Glucose-deficiency transferrin detection method

Abstract

The invention relates to the technical field of biological detection, in particular to a sugar deficiency transferrin detection method. The invention relates to a method for detecting sugar-deficient transferrin. The method comprises the following steps: specifically separating sugar-deficient transferrin in a detection sample by using Fe3O4 magnetic nanoparticles coupled with an anti-sugar-deficient transferrin antibody; washing and eluting; labeling with a fluorescent dye; fluorescence detection; and calculating the concentration of the sugar deficiency transferrin. The Fe3O4 magnetic nanoparticles coupled with the anti-sugar-deletion transferrin antibody are used for performing specific separation on the sugar-deletion transferrin antibody in a detection sample, the antibody on the magnetic nanoparticles can efficiently capture the sugar-deletion transferrin, the detection sensitivity is improved, the sugar-deletion transferrin with lower concentration can be detected, and the detection sensitivity is improved. By utilizing the specific binding capacity of the anti-sugar-deficiency transferrin antibody, the influence of other interfering substances in a sample is effectively eliminated, and the detection specificity is improved.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for detecting sugar-deficient transferrin. Background Art

[0002] Glycodeoxytransferrin is an important biomarker that plays an important role in the diagnosis and monitoring of various diseases. Although traditional methods such as enzyme-linked immunosorbent assay (ELISA) are widely used for the detection of AT, they still have limitations in terms of sensitivity, specificity and rapid detection.

[0003] The current detection methods for sugar-deficient transferrin still have shortcomings. Traditional detection methods such as ELISA, although simple and easy to use, have low sensitivity in low-concentration samples and it is difficult to accurately detect trace amounts of sugar-deficient transferrin. In addition, due to the presence of various interfering substances in the samples, traditional methods are easily affected by nonspecific binding, resulting in false positive results.

[0004] In recent years, magnetic nanoparticles have been widely used in the biomedical field due to their unique physical and chemical properties. Fe3O4 magnetic nanoparticles have become an ideal separation medium due to their good biocompatibility and magnetic responsiveness. Using Fe3O4 magnetic nanoparticles coupled with specific antibodies, highly specific separation of target proteins can be achieved, thereby improving the sensitivity and accuracy of detection. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for detecting sugar-deficient transferrin.

[0006] A method for detecting sugar-deficient transferrin comprises the following steps:

[0007] (1) using Fe3O4 magnetic nanoparticles coupled with anti-glycosylated transferrin antibodies to specifically separate and detect the sugar-deficient transferrin in the sample;

[0008] (2) washing and eluting the separated test sample;

[0009] (3) using fluorescent dye to label the sugar-deficient transferrin in the sample after elution;

[0010] (4) detecting the labeled sample using fluorescence detection technology;

[0011] (5) A standard curve is established using the standard substance, and the concentration of sugar-deficient transferrin in the test sample is calculated based on the standard curve.

[0012] Furthermore, the Fe3O4 magnetic nanoparticles are prepared by chemical coprecipitation and subjected to amination or carboxylation surface modification treatment, wherein amination treatment uses a silane coupling agent, the silane coupling agent includes APTES, and carboxylation treatment uses a surface modifier, the surface modifier includes 3-carboxypropyltrimethoxysilane.

[0013] Furthermore, the surface of the Fe3O4 magnetic nanoparticles is coupled to anti-sugar-deficient transferrin antibodies via biotin-avidin.

[0014] Furthermore, the specific steps of biotin-avidin coupling are:

[0015] (a) Anti-glycosylated transferrin antibody was labeled with biotin;

[0016] (b) Activating the amino- or carboxyl-treated Fe3O4 magnetic nanoparticles using avidin or streptavidin;

[0017] (c) adding the biotin-labeled antibody to the activated Fe3O4 magnetic nanoparticle solution;

[0018] (d) incubating for 4-6 h or overnight at 4°C to allow the antibody to fully bind to the magnetic nanoparticles;

[0019] (e) separating the magnetic nanoparticles using a pH 5.0 citrate buffer and washing them three times with the citrate buffer;

[0020] (f) blocking the magnetic nanoparticles with a blocking buffer;

[0021] (g) The coupled Fe3O4 magnetic nanoparticles were resuspended in storage buffer and stored at 4°C.

[0022] Furthermore, the blocking buffer is PBS containing 5% glycerol and 0.05% Proclin 300, and the storage buffer is PBS containing 0.5% BSA, 0.05% Proclin 300 and 0.05% Tween 20 pH 7.4.

