WO2020047735A1 - 一种基于磁微粒的时间分辨荧光免疫检测方法 - Google Patents

一种基于磁微粒的时间分辨荧光免疫检测方法 Download PDF

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WO2020047735A1
WO2020047735A1 PCT/CN2018/103954 CN2018103954W WO2020047735A1 WO 2020047735 A1 WO2020047735 A1 WO 2020047735A1 CN 2018103954 W CN2018103954 W CN 2018103954W WO 2020047735 A1 WO2020047735 A1 WO 2020047735A1
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antibody
magnetic beads
magnetic
time
beads
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French (fr)
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李根平
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广州源起健康科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • 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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated

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  • the present invention relates to the technical fields of detection such as biomedicine, animal disease, food safety, drug screening, etc. Specifically, the present invention relates to a time-resolved fluorescence detection method based on magnetic particles.
  • TRFIA Time-resolved Fluorescence Immunoassay
  • microplate TRFIA technology is mainly semi-automatic detection, which requires a certain amount of samples to be detected before it is difficult to achieve instant detection.
  • Invention patent CN 105911041 A, which discloses a time-resolved fluorescence detection method, which is characterized by the use of a bidentate or tetradentate ⁇ -diketone compound and europium complex to emit light, not the dissociation enhanced lanthanide fluorescence immunoassay Luminous system.
  • the sensitivity of this patent is low, and the lower detection limit is at the level of 10 -10 to 10 -11 mol / L.
  • a fluorescent synergist is still required, and the purity of the fluorescent synergist has a serious effect on the experimental results.
  • the object of the present invention is to overcome the above technical problems and provide a time-resolved fluorescent immunodetection method based on magnetic particles.
  • the present invention adopts the following technical solutions:
  • a time-resolved fluorescence immunoassay method based on magnetic particles includes the following steps:
  • the immunomagnetic beads are covalent conjugates of superparamagnetic particles with a diameter of 0.1 to 3 ⁇ m modified with a functional group and a monoclonal antibody or an antigen;
  • the magnetic particles are carboxy magnetic beads, amino magnetic beads, hydroxyl magnetic beads, tosyl magnetic beads, NHS magnetic beads, protein A magnetic beads, protein G magnetic beads, anti-mouse IgG magnetic beads, hydrophilic magnetic beads, One or more of the hydrophobic magnetic beads; or the immunomagnetic beads are immunomagnetic beads formed by the reaction of streptavidin magnetic microbeads with biotinylated antibodies.
  • the preparation method of the immunomagnetic beads is prepared by washing, activating, coupling, and blocking magnetic particles with a diameter of 0.1 to 3 ⁇ m and corresponding antibodies or antigens.
  • the chelate formed by the polyamino polycarboxyl group and a rare earth ion as described in step (2) is used to label a specific antigen or antibody, and the manner of forming a fluorescent label is:
  • the rare earth chelate is directly connected to the biological raw material through a chemical bond; or the rare earth chelate is connected to a streptavidin through a chemical bond, and the antigen or antibody is labeled with biotin.
  • the rare earth chelate is bound to the streptavidin through biotin. Heparin reacts with the antigen or antibody.
  • the polyamino polycarboxyl group having no bifunctional group having an aromatic group in step (2) is isothiocyanate phenyl-ethylenediaminetetraacetic acid (EDTA ), Isothionitrile acid-diethylenetriaminetetraacetic acid (ETTA), diethylenetriaminepentaacetic acid (DTPA) or N- (P-isothiohydrobenzyl) -diethylenetriaminetetraacetic acid (DTTA );
  • the rare earth ion is europium (Eu 3+ ), europium (Sm 3+ ), europium (Tb 3+ ), or europium (Dy 3+ ).
  • the preparation process of the rare earth ion chelate-labeled antibody or antigen in step (2) is: the mass ratio of the antigen or antibody to the rare earth ion chelate is 1: 1 ⁇ 10: 1; In 0.05M carbonate buffer at pH9.0, add rare earth ion chelate and dialyzed antigen or antibody, mix and dissolve in 0.05M carbonate buffer at pH9.0, shake at room temperature Overnight; collect the peak tube after purification with dextran gel column; dilute the fluorescent label prepared above, and use it.
  • the temperature of the immune reaction in step (2) is 25 ° C. to 37 ° C., and the humidity is 30 to 75% RH, which is performed under shaking and shaking.
  • the formula of the cleaning solution in step (4) is: 0.8-1.5% Tween-20, 0.05% Proclin300, pH 7.2-7.5 0.025M Tris-Hcl buffer solution .
  • the formula of the fluorescence enhancement solution in step (4) is: 0.0002 to 0.0009% ⁇ -NTA, 0.0024% TOPO, 0.08% acetic acid, and 0.05% Triton X- 100 composition, adjust the pH to 3.0-3.2 with an appropriate amount of potassium hydrogen phthalate.
  • a polyaminopolycarboxylic acid without a bifunctional group having an aromatic group is selected as a chelating agent and a rare earth ion is chelated as a label, and an antigen or an antibody is labeled on the magnetic beads and the rare earth ion chelate, respectively.
  • the immune response of the antigen antibody is analyzed and detected, and the performance of the prepared reagent can reach the level of the equivalent chemiluminescence reagent.
