WO2020024745A1 - 一种荧光免疫检测毛发痕量毒品的试剂盒 - Google Patents
一种荧光免疫检测毛发痕量毒品的试剂盒 Download PDFInfo
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- WO2020024745A1 WO2020024745A1 PCT/CN2019/093739 CN2019093739W WO2020024745A1 WO 2020024745 A1 WO2020024745 A1 WO 2020024745A1 CN 2019093739 W CN2019093739 W CN 2019093739W WO 2020024745 A1 WO2020024745 A1 WO 2020024745A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
Definitions
- the invention belongs to the technical field of biological detection, in particular to a reagent kit for fluorescent immunodetection of hair trace drugs.
- Chinese patent CN101750458B discloses a method for detecting drug residues in humans, which uses HPLC-Chip-MS / MS and HPLC-MS / MS to detect drug residues, which has the advantages of low cost, high accuracy and high stability.
- Chinese patent CN104422762A discloses an ELISA kit and a detection method for detecting drugs in urine, saliva, blood, and hair. The ELISA kit has the characteristics of high specificity and high sensitivity.
- the object of the present invention is to provide a kit for detecting hair trace drugs by fluorescence immunoassay.
- the invention can quickly, accurately and sensitively detect drugs in hair.
- a kit for detecting hair trace drugs by fluorescence immunoassay comprising a sample lysate and a fluorescent immunochromatographic test strip;
- the lysate is used for dissolving small drug molecules from hair
- the fluorescent immunochromatographic test strip includes a PVC liner, a nitrocellulose membrane, a sample pad, and an absorption pad, and the sample pad, the nitrocellulose membrane, and the absorption pad are sequentially staggered and fixed on the PVC liner;
- the sample pad has a drug antibody-quantum dot nanoparticle complex and a rabbit IgG-quantum dot nanoparticle complex;
- the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a drug antigen-bovine serum albumin complex, and the quality control line is coated with a goat anti-rabbit antibody.
- the detection object of the present invention is that the hair is tested for blood, urine, or saliva compared with the conventional detection method, which can overcome the difficulty of obtaining evidence for drug testing and the mismatch of the person being tested.
- the mass ratio of lysate to hair is 100: 1.
- the lysing solution contains a surfactant, a keratinase and a buffer solution.
- a surfactant e.g., keratinase can corrode the surface of the hair and make the hair a rough surface, thereby facilitating the dissolution of small drug molecules from the sample to be tested.
- Surfactants can wash stains on the surface of hair, so that keratinase can fully contact the hair, and further improve the dissolution rate of drugs.
- the buffer solution can neutralize the side-reactants produced by keratinase decomposition of hair, and improve the biological activity of keratinase.
- the surfactant is preferably a non-ionic surfactant, such as polyvinylpyrrolidone (PVP), Triton X 100, Triton X 114, Triton X 405, Tween 20, Tween 60, Tween 80.
- PVP polyvinylpyrrolidone
- Triton X 100 Triton X 114
- Triton X 405 Tween 20, Tween 60, Tween 80.
- non-ionic surfactants do not easily affect the ionic strength, thereby avoiding affecting the immune response in subsequent reagent strips and improving the accuracy of immune detection.
- the buffer solvent is preferably a TRIS (anhydrous sodium tetraborate) -EDTA buffer solution.
- the buffer system of the present invention has a pH range close to 8, a mild nature, and higher solution stability.
- the lysing solution further includes sodium bicarbonate.
- the addition of sodium bicarbonate is mainly to further adjust the pH of the lysate.
- the mass percentage of the surfactant in the lysate is 1-2%, and the content of keratinase is 10,000-50,000 IU / ML.
- the quantum emission nanometer particle has a fluorescence emission wavelength range of 600-620 nm, and more preferably, the emission wavelength is 610 nm.
- the wavelength of the excitation light is 360-430 nm, and more preferably, the wavelength of the excitation light is 365 nm.
- the quantum dot nanoparticles are a quantum dot formed by a single compound or a composite quantum dot assembled by several compounds. More preferably, the compound is selected from one or more of ZnS, CdS, HgS, MgS, CdSe, ZnSe, and ZnCdSe.
- the fluorescence immunochromatography kit of the present invention uses quantum dot nanoparticles as fluorescent emitting substances.
- the quantum dots exhibit wide and continuous excitation spectrum, narrow and symmetrical emission spectrum, high fluorescence intensity, long fluorescence lifetime, and good stability in optical properties. It can overcome the defects of conventional rare earth microspheres, latex microspheres and other fluorescent materials, signal decay blocks, and insignificant signal gradients.