[0023] Furthermore, the step of specifically isolating sugar-deficient transferrin comprises:

[0024] (a) adding the pre-treated test sample into the coupled Fe3O4 magnetic nanoparticle suspension;

[0025] (b) Incubate at room temperature for 10-30 min;

[0026] (c) Separation of magnetic nanoparticles using a magnetic plate;

[0027] (d) washing the magnetic nanoparticles with sodium acetate buffer at pH 5.0;

[0028] (e) Bound sugar-deficient transferrin was eluted using sodium acetate buffer containing 0.1 M NaCl, pH 5.0.

[0029] Furthermore, when a 96-well plate or a multi-well plate is used to process multiple test samples, the Fe3O4 magnetic nanoparticles are separated by placing the well plate on a magnetic plate and waiting for 5-10 minutes to allow the magnetic nanoparticles to be completely adsorbed and retained at the bottom of the well plate.

[0030] Furthermore, the fluorescent dye is Alexa Fluor 488 fluorescent dye, and the labeling conditions are reaction time of 1 hour, room temperature, and pH value of 7.4.

[0031] Furthermore, the fluorescence detection uses an excitation wavelength of 488 nm and an emission wavelength of 519 nm, and sets the detection threshold to 0.035 FU.

[0032] Furthermore, the pretreatment of the test sample specifically includes: treating the serum sample with trichloroacetic acid, centrifuging at 10000 rpm for 5 minutes, collecting the supernatant and diluting it to 1:10 or 1:20 as the test sample.

[0033] The beneficial effects achieved by the present invention are as follows: 1. The present invention uses Fe3O4 magnetic nanoparticles coupled with anti-glycosylated transferrin antibodies to specifically separate the sugar-deficient transferrin antibodies in the test sample. The antibodies on the magnetic nanoparticles can efficiently capture sugar-deficient transferrin, improve the detection sensitivity, and detect lower concentrations of sugar-deficient transferrin. The specific binding ability of the anti-glycosylated transferrin antibodies is utilized to effectively eliminate the influence of other interfering substances in the sample, improve the specificity of the detection, and reduce nonspecific binding. At the same time, the use of magnetic nanoparticles can simplify the separation steps and improve the separation efficiency. The magnetic nanoparticles can be quickly separated by a magnetic plate, reducing the operation steps and improving the degree of automation.

[0034] 2. The present invention uses Alexa Fluor 488 fluorescent dye to label the eluted sugar-deficient transferrin, and uses fluorescence detection technology for detection. The high sensitivity of the fluorescent label is utilized to facilitate the detection of low concentrations of sugar-deficient transferrin. The fluorescent dye has good light stability, can reduce background interference during the detection process, and improve the accuracy of the detection results.

[0035] 3. The detection method of the present invention is suitable for rapid detection of large batches of samples. A multi-well plate is used to process multiple test samples, and magnetic nanoparticles are separated by a magnetic plate to improve work efficiency.

[0036] 4. The present invention reduces background interference and improves the reliability of detection results through specific buffer and washing steps, and optimizes each step from sample dilution, separation, labeling to detection to simplify the operation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The schematic flow chart of the sugar-deficient transferrin detection method used in the present invention;

[0038] Figure 2 The linear range experimental results of the sugar-deficient transferrin detection method used in Examples 1-3 and Comparative Examples of the present invention;

[0039] Figure 3 These are the sensitivity test results of the sugar-deficient transferrin detection method used in Examples 1-3 of the present invention and the comparative example. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with specific embodiments.

[0041] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0042] A method for detecting sugar-deficient transferrin, such as Figure 1 As shown, the following steps are included:

[0043] (1) using Fe3O4 magnetic nanoparticles coupled with anti-glycosylated transferrin antibodies to specifically separate and detect the sugar-deficient transferrin in the sample;

[0044] (2) washing and eluting the separated test sample;

[0045] (3) using fluorescent dye to label the sugar-deficient transferrin in the sample after elution;

[0046] (4) detecting the labeled sample using fluorescence detection technology;

[0047] (5) A standard curve is established using the standard substance, and the concentration of sugar-deficient transferrin in the test sample is calculated based on the standard curve.