  • the liquid phase homogeneous reaction is adopted, and the components are fully mixed, so that the reaction efficiency is higher and the reaction is more sufficient, and the disadvantages of poor repeatability and instability of the heterogeneous reaction are overcome.
  • a chelating agent with a bifunctional group structure is used to connect one end of the chelating agent to one of the rare earth ions Eu (Eu 3+ ), Eu (Sm 3+ ), Eu (Tb 3+ ), and Eu (Dy 3+ ).
  • the other end is connected to a free amino or carboxyl group on the antibody / antigen molecule to form a rare earth ion-labeled antibody / antigen, and an immune complex is formed after an immune reaction. Since the fluorescence intensity of this complex in water is very weak, it is necessary to add a fluorescence enhancing solution, first dissociate the rare earth ions from the complex, and then free the rare earth ions to chelate the fluorescent enhancer (chelating agent) in the fluorescence enhancing solution. Entering into the hydrophobic core of the micelles, the fluorescence of rare earth ions is magnified by a factor of millions.
  • the present invention has the following beneficial effects:
  • magnetic particles have a larger The binding area can fully disperse and react in the liquid phase, which greatly increases the detection range, shortens the reaction time, and improves sensitivity; magnetic particles can be combined with different probe molecules, making it possible to detect different analytes in the same sample.
  • the reaction system of the present invention achieves automation from sample loading, incubation, cleaning, lighting to reporting test results; it can test one or more samples at the same time, and it can test multiple indicators in one experiment, and the operation is simple and fast ( The results can be obtained in 20 minutes), which overcomes the traditional microplate time-resolved fluorescence immunoassay technology that requires a certain amount of samples to be detected, and realizes the instant detection of samples.
  • the present invention also provides a certain basic platform for high-throughput and multi-channel detection of time-resolved immunoassay.
  • Conventional samples can be used to detect multiple item indicators or single reagent strips when used in conjunction with the method.
  • the ability to test the effects of multiple samples at the same time has great potential advantages.
  • FIG. 1 is a schematic structural diagram of a combination reagent strip based on a magnetic particle-based time-resolved fluorescence detection method.
  • Test well number 1 2, Test well number 2, 3, Fluorescence marker number 1, 4, Fluorescence marker number 2, 6, Wash solution, 7, Wash solution, 8, Sample dilution number 1, 9, Sample diluent No. 2, 12, enhancer, 5, 10, 11, 13 are prepared wells.
  • Fig. 2 is a schematic diagram showing the structure of a single test cuvette based on a magnetic particle-based time-resolved fluorescence detection method.
  • Figure 3 is a graph of cTnI dose-response curve.
  • Figure 4 shows the correlation between cTnI kit and Abbott kit.
  • the shape of the combined reagent strip is fan-shaped, and 1 to 13 holes are arranged in sequence from left to right.
  • Wells 1 and 2 are test wells
  • wells 3 and 4 are fluorescent labeled wells
  • wells 6 and 7 are diluent wells
  • wells 8 and 9 are sample diluent wells
  • wells 12 and 12 are enhancement fluid wells.
  • 11, 13 are prepared holes.
  • a magnet can be stored between the 1st and 2nd wells for magnetic separation experiments; the 3rd and 4th wells can be disassembled from the entire reagent strip into separate components, which facilitates the storage and storage of fluorescent labels. See Figure 1 for the structure of the combined reagent strip.
  • the activated magnetic beads are placed on a magnetic stand, the supernatant is discarded, and the activated magnetic beads are collected.
  • the magnetic beads are placed on a magnetic stand, the supernatant is discarded, and the immune magnetic beads are collected.
  • the preservation solution is 0.05M pH 8.0 containing 5% (w / v) BSA, 10% (w / v) trehalose, 2% (w / v) sucrose, and 0.1% (v / v) Tween-20. Tris-HCl buffer.
  • the prepared immunomagnetic beads are diluted so that the final concentration of the cTnI monoclonal antibody is 0.0050 to 0.0100 g / L, and aliquoted into the first well (test well 1) of the reagent strip.
  • the fluorescent substance europium has an excitation wavelength of 340 nm and an emission wavelength of 615 nm.
  • Tris-Hcl buffer containing 0.8 to 1.5% Tween-20, 0.05% Proclin300, pH 7.2 to 7.5, 0.025M Tris-Hcl buffer was prepared, and the prepared solution was dispensed into wells 6 and 7 at 2000 ⁇ L per well.
  • aqueous solution containing 0.0002 to 0.0009% ⁇ -NTA, 0.0024% TOPO, 0.08% acetic acid, and 0.05% Triton X-100 was prepared, and the pH was adjusted to 3.0-3.2 with an appropriate amount of potassium hydrogen phthalate. Dispense the prepared solution at 400 ⁇ L per well to the 12th well (enhancement well) of the reagent strip.
  • the method for preparing the cTnI calibrator is: dilute cTnI antigen to 0, 0.03, 0.1, 0.5, 1, 5, 25, 1.5% Tween-20, 0.02% Proclin300, pH 7.50.02M Tris-Hcl buffer solution. 50ng / ml.
