a reaction solution;

[0013] 3) adding 5 mL of 0.4 mol / L FeCl2 solution to the reaction solution in 2) and stirring the mixture to synthesize gold palladium / iron oxide nanoparticles by self-oxidation-reduction assembly method;

[0014] 4) The reaction product solution in 3) was centrifuged and washed three times at 7500 rpm, and dispersed in deionized water to obtain a gold palladium / iron oxide nanoparticle solution.

[0015] Preferably, the amount of the MYO monoclonal antibody dAb is 5 μg to 12.5 μg.

[0016] The present invention also provides a MYO monoclonal antibody-modified gold-palladium / iron oxide nanoparticle conjugate prepared by the above preparation method.

[0017] An application of the above-mentioned MYO monoclonal antibody-modified gold palladium / iron oxide nanoparticle conjugate is specifically used to prepare a point-of-care detection tool for patients with acute myocardial infarction.

[0018] The present invention also provides an immunochromatographic test strip, comprising the gold-palladium / iron oxide nanoparticle conjugate modified by the MYO monoclonal antibody.

[0019] The present invention also provides a method for preparing the above-mentioned immunochromatographic test strip, comprising the following steps:

[0020] S1. Use a three-dimensional streak instrument to immobilize MYO monoclonal antibody cAb and goat anti-rabbit IgG on a nitrocellulose membrane at a streak volume of 1 μL / cm, and then vacuum dry;

[0021] S2, stick the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad on the back plate in the chromatography direction, and cut them;

[0022] S3. Drop the gold palladium / iron oxide nanoparticle conjugate modified with MYO monoclonal antibody dAb onto the conjugate pad to obtain an immunochromatographic test strip for detecting MYO.

[0023] Preferably, in step S2, the sample pad is completely soaked in 20 mmol / L Tris-HCl in advance and then vacuum dried, wherein the Tris-HCl solution has a pH of 8.0 and contains 5% bovine serum albumin, 1% polyvinyl pyrrolidone and 0.5% Tween-20.

[0024] Preferably, the amount of the MYO monoclonal antibody dAb-modified gold palladium / iron oxide nanoparticle conjugate used in step S3 is 2 μL to 8 μL.

[0025] Compared with the prior art, the specific beneficial effects of the present invention are:

[0026] The present invention firstly synthesizes a gold-palladium / iron oxide nanoparticle conjugate modified with a MYO monoclonal antibody, and uses it as a labeled probe, and utilizes the strong LSPR effect property to construct an immunochromatographic test strip for detecting MYO, thereby solving the shortcomings of the existing methods for detecting MYO, such as cumbersome operation, long time consumption and high price. The chromatographic test strip provided by the present invention has the advantages of fast detection, convenient operation and low cost, and can effectively improve the defect of low sensitivity of the test strip. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic diagram of the structure and working principle of the immunochromatographic test strip provided by the present invention;

[0028] Figure 2 This is a schematic diagram showing the changes in the immunochromatographic test strip obtained in Example 1 as the MYO concentration changes.

[0029] Figure 3 This is a standard curve diagram obtained by detecting MYO using the immunochromatographic test strip obtained in Example 1. DETAILED DESCRIPTION

[0030] In order to make the technical solution of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the specification of the present invention. It should be noted that the following embodiments are only used to better understand the technical solution of the present invention and should not be understood as a limitation to the present invention.

[0032] Step 1. Preparation of MYO monoclonal antibody nanoparticle conjugate:

[0033] 1 mL of the gold palladium / iron oxide nanoparticle solution was mixed with 25 μL of 4% sodium polyacrylate aqueous solution, stirred, and dispersed in 1 mL of 10 mmol / L 2-(N-morpholine)ethanesulfonic acid buffer after centrifugation at 5800 rpm, and 20 μL of 50 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 20 μL of 50 mmol / L N-hydroxysuccinimide were added. After reacting for 30 min, 7.5 μg of MYO monoclonal antibody dAb was added, and the reaction was continued for 3 h. Then, 10 μL of 1% BSA was added for blocking for 40 min, and the MYO monoclonal antibody dAb-modified gold palladium / iron oxide nanoparticle conjugate was obtained by centrifugation; the conjugate was dispersed in 20 mmol / L Tris-HCl solution, wherein the Tris-HCl solution had a pH of 8.0 and contained 20% sucrose, 5% trehalose, 1% BSA and 1% Tween-20.