[0048] The following example is the preparation of Fe3O4 magnetic nanoparticles coupled with anti-glycosylated transferrin antibodies:

[0049] 1. Preparation of Fe3O4 magnetic nanoparticles by chemical coprecipitation

[0050] Add 100 mL of deionized water to a beaker and dissolve 1 mmol of FeCl2·4H2O and 2 mmol of FeCl3·6H2O; slowly add NH4OH (ammonia water) until the pH reaches 10.5, heat and stir in a water bath at 80°C for 30 min, and cool naturally to room temperature; separate the magnetic nanoparticles using a permanent magnet, wash with deionized water three times until the washing liquid is clear, wash once with ethanol, and then dry at 60°C for 24 h;

[0051] 2. Surface modification of Fe3O4 magnetic nanoparticles

[0052] Amination treatment: Add 50 mL of ethanol and 1 mL of APTES to a clean beaker; add HCl to adjust the pH to 3.5, disperse the prepared Fe3O4 magnetic nanoparticles in the above solution, stir overnight at room temperature, separate the magnetic nanoparticles using a permanent magnet, wash with ethanol three times, and dry at 60°C for 24 h;

[0053] Carboxylation treatment: Add 50 mL of ethanol and 1 mL of 3-carboxypropyltrimethoxysilane to a clean beaker, add an appropriate amount of HCl to adjust the pH to 3.5, disperse the prepared Fe3O4 magnetic nanoparticles in the above solution, stir overnight at room temperature, separate the magnetic nanoparticles using a permanent magnet, wash with ethanol three times, and dry at 60°C for 24 h;

[0054] 3. Antibody conjugation of Fe3O4 magnetic nanoparticles

[0055] (a) Anti-glycosylated transferrin antibody was labeled with biotin;

[0056] (b) dispersing the amino or carboxyl-grouped (amino group was used in all examples) Fe3O4 magnetic nanoparticles in a PBS buffer at pH 7.4, adding streptavidin, and stirring at room temperature for 30 min;

[0057] (c) adding the biotin-labeled antibody to the activated Fe3O4 magnetic nanoparticle solution;

[0058] (d) incubating for 5 h to allow the antibody to fully bind to the magnetic nanoparticles;

[0059] (e) separating the magnetic nanoparticles using a pH 5.0 citrate buffer and washing them three times with the citrate buffer;

[0060] (f) The magnetic nanoparticles were blocked with PBS containing 5% glycerol and 0.05% Proclin 300;

[0061] (g) The coupled Fe3O4 magnetic nanoparticles were resuspended in PBS containing 0.5% BSA, 0.05% Proclin 300 and 0.05% Tween20 pH 7.4 and stored at 4°C.

[0062] Example 1: A method for detecting sugar-deficient transferrin, comprising the following steps:

[0063] (1) Sample pretreatment: Take 1 mL of standard solution (sugar-deficient transferrin of known concentration), treat it with trichloroacetic acid, centrifuge it at 10,000 rpm for 5 min, collect the supernatant, and dilute it to 1:10 (supernatant: PBS buffer) as the test sample;

[0064] (2) Separation of sugar-deficient transferrin: Take 500 μL of the pre-treated test sample and add it to 100 μL of a pre-stored Fe3O4 magnetic nanoparticle suspension coupled with anti-sugar-deficient transferrin antibodies; incubate at room temperature for 10 min; separate the magnetic nanoparticles using a magnetic plate; wash the magnetic nanoparticles three times with sodium acetate buffer at pH 5.0, and separate the magnetic nanoparticles again with a magnetic plate after each wash; elute the bound sugar-deficient transferrin using sodium acetate buffer containing 0.1 M NaCl and pH 5.0.

[0065] (3) Fluorescent labeling: Take 100 μL of the eluted sugar-deficient transferrin solution, add Alexa Fluor 488 fluorescent dye (1 mg / mL), and react at room temperature for 1 h, pH 7.4;

[0066] (4) Fluorescence detection: The separated samples were detected using fluorescence detection technology, with an excitation wavelength of 488 nm, an emission wavelength of 519 nm, and a detection threshold of 0.035 FU;

[0067] (5) Calculating concentration: A standard curve is established using the standard substance, and the concentration of sugar-deficient transferrin in the test sample is calculated based on the standard curve.

[0068] Example 2: A method for detecting sugar-deficient transferrin, comprising the following steps:

[0069] (1) Sample pretreatment: Take 1 mL of serum sample, treat it with trichloroacetic acid, centrifuge it at 10,000 rpm for 5 min, collect the supernatant, and dilute it to 1:20 (supernatant: PBS buffer) as the test sample;

[0070] (2) Separation of sugar-deficient transferrin: Take 500 μL of the pre-treated test sample and add it to 100 μL of a pre-stored Fe3O4 magnetic nanoparticle suspension coupled with anti-sugar-deficient transferrin antibodies; incubate at room temperature for 20 min; separate the magnetic nanoparticles using a magnetic plate; wash the magnetic nanoparticles three times with sodium acetate buffer at pH 5.0, and separate the magnetic nanoparticles again with a magnetic plate after each wash; elute the bound sugar-deficient transferrin using sodium acetate buffer containing 0.1 M NaCl and pH 5.0.

