Method for simultaneously determining carbohydrate antigen 125 and human epididymis secretory protein 4

Abstract

The invention discloses a method for simultaneously determining carbohydrate antigen 125 and human epididymal secretory protein 4, and relates to the technical field of tumor marker detection. The determination method comprises the following steps: introducing a double-antibody sandwich immune complex into ICP-MS (Inductively Coupled Plasma Mass Spectrometry) for testing, determining a metal element signal, drawing a working curve according to a linear relationship between the signal frequency of a metal element in the ICP-MS and the concentrations of CA125 and HE4, and calculating the contents of CA125 and HE4 through the working curve, and the double-antibody sandwich immune complex contains ZnO QD (Quantitative Derivative) and SnO2QD (SnO2 Quantitative Derivative). The method has the advantages of high sensitivity, good selectivity, low reagent consumption and the like, and is successfully used for measuring the contents of CA125 and HE4 in human serum.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor marker detection, and in particular to a method for simultaneously measuring carbohydrate antigen 125 and human epididymis secretory protein 4. Background Art

[0002] Carbohydrate antigen 125 (CA125) is a glycoprotein encoded by the MUC16 gene. It was first detected in ovarian cancer tissues by ovarian cancer 125 monoclonal antibodies. The content of CA125 is low in normal human tissues, but high in ovarian cancer. Therefore, CA125 is an important biomarker for the diagnosis of ovarian cancer. Human epididymis protein 4 (HE4) is a secretory glycoprotein product of the WFDC2 gene. HE4 is highly expressed in a variety of tumor cell lines, including ovarian cancer, lung cancer, colon cancer, and breast cancer, and high levels of secretory HE4 have been detected in the serum of ovarian cancer patients. Studies have shown that the combined detection of HE4 and CA125 in serum can improve the clinical diagnosis rate of ovarian cancer and is a good indicator for the diagnosis and differential diagnosis of benign and malignant ovarian tumors. Therefore, the simultaneous detection of the content of CA125 and HE4 in human serum is of great significance for the early diagnosis of ovarian cancer. Currently, the methods reported for detecting CA125 and HE4 in serum include computed tomography, chemiluminescence immunoassay, electrochemical immunoassay, electrochemiluminescence, enzyme-linked immunosorbent assay, and laser-induced breakdown spectroscopy. The above methods require professional operation and are expensive. Therefore, exploring a simple and low-cost detection method has become an important direction for biomarker research.

[0003] Inductively coupled plasma mass spectrometry (ICP-MS) is widely used in elemental labeling combined with ICP-MS to measure RNA, DNA, proteins, cells and viruses due to its advantages of high sensitivity, low detection limit, wide linear range and multiple analysis capabilities. Quantum dots (QD) have received extensive attention in ICP-MS immunoassay due to their good biocompatibility, high sensitivity in ICP-MS and low background in biological samples. Currently, the most studied are CdE (E = S, Se, Te) series quantum dots represented by CdSe. However, the strong toxicity and environmental pollution of Cd series quantum dots limit their application in biology.

[0004] Zinc oxide quantum dots (ZnO QD) have gained wide attention due to their low cost, non-toxicity, good biocompatibility and long-term environmental stability. Tin dioxide quantum dots (SnO2 QD) are widely used in sensors, cytotoxicity and antibacterial activity due to their excellent physical and chemical stability, high photosensitivity and excellent electrical properties. ZnO QD and SnO2 QD overcome the high toxicity defects of commonly used Cd series quantum dots. Magnetic nanoparticles (MNPs) of ferroferric oxide can quickly separate target analytes from complex samples due to their superparamagnetism. The applicant found that the use of amino-functionalized ferroferric oxide magnetic nanoparticles for magnetic separation and the use of synthesized ZnO QD and SnO2 QD as labeled probes for the detection of HE4 and CA125 in serum can improve the detection sensitivity. Summary of the invention

