Dopamine chemiluminescence immunoassay kit

Abstract

The invention discloses a dopamine chemiluminescence immunoassay kit which comprises a dopamine antibody marker, a dopamine standard substance, streptavidin-connected magnetic microspheres, biotin-connected dopamine, a cleaning solution, a luminescent substrate or a pre-excitation solution, an excitation solution and the like. A chemiluminescence competitive immunoassay technology is adopted, dopamine molecules in a sample and biotinylated dopamine compete to be combined with an acridinium ester labeled dopamine antibody, only the biotinylated dopamine molecules can be fixed on the surface of a magnetic particle, the dopamine concentration in the to-be-detected sample is inversely proportional to a luminescence signal, and the detection sensitivity is high. The dopamine content in a sample can be accurately determined by establishing a standard curve. The method does not need to carry out any pretreatment on a to-be-detected sample, can directly measure, has the advantages of rapidness, simplicity, convenience, high accuracy, good repeatability and the like, and has very important significance on clinical diagnosis and curative effect observation of depression.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical detection, and in particular to a dopamine chemiluminescent immunoassay kit. Background Art

[0002] Dopamine is a catecholamine neurotransmitter secreted by the hypothalamus and pituitary gland, which plays a key role in the regulation and signal transduction in the central nervous system. Dopamine is closely related to functions such as motor control, cognitive function, emotional regulation, memory, and is involved in the processing and regulation of visual information. Dopamine is the most important neurotransmitter in the brain related to pleasure. When dopamine levels increase, people will feel happier and more satisfied. When dopamine levels decrease, people will feel depressed, lose interest, and even feel frustrated. Studies have shown that the dopamine level in patients with depression is significantly lower than normal, and the dopamine content is closely related to the condition of depression. In recent years, the incidence of depression has been on the rise, and it has become a type of serious mental illness, which has caused widespread concern for the harm it has caused to the lives of patients and their families. In addition, dopamine plays a very important role in motor control. Therefore, rapid and accurate detection of dopamine is of great significance for the clinical diagnosis, efficacy observation and prognosis of depression.

[0003] At present, the main detection methods of dopamine include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), high pressure liquid chromatography (HPLC) and electrochemical sensor method. Radioimmunoassay (RIA) has been gradually eliminated in recent years due to its radioactive pollution; the accuracy and repeatability of ELISA quantitative detection are poor; HPLC has high cost, low efficiency and low throughput, and electrochemical sensor technology is not mature at present, so it cannot be widely used in clinical detection.

[0004] Patent reports on the development of a dopamine chemiluminescent immunoassay kit can also be seen, but it has not been widely used in the clinical testing market. Since the currently available chemiluminescent assay method requires the dopamine in the sample to be tested to be pre-acylated, the dopamine is connected to biotin, and then the sandwich method is used for detection. This treatment method is relatively cumbersome and complicated, and the protein substances in the blood or urine samples are very complex, which will cause great interference to the biotinylation connection. In addition, dopamine is a small molecule with a molecular weight of only 153.18 Daltons. There is a large steric hindrance effect when using the sandwich method for detection, which affects the accuracy of the determination. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the present invention adopts chemiluminescent competitive immunoassay technology, streptavidin is coated on the surface of magnetic particles, dopamine antigen is connected with biotin, and dopamine antibody is labeled with alkaline phosphatase or acridinium ester. Dopamine molecules in the sample compete with biotinylated dopamine to bind to acridinium ester labeled dopamine antibody, and only biotinylated dopamine molecules can be fixed on the surface of magnetic particles. The dopamine concentration in the sample to be tested is inversely proportional to the luminescent signal. By establishing a standard curve, the dopamine content in the sample can be accurately determined. The present invention does not require any pretreatment of the sample to be tested, and can be directly measured, and has the advantages of being quick and simple, high accuracy, and good repeatability.

[0006] The dopamine chemiluminescent immunoassay kit comprises a dopamine standard, magnetic microspheres connected to streptavidin, dopamine connected to biotin, a dopamine antibody marker, a cleaning solution, a luminescent substrate or a pre-excitation solution and an excitation solution, etc.

[0007] The dopamine standard is prepared by mixing 0.02-0.2M citrate or HEPES buffer with bovine serum in a ratio of 1:1 to 4:1, adding 0.1%-1% casein, 0.1%-1% sucrose, and 0.02%-0.2% preservative as a basic buffer. The basic buffer and dopamine antigen are prepared in a certain ratio to prepare 5-6 standards of different concentrations, with a concentration range of 0 to 10000 ng / mL.