[0071] (3) Fluorescent labeling: Take 100 μL of the eluted sugar-deficient transferrin solution, add Alexa Fluor 488 fluorescent dye (1 mg / mL), and react at room temperature for 1 h, pH 7.4;

[0072] (4) Fluorescence detection: The separated samples were detected using fluorescence detection technology, with an excitation wavelength of 488 nm, an emission wavelength of 519 nm, and a detection threshold of 0.035 FU;

[0073] (5) Calculating concentration: A standard curve is established using the standard substance, and the concentration of sugar-deficient transferrin in the test sample is calculated based on the standard curve.

[0074] Example 3: A method for detecting sugar-deficient transferrin, comprising the following steps:

[0075] (1) Sample pretreatment: Take 1 mL of environmental water sample, treat it with trichloroacetic acid, centrifuge it at 10,000 rpm for 5 min, collect the supernatant, and dilute it to 1:10 (supernatant: PBS buffer) as the test sample;

[0076] (2) Separation of sugar-deficient transferrin: Take 500 μL of the pre-treated test sample and add it to 100 μL of a pre-stored Fe3O4 magnetic nanoparticle suspension coupled with anti-sugar-deficient transferrin antibodies; incubate at room temperature for 30 min; separate the magnetic nanoparticles using a magnetic plate; wash the magnetic nanoparticles three times with sodium acetate buffer at pH 5.0, and separate the magnetic nanoparticles again with a magnetic plate after each wash; elute the bound sugar-deficient transferrin using sodium acetate buffer containing 0.1 M NaCl and pH 5.0.

[0077] (3) Fluorescent labeling: Take 100 μL of the eluted sugar-deficient transferrin solution, add Alexa Fluor 488 fluorescent dye (1 mg / mL), and react at room temperature for 1 h, pH 7.4;

[0078] (4) Fluorescence detection: The separated samples were detected using fluorescence detection technology, with an excitation wavelength of 488 nm, an emission wavelength of 519 nm, and a detection threshold of 0.035 FU;

[0079] (5) Calculating concentration: A standard curve is established using the standard substance, and the concentration of sugar-deficient transferrin in the test sample is calculated based on the standard curve.

[0080] Comparative Example: Conventional sugar-deficient transferrin ELISA kit, the detection method is:

[0081] (1) Use a commercially available ELISA kit and follow the instructions;

[0082] (2) Dilute the sample to 1:10, add it to the ELISA plate, and perform subsequent operations according to the kit instructions;

[0083] (3) Use an enzyme-labeled instrument to detect the absorbance value on the ELISA plate, and calculate the concentration of sugar-deficient transferrin in the sample using a standard curve.

[0084] Test experiments: precision, linear range, sensitivity, specificity

[0085] 1. Precision experiment: Prepare standard samples or samples of known concentration, then measure the same standard sample repeatedly according to the experimental plan, record the results of each measurement, and calculate the coefficient of variation (CV).

[0086] Results As can be seen from Table 1, the CVs of Examples 1-3 were 3.5%, 4.1% and 5.0%, respectively, which were all lower than 5.2%, 6.8% and 7.5% of the comparative example, indicating that the detection method of the present invention has higher precision.

[0087] Table 1 Precision test results

[0088]

[0089] 2. Linear range experiment: Prepare a series of standard solutions with different concentrations (0-10 ng / mL) and measure their response values ​​(OD values).

[0090] Results from Figure 2 It can be seen that the OD values ​​of Examples 1-3 at various concentrations all show a good linear relationship, which is better than the comparative example. Specific data show that the OD values ​​of Examples 1-3 increase linearly with increasing concentrations, and the linear correlation coefficient is relatively high.

[0091] 3. Sensitivity experiment: Prepare a series of low concentration (0.01-0.10 ng / mL) standard solutions and measure their response values ​​(OD values).

[0092] Results from Figure 3It can be seen that Examples 1-3 exhibit higher sensitivity in the low concentration range, and the OD value changes significantly, which is better than the comparative example. Specific data show that the response values ​​of Examples 1-3 at low concentrations are significantly higher than those of the comparative example, indicating that the method has higher sensitivity.