[0005] In view of the above shortcomings, the present invention provides a method for simultaneously determining carbohydrate antigen 125 and human epididymal secretory protein 4, which has the advantages of high sensitivity, good selectivity and low reagent consumption. The specific technical scheme is as follows:

[0006] A method for simultaneously determining carbohydrate antigen 125 and human epididymal secretory protein 4, the determination method comprising introducing a double antibody sandwich immune complex into ICP-MS for testing, determining metal element signals, and drawing a working curve based on the linear relationship between the signal frequency of the metal element in a single particle ICP-MS and the concentrations of CA125 and HE4, and calculating the contents of CA125 and HE4 through the working curve; the double antibody sandwich immune complex comprises ZnO QDs and SnO2 QDs.

[0007] Furthermore, the preparation method of the double antibody sandwich immune complex comprises the following steps:

[0008] (1) Preparation of AMNPs: Disperse ferroferric oxide magnetic nanoparticles in ethanol and ultrapure water, ultrasonicate at 200 W for 30 min, transfer to a three-necked flask, add 3-aminopropyltriethoxysilane while stirring, heat to 50-65° C. for reaction, cool to room temperature after the reaction, separate by external magnetic field, wash with ultrapure water and anhydrous ethanol alternately for 3-5 times, and obtain the AMNPs (amino-modified ferroferric oxide magnetic nanoparticles), dry and set aside;

[0009] (2) Preparation of ZnO QDs: First, 0.1 mol / L zinc acetate methanol solution was added dropwise to 1 mol / L potassium hydroxide methanol solution, and magnetic stirring was performed for 0.5-2 h to control the growth of particles, and then 3-aminopropyltriethoxysilane was added dropwise, and finally ultrapure water was added to cause a sol-gel reaction to obtain the ZnO QDs;

[0010] (3) Preparation of functionalized SnO2 QDs: stannous chloride dihydrate and thiourea were ultrasonically dispersed with ultrapure water at 200 W power, magnetically stirred for 24 h, reacted in a reactor at 150-250 °C, centrifuged at 10,000 rpm for 15 min, and the upper layer liquid was discarded to obtain SnO2 QDs. The SnO2 QDs were added to an ethanol solution of p-aminobenzoic acid, ultrasonicated, and then placed in a hot water bath at 60 °C for reaction, centrifuged, and obtained functionalized SnO2 QDs;

[0011] (4) Preparation of AMNPs-Ab1: AMNPs were dispersed in a PBS buffer solution (0.01 mol / L, pH = 7.4), sonicated, activated by adding EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), and then CA125-Ab1 and HE4-Ab1 were added respectively. The mixture was stirred for reaction, magnetically separated, and washed with a PBS buffer solution to obtain the AMNPs-Ab1.

[0012] (5) Preparation of CA125-Ab2-ZnO QDs: dispersing the ZnO QDs in a PBS buffer solution, adding EDC and NHS, reacting, centrifuging, discarding the supernatant, collecting the lower layer, adding CA125-Ab2 to the lower layer, stirring for reaction, and centrifuging to obtain the CA125-Ab2-ZnO QDs;

[0013] (6) Preparation of HE4-Ab2-SnO2 QDs: dispersing the functionalized SnO2 QDs in a PBS buffer solution, adding EDC and NHS, reacting, centrifuging, discarding the supernatant, collecting the lower layer, pipetting HE4-Ab2 into the lower layer, stirring to react, centrifuging, and obtaining HE4-Ab2-SnO2 QDs;

[0014] (7) Preparation of double antibody sandwich immune complex: adding CA125 and HE4 to AMNPs-Ab1, incubating and reacting, and magnetically separating to obtain an AMNPs-Ab1-CA125 / HE4 complex; respectively adding the CA125-Ab2-ZnO QD and HE4-Ab2-SnO2 QD to the AMNPs-Ab1-CA125 / HE4 complex, incubating and reacting to form a double antibody sandwich AMNPs-Ab1-CA125 / HE4-Ab2-ZnO QD / SnO2 QD complex.