[0008] The preservatives include, but are not limited to, sodium azide, ProClin300, sodium thimerosal, and the like.

[0009] The streptavidin-linked magnetic microspheres are prepared by washing the magnetic microspheres 2-3 times with an equal volume of microsphere linking solution, diluting the magnetic microspheres with the microsphere linking solution to 2-20 mg / mL, and adding streptavidin to the magnetic microsphere suspension to make its concentration 50-500 μg / mL. The magnetic microsphere suspension is fully rotated and mixed on a rotary mixer, and then 0.5-5 mg EDC solution is added for activation connection for 2-8 hours. After the connection is completed, the magnetic microspheres are blocked with a blocking solution, washed with a cleaning solution 2-3 times, and then diluted with a magnetic microsphere preservation solution to make the magnetic beads The concentration is 0.1-0.5 mg / mL, which is the working solution of the magnetic microsphere suspension linked to streptavidin.

[0010] The magnetic microspheres include carboxyl magnetic microspheres, amino magnetic microspheres, toluenesulfonyl magnetic microspheres, etc., with a size of 0.2-5.0 μm.

[0011] The biotin-linked dopamine is prepared by dissolving biotin or its derivatives with an organic solvent to a concentration of 1 to 5 mg / mL, dissolving dopamine with double distilled water to a concentration of 10 to 50 mg / mL, and then diluting it to 0.2 to 2 mg / mL with a biotin linker, adding a biotin solution to the dopamine solution, wherein the molar ratio of biotin to dopamine is 1:1 to 5:1, stirring the reaction for 1 to 5 hours, adding a stop solution, and purifying by high pressure liquid chromatography. Dilute it to 0.1 to 1.0 mg / mL with a dopamine diluent for storage. Dilute it 500 to 2000 times with a dopamine diluent before use, i.e., a biotinylated dopamine working solution.

[0012] The biotin derivatives include biotin succinimidyl ester, biotin hexanoic acid imidate, biotin-PEG imidate and the like.

[0013] The alkaline phosphatase-labeled dopamine antibody is specifically prepared by dissolving sodium periodate with double distilled water to a concentration of 2 to 20 mg / mL, adding sodium periodate solution to alkaline phosphatase to a ratio of 2:1 to 1:5, stirring the reaction for 0.5-1.0 hours, ultrafiltration 1-3 times with 0.01M acetate buffer with a pH of 4.4, mixing the activated alkaline phosphatase with the dopamine antibody in a mass ratio of 1:1 to 3:1, adjusting the pH to 8.0 to 9.5 with a carbonate buffer, and stirring the reaction for 2 to 6 hours. The reaction is terminated with sodium borohydride, and the ratio of sodium borohydride to alkaline phosphatase should be 1:5 to 1:20. Ultrafiltration purification 2 to 4 times. The antibody is diluted to 0.1 to 1.0 mg / mL with a marker diluent and stored for standby use. It is diluted 200 to 2000 times with a marker diluent, which is the alkaline phosphatase antibody marker working solution.

[0014] The acridinium ester-labeled dopamine antibody is prepared by dissolving the acridinium ester or its derivatives in an organic solvent to a concentration of 1 to 5 mg / mL, ultrafiltering the dopamine antibody with a labeled connection liquid 2 to 3 times to make the antibody concentration 0.2 to 2 mg / mL. Add an acridinium ester solution to the antibody, the mass ratio of acridinium ester to antibody is 1:5 to 1:50, stir the reaction for 2 to 6 hours, add the stop solution, stir the reaction for 20 to 60 minutes, and purify with a PD-10 chromatography column. Dilute the antibody to 0.1 to 1.0 mg / mL with a marker diluent for standby use. Dilute it 500-5000 times with a marker diluent, which is the acridinium ester antibody marker working solution.

[0015] The acridinium ester derivatives include acridinium ester (NSP-SA-NHS), acridinium ester (ME-DMAE-NHS), acridinium ester (NSP-DMAE-NHS), etc. The organic solvent includes dimethyl sulfoxide (DMSO), etc.

[0016] The cleaning solution is composed of 0.02-0.2M phosphate buffer or Tris-HCl buffer, with a pH of 6-8, and 0.02%-0.2% of a surfactant, such as Tween-20, Triton-100, S9, etc.