[0093] 4. Specificity experiment: Select several common interfering substances (BSA, IgG, HSA, hemoglobin) and measure the response value (OD value) in the presence of these interfering substances.

[0094] Results From Table 2, it can be seen that the background signals of Examples 1-3 in the presence of interfering substances are lower, indicating good specificity. Specific data show that the OD values ​​of Examples 1-3 under various interfering substances are lower than those of the comparative example, showing good specificity.

[0095] Table 2 Specificity test results

[0096]

[0097] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for detecting sugar-deficient transferrin, characterized in that: The following steps are involved: (1) using Fe3O4 magnetic nanoparticles coupled with anti-glycosylated transferrin antibodies to specifically separate and detect the sugar-deficient transferrin in the sample; (2) washing and eluting the separated test sample; (3) using fluorescent dye to label the sugar-deficient transferrin in the sample after elution; (4) detecting the labeled sample using fluorescence detection technology; (5) A standard curve is established using the standard substance, and the concentration of sugar-deficient transferrin in the test sample is calculated based on the standard curve.

2. A method for detecting sugar-deficient transferrin according to claim 1, characterized in that: The Fe3O4 magnetic nanoparticles are prepared by chemical coprecipitation and subjected to amination or carboxylation surface modification treatment. Amination treatment uses a silane coupling agent, which includes APTES. Carboxylation treatment uses a surface modifier, which includes 3-carboxypropyltrimethoxysilane.

3. A method for detecting sugar-deficient transferrin according to claim 1, characterized in that: The surface of the Fe3O4 magnetic nanoparticles is coupled with anti-sugar-deficient transferrin antibodies via biotin-avidin.

4. A method for detecting sugar-deficient transferrin according to claim 3, characterized in that: The specific steps of biotin-avidin coupling are: (a) Anti-glycosylated transferrin antibody was labeled with biotin; (b) Activating the amino- or carboxyl-treated Fe3O4 magnetic nanoparticles using avidin or streptavidin; (c) adding the biotin-labeled antibody to the activated Fe3O4 magnetic nanoparticle solution; (d) incubating for 4-6 h or overnight at 4°C to allow the antibody to fully bind to the magnetic nanoparticles; (e) separating the magnetic nanoparticles using a pH 5.0 citrate buffer and washing them three times with the citrate buffer; (f) blocking the magnetic nanoparticles with a blocking buffer; (g) The coupled Fe3O4 magnetic nanoparticles were resuspended in storage buffer and stored at 4°C.

5. A method for detecting sugar-deficient transferrin according to claim 4, characterized in that: The blocking buffer was PBS containing 5% glycerol and 0.05% Proclin 300, and the storage buffer was PBS containing 0.5% BSA, 0.05% Proclin 300 and 0.05% Tween 20 pH 7.

4.

6. The method for detecting sugar-deficient transferrin according to claim 1, characterized in that: The step of specifically isolating sugar-deficient transferrin comprises: (a) adding the pre-treated test sample into the coupled Fe3O4 magnetic nanoparticle suspension; (b) Incubate at room temperature for 10-30 min; (c) Separation of magnetic nanoparticles using a magnetic plate; (d) washing the magnetic nanoparticles with sodium acetate buffer at pH 5.0; (e) Bound sugar-deficient transferrin was eluted using sodium acetate buffer containing 0.1 M NaCl, pH 5.

0.

7. A method for detecting sugar-deficient transferrin according to claim 6, characterized in that: When using a 96-well plate or multi-well plate to process multiple test samples, place the well plate on the magnetic plate and wait for 5-10 minutes until the magnetic nanoparticles are completely adsorbed and retained at the bottom of the well plate.

8. The method for detecting sugar-deficient transferrin according to claim 1, characterized in that: The fluorescent dye is Alexa Fluor 488 fluorescent dye, and the labeling conditions are reaction time of 1 hour, room temperature, and pH value of 7.

4.

9. The method for detecting sugar-deficient transferrin according to claim 1, characterized in that: The fluorescence detection used an excitation wavelength of 488 nm and an emission wavelength of 519 nm, and the detection threshold was set at 0.035 FU.

10. The method for detecting sugar-deficient transferrin according to claim 1, characterized in that: The pretreatment of the test sample specifically includes: treating the serum sample with trichloroacetic acid, centrifuging at 10000 rpm for 5 minutes, collecting the supernatant and diluting it to 1:10 or 1:20 as the test sample.

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