  • Each component is dispensed into specific wells of the combined reagent strip, and the semi-finished product is assembled by the following steps: 100 ⁇ L immunomagnetic beads, 200 ⁇ L biotinylated antibody, 100 ⁇ LSA-Eu are dispensed in the first, third, and fourth wells of Example 1, respectively. 3+ , the cleaning solution is placed in wells 6 and 7, and the enhancement solution is placed in well 12.
  • the components of the cTnI kit are packed into different reagent bottles, the reaction place is a reaction cup, and the structure of the reaction cup is shown in FIG. 2.
  • biotinylated cTnI monoclonal antibody at a concentration of 0.0050-0.0200 g / L was dispensed into a reagent bottle, and the SA-magnetic beads were dispensed into another reagent bottle. The two are linked together in a reaction cup via streptavidin-biotin.
  • Each component is dispensed into different reagent bottles.
  • the combination reagent strip and the reagent bottle are coated with a product information identifier that can be scanned and identified by a full-automatic fluorescence detection analyzer, including an enterprise standard curve, a batch, a production date, and an expiration date.
  • Detection steps of the kit Add immunomagnetic beads, samples, biotinylated cTnI mAb, SA-Eu 3+ or SA-magnetic beads, biotinylated cTnI mAb, The sample and target cTnI antibody were incubated for 15 min at 37 ° C with shaking, and then the magnetic beads were adsorbed under the action of a magnetic field. The free SA-Eu 3+ was removed by washing with a washing solution, and then an enhancement solution was added and incubated for 5 min. The excitation wavelength was 340 nm. The fluorescence signal was collected at an emission wavelength of 615 nm, and the cTnI protein concentration of the test sample was obtained according to the calibration curve.
  • Example 1 the cTnI kit prepared by using the present invention was used to determine the calibrator cTnI.
  • the detection range was 0.03 to 50 ng / ml.
  • a commercially available high-sensitivity troponin-I assay kit (chemiluminescence microparticle immunoassay) (Abbott Trading Co., Ltd.) and the method in Example 1 were used to test the samples, and the test was repeated three times to verify the accuracy of the test results of the present invention. Table 2 below.
  • the deviation of this reagent is less than ⁇ 10%, and the test results of this reagent are accurate and reliable.
  • the test results of 100 cases of clinical serum were compared with the Abbott kit.
  • the positive coincidence rate was 96.05% and the negative coincidence rate was 83.33%.
  • the total coincidence rate of the two was 93.00%, and the correlation with the Abbott reagent was 0.996 ( Figure 4 ).
  • the magnetic particle-based time-resolved fluorescence detection method of the present invention has the following advantages: 1) high sensitivity; 2) short detection time, 20min report; 3) less sample demand, only 50 ⁇ L for one sample loading; 4) simple operation ,save human effort. Achieve remote result query, equipment maintenance, troubleshooting, quality monitoring, improve after-sales maintenance efficiency, and reduce maintenance costs.
  • a magnetic particle-based time-resolved fluorescent immunoassay method is used to prepare a swine fever virus antibody detection kit, which contains the main components: a reagent strip, a negative control serum and a positive control serum.
  • the structure diagram of the reagent strip is shown in FIG. 1.
  • the main components of the reagent strip include immunomagnetic beads of the recombinant antigen E2 protein of classical swine fever virus, anti-pig antibody of standard sheep, sample dilution solution, cleaning solution, and enhancement solution;
  • the activated magnetic beads are placed on a magnetic stand, the supernatant is discarded, and the activated magnetic beads are collected.
  • the magnetic beads are placed on a magnetic stand, the supernatant is discarded, and the immune magnetic beads are collected.
  • the magnetic beads were placed on a magnetic stand, the supernatant was discarded, and 100 ⁇ L of immunomagnetic bead preservation solution was added.
  • the preservation solution is a 0.05 M pH 8.0 Tris-HCl buffer solution containing 6% (w / v) BSA, 0.1% (v / v) Tween-20 and 0.1% (w / v) PEG20000.
  • the prepared immunomagnetic beads are diluted in a certain proportion so that the final concentration of the classical swine fever recombinant E2 protein is 0.0025-0.0080 g / L, and 100 ⁇ L / well is dispensed into the first well (test well 1) of the reagent strip.
  • sample dilution solution is 0.01M pH 7.4PBS, and 620 ⁇ L / well is dispensed into the 8th well (sample dilution solution well) of the reagent strip.
  • Example 5 Comparison of the coincidence rate of detection results between the swine fever virus antibody detection kit and the United States IDEXX swine fever antibody detection kit
  • This method compares the test results of 100 clinical sera with IDEXX swine fever antibody detection kit.
  • the positive coincidence rate is 96.49%
  • the negative coincidence rate is 97.67%
  • the total coincidence rate of the two is 97.00%.
  • Table 4 shows the test results. From the results, it can be known that the swine fever virus antibody detection kit prepared by the magnetic particle-based time-resolved fluorescent immunodetection method has a good correlation with the imported kit.