[0017] The dopamine chemiluminescent immunoassay kit has the following specific steps of adding samples: a. adding 10-100 µL of magnetic microsphere suspension working solution, 10-100 µL of dopamine standard or sample to be tested, 10-50 µL of biotinylated dopamine solution, and 10-50 µL of dopamine antibody marker working solution to the corresponding reaction cup; b. incubating at room temperature or 37°C for 10-30 minutes, magnetically separating, and washing 3-5 times; c. adding luminescent substrate or pre-excitation solution and excitation solution to detect luminescent signals. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the standard curve of alkaline phosphatase labeled dopamine chemiluminescent immunoassay kit.

[0019] Figure 2 This is the standard curve of the acridinium ester labeled dopamine chemiluminescent immunoassay kit. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is described in more detail below through specific embodiments.

[0021] The dopamine antigen used in the present invention was purchased from SIGMA; mouse anti-human dopamine monoclonal antibody was prepared by our company; acridinium ester and its derivatives, biotin and its derivatives, streptavidin, etc. were purchased from Dalian Meilun Biotechnology Co., Ltd.; alkaline phosphatase was purchased from BBI Solutions. Components not described in detail in the specification, such as alkaline phosphatase luminescent substrate, acridinium ester luminescent pre-excitation solution and excitation solution, are conventional reagents and can be purchased commercially and meet relevant industry standards. Example 1

[0022] Preparation method of dopamine immunoassay kit based on alkaline phosphatase chemiluminescence system: Dopamine standards were prepared using standard base buffer and dopamine antigen with 6 different concentrations of dopamine standards, the concentrations were 5, 25, 120, 600, 2500, 10000 ng / mL. The standard base solution was prepared by mixing 0.02M, pH=7.3 citrate buffer with fetal bovine serum in a ratio of 3:1, adding 0.5% casein, 0.2% sucrose, and 0.1% sodium azide.

[0023] Preparation of magnetic microspheres linked to streptavidin: Wash 1.0µm carboxyl magnetic microspheres 3 times with an equal volume of microsphere linking solution, dilute them to 10mg / mL with microsphere linking solution, and add streptavidin to the magnetic microsphere suspension to make its concentration 200µg / mL. The magnetic microsphere suspension is fully rotated and mixed on a rotating mixer, and then 2mg EDC solution is added, and the connection is activated for 4 hours on a rotating mixer. After the connection is completed, the magnetic microspheres are blocked with blocking solution, washed 3 times with cleaning solution, and then diluted with magnetic microsphere storage solution to make the magnetic beads The concentration is 0.2mg / mL, which is the working solution of magnetic microsphere suspension linked to streptavidin.

[0024] The preparation method of biotin-linked dopamine is as follows: biotin succinimidyl ester is dissolved in DMF to a concentration of 5 mg / mL, dopamine is dissolved in double distilled water to a concentration of 20 mg / mL, and then it is diluted to 1 mg / mL with a biotin linker solution, and a biotin solution is added to the dopamine solution, the molar ratio of biotin to dopamine is 3:1, the reaction is stirred for 4 hours, a stop solution is added, and it is purified by high pressure liquid chromatography. It is diluted to 1.0 mg / mL with a dopamine diluent for standby use. It is diluted to 0.2 µg / mL with a dopamine diluent before use, which is the biotinylated dopamine working solution.

[0025] Preparation method of dopamine antibody labeled with alkaline phosphatase: Sodium periodate is dissolved in double distilled water to a concentration of 20 mg / mL, sodium periodate solution is added to alkaline phosphatase to make the mass ratio of the two to be 1:1, stirring reaction for 0.5 hours, ultrafiltration 3 times with 0.01M acetate buffer with a pH of 4.4, activated alkaline phosphatase and dopamine antibody are mixed at a mass ratio of 2:1, and the pH is adjusted to 9.0 with carbonate buffer, and stirring reaction is performed for 4 hours. The reaction is terminated with sodium borohydride, and the ratio of sodium borohydride to alkaline phosphatase should be 1:10. Ultrafiltration purification 3 times. The antibody is diluted to 1.0 mg / mL with a marker diluent and stored for standby use. It is diluted 4000 times with a marker diluent, which is the alkaline phosphatase antibody marker working solution.

[0026] The washing solution used was 0.02M PBS buffer, pH 7.3, with 0.05% Tween-20 added.