[0015] Furthermore, in step (4), the volume ratio of CA125-Ab1, HE4-Ab1 and AMNPs is 1:1:1-2.

[0016] Furthermore, in step (7), the volume ratio of CA125, HE4 and AMNPs-Ab1 is 1:1:55-60.

[0017] Furthermore, in step (7), the volume ratio of the CA125-Ab2-ZnO QD, HE4-Ab2-SnO2 QD and AMNPs-Ab1-CA125 / HE4 complex is 1:1:2.5-3.5.

[0018] Furthermore, the linear equation of the signal frequency (Y) of the metal element and the concentration (X) of CA125 is Y=3.54X+9.22. 64 The linear relationship equation between Zn signal frequency (Y) and CA125 concentration (X) is Y=3.54X+9.22.

[0019] Furthermore, the linear equation of the signal frequency (Y) of the metal element and the concentration (X) of HE4 is Y=5.59X+13.77. 120 The linear relationship equation between the signal frequency (Y) of Sn and the concentration (X) of HE4 is Y=5.59X+13.77.

[0020] Furthermore, the double antibody sandwich immune complex is diluted with PBS buffer solution before being introduced into ICP-MS for testing.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention establishes a new method for analyzing ovarian cancer biomarkers CA125 and HE4 in human serum by combining amino-functionalized zinc oxide quantum dots (ZnO QD) and tin oxide quantum dots (SnO2QD) labeled antibodies with magnetic immune single particle inductively coupled plasma mass spectrometry. Amino-functionalized ferroferric oxide magnetic nanoparticles are synthesized to combine CA125 and HE4 primary antibodies for capturing CA125 and HE4 antigens, and independently synthesized ZnO QD and SnO2 QD are used to label CA125 and HE4 secondary antibodies, respectively. The signal frequencies of zinc and tin are determined by ICP-MS to achieve quantitative analysis of CA125 and HE4. The detection method of the present invention provides an effective new approach for the analysis of CA125 and HE4, and the detection is convenient and rapid, which is significantly improved compared to the traditional enzyme-linked immunosorbent assay method and can be used for clinical analysis.

[0023] 2. The detection method of the present invention has good selectivity and high sensitivity. Under the optimal experimental conditions, the linear ranges of CA125 and HE4 are 0.02-200 U / mL and 0.02-100 ng / mL, respectively. The detection limits are 0.004 U / mL and 0.006 ng / mL (3σ), respectively. The RSDs are 2.2% and 3.5% (n=6), respectively, which can meet the actual detection needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0026] Figure 1 is a flow chart of the sandwich immune response of the present invention;

[0027] Figure 2 TEM image of the double-antibody sandwich AMNPs-Ab1-CA125 / HE4-Ab2-ZnO QD / SnO2 QD complex prepared in Example 1;

[0028] Figure 3 It is the linear curve graph of CA125 in Example 2;

[0029] Figure 4 This is the linear curve diagram of HE4 in Example 2. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0031] Example 1

[0032] like Figure 1 As shown, the preparation method of the double antibody sandwich immune complex in this embodiment includes the following steps:

[0033] (1) Preparation of AMNPs: Disperse ferroferric oxide magnetic nanoparticles in ethanol and ultrapure water, ultrasonicate at 200 W for 30 min, transfer to a three-necked flask, add 3-aminopropyltriethoxysilane while stirring, heat at 55° C. for reaction, cool to room temperature after the reaction, separate by external magnetic field, wash with ultrapure water and anhydrous ethanol alternately for 5 times, and obtain the AMNPs (amino-modified ferroferric oxide magnetic nanoparticles), dry and set aside;