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Abstract

一种基于磁微粒的时间分辨荧光免疫检测方法，包括将特异性的抗原或抗体包被在磁珠上，形成免疫磁珠；将聚氨基多羧基与稀土离子形成的螯合物标记特异性的抗原或抗体，形成荧光标记物；加入样本，发生免疫反应，使免疫磁珠、被测样本和荧光标记物形成复合物，清洗后再加入荧光增强物，使用解离-增强技术使荧光值增强百万倍，最后检测，获得被测样本的浓度等信息。

Description

一种基于磁微粒的时间分辨荧光免疫检测方法 技术领域
本发明涉及生物医学、动物疫病、食品安全、药物筛选等检测技术领域，具体地说，本发明涉及一种基于磁微粒的时间分辨荧光检测方法。
背景技术
时间分辨荧光免疫分析(Time-resolved Fluorescence immunoassay，TRFIA)技术是继放射免疫分析之后标记物发展的一个新里程碑，已成为生物医学研究和临床超微量生化检验中一项最有发展前景的分析手段。TRFIA以荧光寿命长、Stokes位移大的稀土离子作为标记物，具有制备简便、储存时间长、无放射性污染、标准曲线范围宽、不受样品自然荧光干扰、多标记等优点。它克服了放射免疫分析法易对环境造成污染；胶体金法、荧光免疫层析法灵敏度低，重复性差；酶联免疫法酶标记不稳定；化学发光法只能一次发光，不能实现多标记等缺点。
目前国内外厂家均采用基于微孔板TRFIA技术，由于微孔板的固液相反应面积小，需要的免疫反应时间较久；微孔板包被抗体或抗原是通过物理吸附包被，包被抗体或抗原很难标准化，使得检测结果精密度较差；微孔板TRFIA技术主要为半自动检测，使得样本需要积累到一定量后才能检测，难以实现即时检测。
自动化和手工误差是制约微孔板TRFIA的两大难题。基于行业对自动化、信息化、智能化、大数据化检测系统的迫切需求，实现检测结果的高精密度、高准确度，从根本上解决试剂质量问题的困扰，同时实现各领域系统大数据的建立。“精准化、自动化、云端化”时间分辨荧光免疫分析系统将会是市场未来的发展趋势。
发明专利：CN 105911041 A，公开了一种时间分辨荧光检测方法，其特征是应用二齿或四齿β-二酮类化合物与铕的配合物发光，不是应用解离增强镧系元素荧光免疫分析发光体系。该专利的灵敏度低，检出下限在10 -10～10 -11mol/L水平，仍然需要荧光协同剂，荧光协同剂的纯度对实验结果影响严重。
发明内容
本发明的目的是克服上述技术问题，提供了一种基于磁微粒的时间分辨荧光 免疫检测方法。
为实现上述目的，本发明采用了如下技术方案：
一种基于磁微粒的时间分辨荧光免疫检测方法，包含如下步骤：
(1)将特异性的抗原或抗体包被在磁珠上，形成免疫磁珠；
(2)将不带芳香基的双功能基团的聚氨基多羧基与稀土离子形成的稀土离子螯合物标记特异性的抗原或抗体，形成荧光标记物；
(3)加入被测样本，发生免疫反应，使免疫磁珠、被测样本和荧光标记物形成复合物；
(4)通过外加磁场完成对免疫磁珠分离和固定，使用清洗液将游离荧光标记物洗去，再加入荧光增强液来增强荧光值；
(5)最后检测荧光，根据内置的待测物浓度与荧光信号值拟合的剂量-反应曲线通过软件计算出被测样本浓度。
作为优选的，在上述的时间分辨荧光免疫检测方法中，所述的免疫磁珠为带有官能团修饰的直径0.1～3μm的超顺磁微粒与单克隆抗体或者抗原的共价偶联物；其中所述的磁微粒为羧基磁珠、氨基磁珠、羟基磁珠、甲苯磺酰基磁珠、NHS磁珠、蛋白A磁珠、蛋白G磁珠、抗小鼠IgG磁珠、亲水磁珠、疏水磁珠中的一种或几种；或者是免疫磁珠为通过链酶亲和素磁微珠与生物素化抗体反应形成的免疫磁珠。
作为优选的，在上述的时间分辨荧光免疫检测方法中，所述免疫磁珠的制备方法为直径0.1～3μm的磁微粒与对应抗体或抗原经洗涤、活化、偶联、封闭制备得到。
作为优选的，在上述的时间分辨荧光免疫检测方法中，步骤(2)所述将聚氨基多羧基与稀土离子形成的螯合物标记特异性的抗原或抗体，形成荧光标记物的方式有：直接把稀土螯合物通过化学键与生物原料连接起来；或者把稀土螯合物通过化学键与链酶亲和素连接起来，抗原或抗体标记生物素，最后稀土螯合物通过生物素与链酶亲和素反应，与抗原或抗体连接起来。
作为优选的，在上述的时间分辨荧光免疫检测方法中，步骤(2)所述不带芳香基的双功能基团的聚氨基多羧基为异硫腈酸苯基-乙二胺四乙酸(EDTA)，异硫腈酸-二乙三胺基四乙酸(ETTA)、二乙三胺基五乙酸(DTPA)或N-(P-异硫氢基 苄基)-二乙烯三胺四乙酸(DTTA)；所述稀土离子为铕(Eu 3+)、钐(Sm 3+)，铽(Tb 3+)或镝(Dy 3+)。