[0027] The specific sample loading steps of the dopamine immunoassay kit based on the alkaline phosphatase chemiluminescence system are as follows: a. Add 50µL of magnetic microsphere suspension working solution, 50µL of dopamine standard or test sample, 25µL of biotinylated dopamine solution, and 50µL of dopamine antibody marker working solution to the corresponding reaction cup; b. Incubate at room temperature or 37°C for 10 minutes, magnetically separate, and wash 3 times; c. Add 100µL of luminescent substrate and detect the luminescent signal. Establishment of standard curve:

[0028] According to the operating instructions of the alkaline phosphatase chemiluminescence kit, each standard product was tested twice, and then the average value was calculated. The standard curve was established with the standard product concentration as the horizontal axis and the average luminescence value as the vertical axis, and four parameters were fitted. The test results are shown in the following table, and the standard curve is attached. Figure 1 .

[0029]

[0030] The minimum detection limit of the kit is determined by: Experimental method: Use zero calibrator as sample for 20 measurements, calculate the average (X) and standard deviation (SD) of the 20 relative luminescence values ​​RLU, calculate the RLU value corresponding to X-2SD, and use the RLU value to obtain the corresponding concentration value from the standard curve, which is the minimum detection limit of this kit.

[0031] Kit Accuracy Determination: Prepare a high-concentration DA sample to a concentration of 2000 ng / mL; prepare another low-value human serum sample B close to S0; mix sample A and sample B at a volume ratio of 1: 99 and 1: 9 to prepare two samples with theoretical concentrations of approximately 20 and 200 ng / mL, respectively, and test according to the kit operating method, repeat the test 3 times, and take the average value X. The ratio between X and the theoretical value is calculated as the recovery rate of this kit.

[0032] Measurement results:

[0033] Kit specificity assessment: The interference of adrenaline, noradrenaline, levodopa and 5-hydroxytryptamine on the kit of the present invention was determined, adrenaline, noradrenaline and 5-hydroxytryptamine samples with a concentration of 2000 ng / mL were prepared, and the kit of the present invention was used for detection, and the poor reaction rate was calculated.

[0034]

[0035] From the above results, it can be seen that the cross-reaction rates of this kit with epinephrine, norepinephrine, levodopa and serotonin are all less than 0.05%. Example 2

[0036] Preparation method of dopamine immunoassay kit based on acridinium ester chemiluminescence system: The preparation method of acridinium ester labeled dopamine antibody is to dissolve the acridinium ester derivative NSP-SA-NHS in DMF to a concentration of 5 mg / mL, and ultrafilter the dopamine antibody twice with the labeled connection liquid to make the antibody concentration 1 mg / mL. Add acridinium ester solution to the antibody, the mass ratio of acridinium ester to antibody is 1:10, stir and react for 3 hours, add the stop solution, stir and react for 30 minutes, and purify with a PD-10 chromatography column. Dilute the antibody to 1.0 mg / mL with a marker diluent for use. Before use, dilute it with a marker diluent and dilute it 4000 times with a marker diluent, which is the acridinium ester antibody marker working solution.

[0037] The preparation methods of other components of the kit, such as dopamine standard, streptavidin-linked magnetic microspheres, and biotin-linked dopamine, are the same as those in Example 1, and thus will not be described again.

[0038] The specific steps of adding samples to the dopamine immunoassay kit based on the acridinium ester chemiluminescence system are as follows: a. Add 50µL of magnetic microsphere suspension working solution, 50µL of dopamine standard or sample to be tested, 25µL of biotinylated dopamine solution, and 50µL of dopamine antibody marker working solution to the corresponding reaction cup; b. Incubate at room temperature or 37°C for 10 minutes, separate magnetically, and wash 3 times; c. Add 100µL each of pre-excitation solution and excitation solution, and detect the luminescence signal. Establishment of standard curve:

[0039] The acridinium ester chemiluminescence kit was used according to its operating instructions to perform two replicate tests on each standard product, and then the average value was calculated. A standard curve was established with the standard product concentration as the horizontal axis and the average luminescence value as the vertical axis, and four-parameter fitting was performed. The test results are shown in the following table, and the standard curve is attached. Figure 2 .

[0040]

[0041] The minimum detection limit of the kit is determined by: Experimental method: Use zero calibrator as sample for 20 measurements, calculate the average (X) and standard deviation (SD) of the 20 relative luminescence values ​​RLU, calculate the RLU value corresponding to X-2SD, and use the RLU value to obtain the corresponding concentration value from the standard curve, which is the minimum detection limit of this kit.