[0034] Step 2: Preparation of immunochromatographic test strips:

[0035] MYO monoclonal antibody cAb and goat anti-rabbit IgG were immobilized on nitrocellulose membrane using a three-dimensional streak instrument at a streak volume of 1 μL / cm and placed in a vacuum dryer at 30°C for 4 h;

[0036] 1) The sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are pasted on the back plate in the chromatography direction and cut into 4 mm wide test strips. The sample pad is pre-treated with 20 mmol / L Tris-HCl solution, the pH of the Tris-HCl solution is 8.0, containing 5% bovine serum albumin, 1%

[0037] Polyvinyl pyrrolidone and 0.5% Tween-20. After complete immersion, vacuum dry at 30°C for 4h;

[0038] 2) Drop 2-8 μL of gold palladium / iron oxide nanoparticle conjugate modified with MYO monoclonal antibody dAb onto the conjugate pad to obtain a colorimetric test strip for detecting MYO.

[0039] Step 3: MYO test:

[0040] Add 80 μL of the sample solution to be tested onto the sample pad, let it stand for 15 minutes until the test strip is fully reacted, and then collect the grayscale signal to prepare a standard curve to obtain the MYO content in the sample to be tested.

[0041] Figure 1 The structure and working principle of the immunochromatographic test strip provided by the present invention are shown in FIG. Figure 2-3 As shown. Among them, Figure 2The picture of the immunochromatographic test strip for detecting MYO obtained in this example changes with the change of MYO concentration can prove that the T / C line on the MYO detection test strip changes sensitively with the MYO concentration visible to the naked eye. Figure 3 The standard curve diagram obtained by detecting MYO using the immunochromatographic test strip for detecting MYO obtained in this embodiment can prove that the test strip provided by the present invention has high sensitivity and can achieve more accurate detection of MYO.

[0042] The above-described embodiments merely express the implementation methods of the present invention, but they cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing a MYO monoclonal antibody nanoparticle conjugate, characterized in that: The steps include: The gold palladium / iron oxide nanoparticle solution and the sodium polyacrylate aqueous solution were mixed and stirred, dispersed in 2-(N-morpholine)ethanesulfonic acid buffer after centrifugation, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added, and after sufficient reaction, MYO monoclonal antibody dAb was added to continue the reaction, and finally BSA was added for blocking and centrifugation to obtain MYO monoclonal antibody dAb-modified gold palladium / iron oxide nanoparticle conjugates, which were dispersed in Tris-HCl solution.

2. The method for preparing the MYO monoclonal antibody-modified gold palladium / iron oxide nanoparticle conjugate according to claim 1, characterized in that: The Tris-HCl solution has a pH of 8.0 and contains 20% sucrose, 5% trehalose, 1% BSA and 1% Tween-20.

3. The method for preparing the MYO monoclonal antibody-modified gold palladium / iron oxide nanoparticle conjugate according to claim 1, characterized in that: The amount of the MYO monoclonal antibody dAb is 5 μg to 12.5 μg.

4. A MYO monoclonal antibody-modified gold palladium / iron oxide nanoparticle conjugate prepared by the preparation method according to any one of claims 1 to 3.

5. An application of the MYO monoclonal antibody modified gold palladium / iron oxide nanoparticle conjugate as claimed in claim 4, characterized in that: Used to prepare instant detection tools for patients with acute myocardial infarction.

6. An immunochromatographic test strip, characterized in that: The invention comprises the gold palladium / iron oxide nanoparticle conjugate modified with the MYO monoclonal antibody as claimed in claim 4.

7. A method for preparing the immunochromatographic test strip according to claim 6, characterized in that: The following steps are included: S1. Use a three-dimensional streak instrument to immobilize MYO monoclonal antibody cAb and goat anti-rabbit IgG on a nitrocellulose membrane at a streak volume of 1 μL / cm, and then vacuum dry; S2, stick the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad on the back plate in the chromatography direction, and cut them; S3. Drop the gold palladium / iron oxide nanoparticle conjugate modified with MYO monoclonal antibody dAb onto the conjugate pad to obtain an immunochromatographic test strip for detecting MYO.

8. The method for preparing the immunochromatographic test strip according to claim 7, characterized in that: The amount of the MYO monoclonal antibody dAb modified gold palladium / iron oxide nanoparticle conjugate used in step S3 is 2 μL to 8 μL.

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