作为优选的，在上述的时间分辨荧光免疫检测方法中，步骤(2)所述稀土离子螯合物标记抗体或抗原制备过程为：抗原或抗体与稀土离子螯合物的质量比为1:1～10:1；在0.05M pH9.0的碳酸盐缓冲液下，加入稀土离子螯合物与已透析抗原或抗体，混合后溶于0.05M pH9.0的碳酸盐缓冲液，室温震荡过夜；用葡聚糖凝胶柱纯化后收集峰管；将上述制备好的荧光标记物稀释，备用。
作为优选的，在上述的时间分辨荧光免疫检测方法中，步骤(2)所述免疫反应的温度为25℃～37℃，湿度为30～75％RH，在摇动、振荡下进行。
作为优选的，在上述的时间分辨荧光免疫检测方法中，步骤(4)所述清洗液的配方为：0.8～1.5％Tween-20，0.05％Proclin300，pH 7.2～7.5 0.025M Tris-Hcl缓冲液。
作为优选的，在上述的时间分辨荧光免疫检测方法中，步骤(4)所述荧光增强液的配方为：0.0002～0.0009％β-NTA、0.0024％TOPO、0.08％醋酸、0.05％的Triton X-100组成，用适量邻苯二甲酸氢钾调整pH为3.0-3.2的水溶液。
本发明选取不带芳香基的双功能基团的聚氨基多羧酸类为螯合剂与稀土离子螯合作为标记物，分别在磁珠和稀土离子螯合物上进行抗原或抗体的标记，利用抗原抗体的免疫反应进行分析检测，所制备的试剂性能可达同等化学发光试剂的水平。同时，采用液态均相反应，各组分充分混合，使得反应效率更高且反应更充分，克服了非均相反应重复性差和不稳定的缺点。免疫反应后，通过外加磁场完成对免疫磁珠分离和固定，使用清洗液将多余组分完全去除后，再加入荧光增强液读取荧光值，整个反应过程全程仪器操作，实现了自动化、精准化、智能化，解决了传统板式时间分辨荧光免疫分析技术难以自动化和手工误差大的问题。采用具有双功能基团结构的螯合剂，使其一端与稀土离子铕(Eu 3+)、钐(Sm 3+)、铽(Tb 3+)、镝(Dy 3+)中的1种连接，另一端与抗体/抗原分子上的自由氨基或羧基连接，形成稀土离子标记的抗体/抗原，经过免疫反应之后生成免疫复合物。由于这种复合物在水中的荧光强度非常弱，因此需加入荧光增强液，首先把稀土离子从复合物上解离下来，然后自由稀土离子与荧光增强液中荧光增强物(螯合剂)螯合进入胶束的疏水内核，使稀土离子的荧光成百万倍的放大。
与现有技术相比，本发明具有如下有益效果：
(1)首次将免疫磁微粒技术与时间分辨荧光技术融合到产品中，解决POCT行业快而不准的痛点。由于磁微粒与抗原或抗体为共价偶联，克服了物理吸附的不稳定性，因此免疫磁微粒保存时间久且更稳定；磁微粒的外观和包被过程的灵活性更大，可以根据不同的实验要求进行选择，生产质量可控；采用磁分离技术，形成的免疫复合物在外加磁场中直接沉淀，不需离心即可以将免疫复合物与未结合物质分离；因磁微粒具有更大的结合面积与能在液相中分散而充分反应，大大提高检测范围，缩短反应时间，提高灵敏度；磁微粒可以和不同的探针分子结合，使检测同一样本中不同的待测物成为可能。
(2)本发明的反应体系从加样、孵育、清洗、采光到报告检测结果全过程实现了自动化；可同时检测一份或多份样本、并且一次实验可检测多个指标、操作简便快速(20分钟即可出结果)，克服了传统微孔板式时间分辨荧光免疫技术需要样本累积到一定数量才能检测，实现了样本的即时检测。
(3)采用时间分辨荧光免疫分析技术，采用镧系螯合物铕、铽、钐、镝作为标记物，其具有较宽的激发光谱、较窄发射光谱，有利于降低本底，提高灵敏度；紫外光激发具有较高量子产率、较大Stokes位移，避免激发光谱和荧光发射光谱以及生物基质发射的光谱重合，荧光衰变时间长等优点，比传统荧光物质检测范围更宽、特异性更好。由于是液相反应系统，反应更加充分，加上使用稀土螯合物荧光示踪物和磁微粒的放大系统，使得检测灵敏度和检测重复性要远远好于传统微孔板式TRFIA和层析方法。
(4)本发明同时也为时间分辨免疫分析法的高通量以及多通道检测提供了一定的基础平台，常规的样本在配合本发明的方法时，能实现检测多个项目指标或者单一试剂条可以同时检测多份样本的效果，有着非常大的潜在优势。
附图说明
图1为基于磁微粒的时间分辨荧光检测方法的组合试剂条的结构示意图。
1、测试孔一号，2、测试孔二号，3、荧光标记物一号，4、荧光标记物二号，6、洗液，7、洗液，8、样本稀释液一号，9、样本稀释液二号，12、增强液，5、10、11、13均为预备孔。
图2为基于磁微粒的时间分辨荧光检测方法的单个测试的反应杯的结构示 意图。
图3为cTnI剂量-反应曲线图。
图4为cTnI试剂盒与雅培试剂盒相关性。
具体实施方式
下面结合具体实施方式对本发明的实质性内容作进一步详细的描述。