[0034] (2) Preparation of ZnO QDs: First, 0.1 mol / L zinc acetate methanol solution was added dropwise to 1 mol / L potassium hydroxide methanol solution, and magnetic stirring was performed for 1 h to control the growth of particles, and then 3-aminopropyltriethoxysilane was added dropwise, and finally ultrapure water was added to cause a sol-gel reaction to obtain the ZnO QDs;

[0035] (3) Preparation of functionalized SnO2 QDs: stannous chloride dihydrate and thiourea were ultrasonically dispersed with ultrapure water at a power of 200 W, magnetically stirred for 24 h, reacted in a reactor at 200 °C, centrifuged at 10,000 rpm for 15 min, and the upper layer liquid was discarded to obtain SnO2 QDs. The SnO2 QDs were added to an ethanol solution of p-aminobenzoic acid, ultrasonicated, and then placed in a hot water bath at 60 °C for reaction, centrifuged, and obtained functionalized SnO2 QDs;

[0036] (4) Preparation of AMNPs-Ab1: 15 mg of AMNPs were dispersed in a PBS buffer solution (0.01 mol / L, pH = 7.4), sonicated, activated by adding EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), and then 10 μL of CA125-Ab1 and 10 μL of HE4-Ab1 were added respectively, stirred for reaction, magnetically separated, and washed with PBS buffer solution to obtain the AMNPs-Ab1;

[0037] (5) Preparation of CA125-Ab2-ZnO QDs: dispersing the ZnO QDs in a PBS buffer solution, adding EDC and NHS, reacting, centrifuging, discarding the supernatant, and collecting the lower layer; adding CA125-Ab2 to the lower layer, stirring for reaction; centrifuging, discarding the supernatant, and obtaining the CA125-Ab2-ZnO QDs;

[0038] (6) Preparation of HE4-Ab2-SnO2 QDs: dispersing the functionalized SnO2 QDs in a PBS buffer solution, adding EDC and NHS, reacting, centrifuging, discarding the supernatant, and collecting the lower layer; pipetting HE4-Ab2 into the lower layer, stirring to react, centrifuging, and discarding the supernatant to obtain HE4-Ab2-SnO2 QDs;

[0039] (7) Preparation of double antibody sandwich immune complex: 5 μL CA125 and 5 μL HE4 were added to 300 μL AMNPs-Ab1, incubated, and magnetically separated to obtain AMNPs-Ab1-CA125 / HE4 complex; 100 μL CA125-Ab2-ZnO QD and 100 μL HE4-Ab2-SnO2 QD were respectively added to 300 μL AMNPs-Ab1-CA125 / HE4 complex, incubated, and formed a double antibody sandwich AMNPs-Ab1-CA125 / HE4-Ab2-ZnO QD / SnO2 QD complex. Figure 2 Shown are double-antibody sandwich AMNPs-Ab1-CA125 / HE4-Ab2-ZnO QD / SnO2 QD composite particles under high-resolution transmission electron microscopy.

[0040] Example 2

[0041] Citric acid was used as an eluent to elute the dual-antibody sandwich AMNPs-Ab1-CA125 / HE4-Ab2-ZnO QD / SnO2 QD complex prepared in Example 1, and ultrasonic and magnetic separation were performed to collect the eluate; 10 μL of the eluate was diluted to 1 mL with PBS buffer solution and introduced into ICP-MS test; a blank test was used to eliminate the interference of Zn in the sample.

[0042] The operating parameters of the ICP-MS instrument were as follows: RF power of 1500 W, nebulizer gas flow rate of 1.0 L / min, auxiliary gas flow rate of 0.8 L / min, plasma gas flow rate of 14 L / min, RPq value of 0.45, dwell time of 0.1 ms, helium collision gas flow rate of 1.2 mL / min, and the monitored elements were 64 Zn and 120 Sn.