- the fluorescent signal displayed by the fluorescent immunoassay strip of the present invention can not be significantly attenuated within 2 hours, and the gradient of the signal is very obvious, which can be effectively stratified, making the tested fluorescent signal more stable and convenient for detection, improving detection sensitivity and Accuracy.
- the properties of quantum dots with stable fluorescence, strong signals, and obvious changes also make the fluorescence signal magnitude conversion effect good, which is conducive to quantitative analysis.
- the detection principle of the fluorescent immunochromatographic test strip of the present invention is a competition method.
- the T-line (detection line) -coated antigen and the drug antigen in the sample to be tested compete to bind the drug antibody-quantum dot nanoparticle complex.
- the drug antibody-quantum dot nanocomposite first binds to the T-coated drug antigen-bovine serum albumin complex during swimming, and the T-line forms a fluorescent signal that can be detected by a hair fluorescence immunoanalyzer.
- the drug antigen, coated drug antigen, and drug antigen-bovine serum albumin complex compete for binding, and the signal intensity at the T line will follow the drug antigen in the sample. It increases and gradually weakens; while the rabbit IgG-quantum dot nanoparticle complex will continue to swim and bind to the quality control line (C line) -bound sheep anti-rabbit antibody, forming a stable signal at the C line; if the C line No signal is detected anywhere, the test strip is scrapped and can no longer be used.
- C line quality control line
- the present invention can adopt a matching fluorescence detection analyzer, which can import the relevant quantity curve according to each reagent batch, quickly inject the detection readings, and control the time within 10 seconds, which can convert the fluorescent signal into the corresponding drug or metabolism
- the concentration value of the component is clearly displayed on the display screen.
- the instrument can save more than 500 sets of test results, and the relevant results can be printed immediately, including the corresponding reagent lot number, specimen number, test time, and tester code.
- the detectable drugs of the present invention include morphine, virus, ketamine and the like.
- the kit of fluorescent immunodetection drugs of the present invention uses hair as a detection sample to quantitatively detect the drug content in the hair, the sampling is simple, and the problem of drug detection and sampling difficulties is overcome;
- the present invention uses a lysate composed of a surfactant, keratinase and a buffer solution to dissolve drugs in the hair.
- a lysate composed of a surfactant, keratinase and a buffer solution to dissolve drugs in the hair.
- the combined action of the surfactant, keratinase and a buffer solution can effectively improve the dissolution rate and dissolution of drugs in the hair. speed.
- the present invention uses quantum dots for fluorescent labeling.
- the fluorescence is stable, the signal is strong and the change is obvious.
- the fluorescence signal can be converted into a good quantity, the drug content can be quantitatively analyzed, and it can be obtained quickly by a fluorescence analyzer. result.
- the kit of the present invention is used to detect the drug content in hair.
- the test results have high accuracy, high precision, wide detection range, high stability, and anti-interference (such as drugs, shampoo, conditioner, hair gel, etc.) strong ability.
- 1 is a schematic structural diagram of a fluorescent immunochromatographic test strip according to the present invention, wherein 1 is a PVC liner, 2 is a sample pad, 3 is a nitrocellulose membrane, 4 is an absorption pad, 5 is a detection line, and 6 is a quality control line.
- the kit includes sample lysate, fluorescent immunochromatographic test strip (see Figure 1 for the structure); the lysate is used to dissolve small drug molecules from the sample to be tested; the fluorescent immunochromatographic test strip contains PVC liner 1, nitric acid Fiber membrane 3, sample pad 2 and absorption pad 4, the sample pad, nitrocellulose membrane, and absorption pad are sequentially staggered and fixed on the PVC liner; the sample pad has drug antibody-quantum dot nanoparticle complex and rabbit IgG -Quantum dot nanoparticle complex; a detection line and a quality control line are provided on the nitrocellulose membrane, the detection line is coated with a drug antigen-bovine serum albumin complex, and the quality control line is coated with a goat anti-rabbit antibody.
- Fluorescent microspheres that is, quantum dot nanoparticles formed by one or more of ZnS, CdS, HgS, MgS, CdSe, ZnSe, and ZnCdSe are formulated into a 5 wt% working concentration;
- the blocking solution is a 5 wt% casein solution
- the precipitate is re-dissolved with a polymer, and a ratio of 0.1 ml of the precipitate is added to 1 ml of a polymer solution.