实施例1：
利用本发明的基于磁微粒的时间分辨荧光免疫检测方法制备cTnI试剂盒；
其中，组合试剂条的形状为扇形，从左到右依次排列1～13个孔。第1、2孔为测试孔，第3、4孔为荧光标记孔，第6、7孔为稀释液孔，第8、9为样本稀释液孔，第12为增强液孔，第5、10、11、13为预备孔。第1与2孔之间可以存放磁铁进行磁分离实验；第3、4孔可以从整个试剂条上拆卸成为独立的组分，便于对荧光标记物进行分装储存。组合试剂条的结构见附图1。
具体工艺如下：
(1)cTnI免疫磁珠的制备方法：
①洗涤磁珠
吸取1mg直径1μm羧基磁珠到1.5ml离心管中，将离心管放置在磁力架中，利用磁力架进行磁珠和缓冲液的分离，用1ml 0.1M pH6.0的2-(N-吗啡啉)乙磺酸(MES)洗涤磁珠3～5次。
②磁珠活化
向上述洗涤好的磁珠中加入1ml 0.1M pH6.0的MES，加入20μL 20mg/mL的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸溶液(EDC)和20μL 20mg/ml N-羟基琥珀酰亚胺(NHS)，室温震荡反应0.5～1小时。
③cTnI抗体与磁珠的偶联
上述活化好的磁珠置于磁力架上，弃上清，收集活化好的磁珠。加入0.05～1mg已经用MES透析好的cTnI单克隆抗体，室温持续旋转孵育3～5小时，反应时间取决于磁珠的配位基与浓度。
④封闭
上述磁珠置于磁力架上，弃上清，收集免疫磁珠。加入含有1％BSA 0.1M pH 8.0的Tris-HCl缓冲液，重悬磁珠，封闭未偶联cTnI单克隆抗体的活化羧基位点， 反应30～60min。
⑤储存
上述磁珠置于磁力架上，弃上清，加入500μL免疫磁珠保存液。
所述的保存液为含5％(w/v)BSA，10％(w/v)海藻糖，2％(w/v)蔗糖，0.1％(v/v)Tween-20的0.05M pH 8.0的Tris-HCl缓冲液。
将上述制备好的免疫磁珠稀释，使cTnI单克隆抗体的终浓度为0.0050～0.0100g/L，分装至试剂条第1孔(测试孔1)中。
(2)生物素化cTnI单克隆抗体的制备方法：
①用0.1M pH 8.0碳酸氢钠缓冲液对1mg cTnI单克隆抗体充分透析。
②用1ml DMSO溶解1mg生物素。
③向1mlcTnI单克隆抗体溶液(即含抗体量为1mg)加入120μL生物素溶液(即含生物素120μg),在室温下持续搅拌，保温2～4小时。
④在4℃，对PBS充分透析，以除去游离的生物素。
⑤样品加入5％(w/v)BSA，将结合产物置4℃，避光保存。
将上述制备好的生物素化cTnI单克隆抗体稀释，使cTnI单克隆抗体的终浓度为0.001～0.005g/L，分装至试剂条第3孔中。
(3)SA标记铕制备方法：
①SA-Eu 3+的制备
(1)取出1mg SA置于10KD超滤离心管，10000rpm离心10min，弃去滤液。
(2)加入标记缓冲液(0.05M pH9.0碳酸盐缓冲液)200μL，10000rpm离心10min，弃去滤液。重复此操作4～5次。
(3)将离心管滤膜反转，3000rpm离心6min，收集浓缩的抗体。加入200μL标记缓冲液，静置3～5min，再将离心管滤膜反转，3000rpm离心6min，收集浓缩的SA。
(4)按照质量比SA：铕螯合物DTTA-Eu＝3:1的比例充分混匀，放入旋转培养器，室温反应16～24小时。
②SA-Eu 3+的纯化
用Sepharose CL-6B凝胶柱纯化SA-Eu 3+，然后加入SA-Eu 3+保存剂(15％(w/v)BSA+5％(w/v)Proclin300)，使BSA和Proclin300终浓度为0.2％，经0.22μm滤膜过滤，4℃储存备用。
将上述制备好的SA-Eu 3+稀释，使SA的终浓度为0.005～0.010g/L，分装至试剂条第4孔中。
所述的荧光物质铕的激发波长为340nm，发射波长为615nm。
(4)清洗液的制备方法：
配制含有0.8～1.5％Tween-20，0.05％Proclin300，pH 7.2～7.5 0.025M Tris-Hcl缓冲液，将配制好的溶液以每孔2000μL分装至洗液孔6和7中。
(5)增强液的制备方法：
配制含有0.0002～0.0009％β-NTA、0.0024％TOPO、0.08％醋酸、0.05％的Triton X-100组成，用适量邻苯二甲酸氢钾调整pH为3.0-3.2的水溶液。将配制好的溶液以每孔400μL分装至试剂条第12孔(增强液孔)。
(6)cTnI校准品的制备方法：