[0043] Single particle ICP-MS data processing: Use an iterative algorithm based on 3σ to determine the signal and background signal of the nanoparticles. First calculate the average and standard deviation (σ) of all data, collect all data that are greater than the average plus 3σ; recalculate the standard deviation of the remaining data, and then recollect all data that are greater than the new average plus 3σ; repeat until no other data points are greater than the sum of the average and 3σ. The remaining data represents the background signal, and the collected data is the signal of the nanoparticle.

[0044] Under the optimal experimental conditions, this method was used to detect different concentrations of CA125 and HE4. 64 Zn and 120The signal frequency (Y) of Sn was linearly related to the concentration (X) of CA125 and HE4, respectively. The linear range of CA125 was 0.02-200 U / mL, the linear equation was Y=3.54X+9.22, and the linear correlation coefficient R 2 =0.9994, the detection limit was 0.004U / mL (3σ), and the relative standard deviation (RSD) was 2.2% (n=6); the linear range of HE4 was 0.02-100ng / mL, the linear equation was Y=5.59X+13.77, and the linear correlation coefficient R 2 =0.9995, the detection limit is 0.006ng / mL (3σ), and the RSD is 3.5% (n=6). The linear curve of CA125 is as follows Figure 3 As shown, the linear curve of HE4 is Figure 4 shown.

[0045] Detection example of clinical samples: 5 serum samples were collected from the Affiliated Hospital of Guangxi University of Chinese Medicine, and the double-antibody sandwich AMNPs-Ab1-CA125 / HE4-Ab2-ZnO QD / SnO2 QD complex prepared in Example 1 was used for detection according to the method of the present application. The contents of CA125 and HE4 in each sample were calculated according to the above linear equation, and then compared with the results obtained by the enzyme-linked immunosorbent assay used in the hospital's clinical diagnosis. The results are shown in Table 1. In the enzyme-linked immunosorbent assay, a normal value of CA125 (U / mL) greater than or equal to 35U / mL is considered a positive result (+), otherwise it is considered a negative result (-); a normal value of HE4 greater than or equal to 2.5ng / mL is considered a positive result (+), otherwise it is considered a negative result (-).

[0046] Table 1 Comparison of CA125 and HE4 content in each sample

[0047]

[0048] The above table shows that the detection results of the detection method of the present invention are basically consistent with those of the conventional method, and can meet the actual detection needs.

[0049] In summary, the present invention has established a new method for analyzing ovarian cancer biomarkers CA125 and HE4 in human serum by combining amino-functionalized zinc oxide quantum dots (ZnO QD) and tin oxide quantum dots (SnO2 QD) labeled antibodies with magnetic immunosingle particle inductively coupled plasma mass spectrometry. This method can provide an effective new approach for the analysis of CA125 and HE4, and the detection is convenient and rapid, and can be used for clinical analysis.

[0050] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A method for simultaneously determining carbohydrate antigen 125 and human epididymis secretory protein 4, characterized in that: The determination method is to introduce a double antibody sandwich immune complex into ICP-MS for testing, determine the metal element signal, and draw a working curve based on the linear relationship between the signal frequency of the metal element in a single particle ICP-MS and the concentration of CA125 and HE4, and calculate the content of CA125 and HE4 through the working curve; the double antibody sandwich immune complex contains ZnO QD and SnO2 QD.