- the polymer may be polystyrene, polybutene, or the like.
- the composite plastic sheet is placed on a cutting machine and cut into a single piece of test paper.
- the fluorescence detection analyzer detects the fluorescence signal of the test strip, converts the fluorescence signal into an electrical signal, and then automatically calculates the concentration based on the standard curve information on the ID card of each batch of test strips and presents it on the display of the instrument.
- the fluorescent label is: particles formed by quantum dots ZnCdSe / ZnS embedded in polystyrene microspheres.
- the lysate formulation is:
- Dilute morphine enterprise reference products to 0ng / mg, 0.05ng / mg, 0.1ng / mg, 1ng / mg, 2.5ng / mg, 5ng / mg, 10ng / mg, 15ng / mg, 20ng / mg, and then follow the instructions
- the detection step three batches of morphine / methamphetamine / ketamine hair rapid detection reagent products were tested respectively, and each concentration was tested for 3 samples and the average value was obtained. It is required that the linear correlation coefficient R ⁇ 0.99, and the relative deviation is ⁇ 10%.
- Table 1 The results are shown in Table 1 below.
- the relative deviation is greater than 10% when the concentration is ⁇ 0.1ng / mg, and the relative deviation is greater than 10% when the concentration is greater than 10ng / mg, which does not meet the requirements; within the range of 0.1-10ng / ml, the correlation coefficient R of the product linearity ⁇ 0.99, the relative deviation is within ⁇ 10% so the linear range of the three products is 0.1-10ng / mg.
- the precision CV of the morphine hair rapid detection reagent batch is ⁇ 10%.
- the minimum detection limit (X + 3SD) of morphine hair rapid detection reagent is ⁇ 0.1ng / mg.
- the morphine hair rapid detection reagent has no interference when the interference substance concentration is 10 ug / ml.
- test buffer is stored at 2-8 ° C.
- the entire test buffer is changed from 2- Take it out of the refrigerator at 8 °C, open the bottle, and return to room temperature for testing. After the test, return the test buffer to the ambient conditions of 2-8 °C.
- the test time is 0, 3, 6, 9, 12, 18, 24, 30 and 31 months. (If the product is found to be unstable during the test, you need to use the test buffer prepared for verification, indicating whether it is the stability of the finished product or the reason for the test buffer).
- the detection items are as follows:
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Abstract
Description
化剂名称 | 用量g/L |
无水四硼酸钠 | 10 |
角蛋白酶 | 5 |
PVP-10 | 10 |
曲拉通-100 | 15 |
碳酸氢钠 | 20 |
EDTA | 5 |
Claims (10)
- 一种荧光免疫检测毛发痕量毒品的试剂盒,其特征在于,包括样品裂解液,荧光免疫层析试纸条;所述裂解液用于将毒品小分子从毛发中溶出;所述荧光免疫层析试纸条包含PVC衬板、硝酸纤维膜、样品垫和吸收垫,所述样品垫、硝酸纤维膜和吸收垫依次交错固定于PVC衬板之上;所述样品垫上有毒品抗体-量子点纳米颗粒复合物以及兔IgG-量子点纳米颗粒复合物;所述硝酸纤维膜上设有检测线和质控线,检测线包被有毒品抗原-牛血清白蛋白复合物,质控线包被有羊抗兔抗体。
- 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒,其特征在于,裂解液和毛发的质量比为100:1。
- 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒,其特征在于,所述裂解液包含表面活性剂、角蛋白酶和缓冲溶液。
- 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒,其特征在于,所述表面活性剂优选非离子表面活性剂。
- 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒,其特征在于,所述缓冲溶剂优选为TRIS(无水四硼酸钠)-EDTA缓冲溶液。
- 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒,其特征在于,所述裂解液还包括碳酸氢钠。
- 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒,其特征在于,所述裂解液中表面活性剂的质量百分比为1-2%、角蛋白酶的含量为10000-50000IU/ML。
- 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒,其特征在于,所述量子点纳米颗粒的荧光发射波长范围为600-620nm,更优选的,发射波长为610nm。
- 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒,其特征在于,所述量子点纳米颗粒为单一化合物形成的量子点或是几种化合物组装成的复合物量子点。
- 根据权利要求1所述的一种荧光免疫检测毛发痕量毒品的试剂盒,其特征在于,所述化合物选自ZnS、CdS、HgS、MgS、CdSe、ZnSe、ZnCdSe中的一种或多种。
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