所述cTnI校准品的制备方法为：用含1.5％Tween-20，0.02％Proclin300，pH 7.50.02M Tris-Hcl缓冲液将cTnI抗原稀释至0、0.03、0.1、0.5、1、5、25、50ng/ml。
各组分分装至组合试剂条的特定孔中，半成品通过以下工序组装而成：在实施例1第1、3、4孔分别分装100μL免疫磁珠、200μL生物素化抗体、100μLSA-Eu 3+，清洗液放置在第6、7孔，增强液放置在第12孔。
实施例2
在该实施例中，cTnI试剂盒各组分装到不同试剂瓶中，反应场所为反应杯，反应杯的结构见附图2。
(1)cTnI免疫磁珠的制备方法：
将浓度0.0050～0.0200g/L为生物素化cTnI单克隆抗体分装至试剂瓶中，将SA-磁珠分装至另一试剂瓶中。二者在反应杯中通过链酶亲和素-生物素连接在一起。
(2)铕标cTnI抗体制备方法：
①铕标cTnI单抗的制备
a.取出1mg cTnI单克隆抗体置于30KD超滤离心管，10000rpm离心10min，弃去滤液。
b.加入标记缓冲液(0.05M pH9.0碳酸盐缓冲液)200μL，10000rpm离心10min，弃去滤液。重复此操作4～5次。
c.将离心管滤膜反转，3000rpm离心6min，收集浓缩的抗体。加入200μL标记缓冲 液，静置3～5min，再将离心管滤膜反转，3000rpm离心6min，收集浓缩的cTnI单抗。
d.按照质量比cTnI单克隆抗体：铕螯合物DTTA-Eu＝5:1的比例充分混匀，放入旋转培养器，室温反应16～24小时。
②铕标cTnI单抗的纯化
用SepHadex TM G-75凝胶柱纯化铕标cTnI单抗，然后加入铕标cTnI单抗保存剂(15％(w/v)BSA+5％(w/v)Proclin300)，使BSA和Proclin300终浓度为0.3％，经0.22μm滤膜过滤，4℃储存备用。
各组分分装到不同的试剂瓶中。所述的组合试剂条和试剂瓶上涂有可供全自动荧光检测分析仪扫描识别的产品信息标识包括企业标准曲线、批次、生产日期、有效期。
试剂盒的检测步骤：在组合试剂条第1孔中加入免疫磁珠、样本、生物素化cTnI单抗、SA-Eu 3+或者单个反应杯中SA-磁珠、生物素化cTnI单抗、样本、铕标cTnI抗体，37度振荡孵育15min，然后在磁场的作用下吸附磁珠，用清洗液清洗去除游离的SA-Eu 3+，然后加入增强液，孵育5min，在激发波长为340nm，发射波长为615nm条件下采集荧光信号，根据校准曲线得到待测样本cTnI蛋白浓度。
实施例3 应用本发明制备cTnI试剂盒与市售试剂盒的比较
实施例1中应用本发明制备cTnI试剂盒测定校准品cTnI，其检测范围为0.03～50ng/ml，校准品浓度与荧光值拟合双对数剂量-反应曲线，标准曲线为Y＝1.011X+4.9268R＝0.999，试剂盒性能良好，见图3。
表1全自动磁微粒时间分辨荧光免疫分析系统测定cTnI标准曲线
浓度ng/ml 荧光值 荧光值 荧光值 均值 变异系数
0 801 812 831 815 2％
0.03 2236 2501 2304 2347 6％
0.1 8439 8521 8301 8420 1％
0.5 40088 40056 41023 40389 1％
1 87569 86127 88156 87284 1％
5 478965 469875 458791 469210 2％
25 2256890 2156910 2069884 2161228 4％
50 4178144 4152601 4203108 4177951 1％
采用市售的高敏肌钙蛋白-I测定试剂盒(化学发光微粒子免疫检测法)(雅 培贸易有限公司)与实施例1中的方法检测样品，重复三次，验证本发明检测结果准确性，结果见下表2。
表2样本检测结果
Figure PCTCN2018103954-appb-000001
与市售试剂比较，本试剂偏差均小于±10％，本试剂的检测结果准确可靠。
采用市售的高敏肌钙蛋白-I测定试剂盒(化学发光微粒子免疫检测法)(雅培贸易有限公司)与实施例1中的试剂检测100例临床样本，检测结果见图4、表3。
表3临床样本检测结果
Figure PCTCN2018103954-appb-000002
本试剂对100例临床血清的检测结果与雅培试剂盒进行比较，阳性符合率96.05％，阴性符合率83.33％，二者的总符合率为93.00％，与雅培试剂的相关性为0.996(图4)。
可见，本发明基于磁微粒的时间分辨荧光检测方法具有如下优势：1)灵敏度高；2)检测时间短，20min出报告；3)样品需求量少，一次上样只需50μL；4)操作简单，节省人力。实现远程结果查询、设备维护、故障处理、质量监控，提高售后维护效率、降低维护成本。