[0042]

[0043] Kit Accuracy Determination: The recovery rate of the kit was determined using the method and sample described in Example 1, and the test results are as follows:

[0044] Kit specificity assessment: The detection method is the same as in Example 1, and the results are as follows.

[0045]

[0046] From the above results, it can be seen that the cross-reaction rates of this kit with epinephrine, norepinephrine, levodopa and serotonin are all less than 0.05%.

Claims

1. A dopamine chemiluminescent immunoassay kit, characterized in that: Including dopamine antibody markers, dopamine standards, streptavidin-linked magnetic microspheres, biotin-linked dopamine, cleaning solution, luminescent substrate or pre-excitation solution and excitation solution, etc.; The antibody marker includes alkaline phosphatase or acridinium ester labeled mouse anti-human dopamine antibody, and the preparation method thereof is as follows: The preparation method of the dopamine antibody labeled with alkaline phosphatase is as follows: first, the alkaline phosphatase is activated with sodium periodate for 0.5-1.0 hour, ultrafiltration is performed 1-3 times, the activated alkaline phosphatase and the dopamine antibody are mixed at a mass ratio of 1:1-3:1, and the reaction is stirred for 2-6 hours; the reaction is terminated with sodium borohydride; ultrafiltration is performed 2-4 times; the antibody is diluted to 0.1-1.0 mg / mL with a marker diluent for standby use; the preparation method of the dopamine antibody labeled with acridinium ester is as follows: the acridinium ester or its derivative is dissolved with an organic solvent to a concentration of 1-5 mg / mL, and the dopamine antibody is ultrafiltered with a marker connection solution 2-3 times to a concentration of 0.2-2 mg / mL; Add acridinium ester solution to the antibody, with the mass ratio of acridinium ester to antibody being 1:5-1:50, stir and react for 2-6 hours, then add stop solution; dilute the antibody to 0.1-1.0 mg / mL with marker diluent for later use; The streptavidin-linked magnetic microspheres are prepared by washing the magnetic microspheres 2-3 times with an equal volume of microsphere linking solution, diluting the magnetic microspheres with the microsphere linking solution to 2-20 mg / mL, adding streptavidin to the magnetic microsphere suspension to make its concentration 50-500 μg / mL; fully rotating and mixing the magnetic microsphere suspension on a rotary mixer, and then adding an EDC solution for activation and linking; after the linking is completed, blocking the magnetic microspheres with a blocking solution, washing them with a cleaning solution for 2-3 times, and then diluting and storing them with a magnetic microsphere storage solution; The preparation method of the biotin-linked dopamine is as follows: dissolving biotin or its derivatives with an organic solvent to a concentration of 1-5 mg / mL, dissolving dopamine with double distilled water to a concentration of 10-50 mg / mL, and then diluting it to 0.2-2 mg / mL with a biotin linker, adding a biotin solution to the dopamine solution, wherein the molar ratio of biotin to dopamine is 1:1-5:1, stirring the reaction for 1-5 hours, adding a stop solution, and purifying by high pressure liquid chromatography.

2. The dopamine chemiluminescent immunoassay kit according to claim 1, characterized in that: Dopamine in blood and urine can be directly detected quantitatively.

3. The dopamine chemiluminescent immunoassay kit according to claim 1, characterized in that: This kit adopts the competitive method for determination. The surface of magnetic microparticles is coated with streptavidin, and dopamine antigen is linked with biotin. Alkaline phosphatase or acridinium ester is used to label dopamine antibody. Dopamine molecules in the sample compete with biotinylated dopamine for binding to acridinium ester-labeled dopamine antibody. Only biotinylated dopamine molecules can be fixed on the surface of magnetic microparticles. After magnetic separation and washing of magnetic microspheres, luminescent substrate or excitation solution and pre-excitation solution are added for detection. The dopamine concentration in the sample to be tested is inversely proportional to the luminescent signal.

4. The dopamine chemiluminescent immunoassay kit according to claim 1, characterized in that: The specific steps of adding samples are as follows: a. Add 10~100µL magnetic microsphere suspension, 10~100µL dopamine standard or test sample, 10~50µL biotinylated dopamine solution, and 10~50µL dopamine antibody marker to the corresponding reaction cup; b. Incubate at room temperature or 37°C for 10~30min, magnetically separate, and wash 3~5 times; c. Add luminescent substrate or pre-excitation solution and excitation solution to detect luminescent signal.

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