2. The method for simultaneously determining carbohydrate antigen 125 and human epididymis secretory protein 4 according to claim 1, characterized in that: The method for preparing the double antibody sandwich immune complex comprises the following steps: (1) Preparation of AMNPs: Dispersing ferroferric oxide magnetic nanoparticles in ethanol and ultrapure water, sonicating, transferring to a three-necked flask, adding 3-aminopropyltriethoxysilane while stirring, heating at 50-65° C. for reaction, cooling to room temperature after the reaction, separating by an external magnetic field, washing with ultrapure water and anhydrous ethanol alternately for 3-5 times to obtain the AMNPs, drying, and setting aside; (2) Preparation of ZnO QDs: First, zinc acetate methanol solution was added dropwise to potassium hydroxide methanol solution, and magnetic stirring was performed for 0.5-2 h to control the growth of particles, and then 3-aminopropyltriethoxysilane was added dropwise, and finally ultrapure water was added to cause a sol-gel reaction to obtain the ZnO QDs; (3) Preparation of functionalized SnO2 QDs: taking stannous chloride dihydrate and thiourea, ultrasonically dispersing them with ultrapure water, magnetically stirring them, reacting them in a reactor at 150-250°C, centrifuging them, and obtaining SnO2 QDs; adding the SnO2 QDs to an ethanol solution of p-aminobenzoic acid, ultrasonically treating them, and then reacting them in a hot water bath, centrifuging them, and obtaining the functionalized SnO2 QDs; (4) preparing AMNPs-Ab1: dispersing AMNPs in a PBS buffer solution, sonicating, adding EDC and NHS for activation, adding CA125-Ab1 and HE4-Ab1 respectively, stirring for reaction, magnetic separation, and washing with a PBS buffer solution to obtain the AMNPs-Ab1; (5) Preparation of CA125-Ab2-ZnO QDs: dispersing the ZnO QDs in a PBS buffer solution, adding EDC and NHS, reacting, centrifuging, discarding the supernatant, and collecting the lower layer; adding CA125-Ab2 to the lower layer, stirring for reaction; centrifuging to obtain the CA125-Ab2-ZnO QDs; (6) Preparation of HE4-Ab2-SnO2 QDs: dispersing the functionalized SnO2 QDs in a PBS buffer solution, adding EDC and NHS, reacting, centrifuging, discarding the supernatant, and collecting the lower layer; pipetting HE4-Ab2 into the lower layer, stirring to react, and centrifuging to obtain HE4-Ab2-SnO2 QDs; (7) Preparation of double antibody sandwich immune complex: adding CA125 and HE4 to AMNPs-Ab1, incubating and reacting, and magnetically separating to obtain an AMNPs-Ab1-CA125 / HE4 complex; respectively adding the CA125-Ab2-ZnO QD and HE4-Ab2-SnO2 QD to the AMNPs-Ab1-CA125 / HE4 complex, incubating and reacting to form a double antibody sandwich AMNPs-Ab1-CA125 / HE4-Ab2-ZnO QD / SnO2 QD complex.

3. The method for simultaneously determining carbohydrate antigen 125 and human epididymis secretory protein 4 according to claim 2, characterized in that: In step (4), the volume ratio of CA125-Ab1, HE4-Ab1 and AMNPs is 1:1:1-2.

4. The method for simultaneously determining carbohydrate antigen 125 and human epididymis secretory protein 4 according to claim 2, characterized in that: In step (7), the volume ratio of CA125, HE4 and AMNPs-Ab1 is 1:1:55-60.

5. The method for simultaneously determining carbohydrate antigen 125 and human epididymis secretory protein 4 according to claim 2, characterized in that: In step (7), the volume ratio of the CA125-Ab2-ZnO QD, HE4-Ab2-SnO2 QD and AMNPs-Ab1-CA125 / HE4 complex is 1:1:2.5-3.

5.

6. The method for simultaneously determining carbohydrate antigen 125 and human epididymis secretory protein 4 according to claim 1, characterized in that: The linear equation of the signal frequency (Y) of the metal element and the concentration (X) of CA125 is Y=3.54X+9.

22.

7. The method for simultaneously determining carbohydrate antigen 125 and human epididymis secretory protein 4 according to claim 1, characterized in that: The linear equation of the signal frequency (Y) of the metal element and the concentration (X) of HE4 is Y=5.59X+13.

77.

8. The method for simultaneously determining carbohydrate antigen 125 and human epididymis secretory protein 4 according to claim 1, characterized in that: The double antibody sandwich immune complex was diluted with PBS buffer solution before being introduced into ICP-MS for testing.

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