实施例4
基于磁微粒的时间分辨荧光免疫检测方法制备猪瘟病毒抗体检测试剂盒，其 包含的主要组分有：试剂条、阴性对照血清和阳性对照血清。所述试剂条的结构示意图见附图1。
所述的试剂条主要组分有猪瘟病毒重组抗原E2蛋白的免疫磁珠、铕标羊抗猪抗体、样本稀释液、清洗液、增强液；
(1)所述免疫磁珠制备
①洗涤磁珠
吸取1mg直径3μm羧基磁珠到1.5ml离心管中，将离心管放置在磁力架中，利用磁力架进行磁珠和缓冲液的分离，用1ml 0.1M pH6.0的2-(N-吗啡啉)乙磺酸(MES)洗涤磁珠3～5次。
②磁珠活化
向上述洗涤好的磁珠中加入1ml 0.1M pH6.0的MES，加入20μL 10mg/mL的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸溶液(EDC)和20μL 50mg/ml N-羟基琥珀酰亚胺(NHS)，室温震荡反应0.5～1小时。
③猪瘟重组E2蛋白与磁珠的偶联
上述活化好的磁珠置于磁力架上，弃上清，收集活化好的磁珠。加入0.5～1mg猪瘟E2蛋白，室温持续旋转孵育30～240分钟，反应时间取决于磁珠的配位基与浓度。
④封闭
上述磁珠置于磁力架上，弃上清，收集免疫磁珠。加入含有5％BSA 0.05M pH8.0的Tris-HCl缓冲液，重悬磁珠，封闭未偶联猪瘟重组E2蛋白的活化羧基位点，反应30～60min。
⑤储存
上述磁珠置于磁力架上，弃上清，加入100μL免疫磁珠保存液。
所述的保存液为含6％(w/v)BSA，0.1％(v/v)Tween-20和0.1％(w/v)PEG20000的0.05M pH 8.0的Tris-HCl缓冲液。
将上述制备好的免疫磁珠按一定比例稀释，使猪瘟重组E2蛋白的终浓度为0.0025～0.0080g/L，100μL/孔分装至试剂条第1孔(测试孔1)中。
(2)所述铕标抗体，将DTPA-Eu作为标记物，在0.05M pH9.0的碳酸盐缓冲液下，加入DTPA-Eu与羊抗猪二抗，按质量比1:2混合后溶于300μL 0.05M pH9.0 的碳酸盐缓冲液，室温震荡过夜。用葡聚糖凝胶柱过滤后收集峰管。将上述制备好的铕标二抗按一定比例稀释，使羊抗猪抗体的终浓度为0.0060～0.0075g/L，100μL/孔分装至试剂条第3孔(标记物1)中。
(3)所述的样本稀释液为0.01M pH7.4PBS，620μL/孔分装至试剂条第8孔(样本稀释液孔)中。
实施例5 猪瘟病毒抗体检测试剂盒与美国IDEXX猪瘟抗体检测试剂盒检测结果符合率比较
本方法对100例临床血清的检测结果与IDEXX猪瘟抗体检测试剂盒进行比较，阳性符合率96.49％，阴性符合率97.67％，二者的总符合率为97.00％。检测结果见下表4。由结果可知基于磁微粒的时间分辨荧光免疫检测方法制备的猪瘟病毒抗体检测试剂盒与进口试剂盒具有良好的相关性。
表4：本试剂盒与IEDXX试剂盒比对结果
Figure PCTCN2018103954-appb-000003

Claims (9)

  1. 一种基于磁微粒的时间分辨荧光免疫检测方法，其特征在于包含如下步骤：
    (1)将特异性的抗原或抗体包被在磁珠上，形成免疫磁珠；
    (2)将不带芳香基的双功能基团的聚氨基多羧基与稀土离子形成的稀土离子螯合物标记特异性的抗原或抗体，形成荧光标记物；
    (3)加入被测样本，发生免疫反应，使免疫磁珠、被测样本和荧光标记物形成复合物；
    (4)通过外加磁场完成对免疫磁珠分离和固定，使用清洗液将游离荧光标记物洗去，再加入荧光增强液来增强荧光值；
    (5)最后检测荧光，根据内置的待测物浓度与荧光信号值拟合的剂量-反应曲线通过软件计算出被测样本浓度。
  2. 根据权利要求1所述的时间分辨荧光免疫检测方法，其特征在于，所述的免疫磁珠为带有官能团修饰的直径0.1～3μm的超顺磁微粒与单克隆抗体或者抗原的共价偶联物；其中所述的磁微粒为羧基磁珠、氨基磁珠、羟基磁珠、甲苯磺酰基磁珠、NHS磁珠、蛋白A磁珠、蛋白G磁珠、抗小鼠IgG磁珠、亲水磁珠、疏水磁珠中的一种或几种；或者是免疫磁珠为通过链酶亲和素磁微珠与生物素化抗体反应形成的免疫磁珠。
  3. 根据权利要求2所述的时间分辨荧光免疫检测方法，其特征在于，所述免疫磁珠的制备方法为直径0.1～3μm的磁微粒与对应抗体或抗原经洗涤、活化、偶联、封闭制备得到。
  4. 根据权利要求1所述的时间分辨荧光免疫检测方法，其特征在于，步骤(2)所述将聚氨基多羧基与稀土离子形成的螯合物标记特异性的抗原或抗体，形成荧光标记物的方式有：直接把稀土螯合物通过化学键与生物原料连接起来；或者把稀土螯合物通过化学键与链酶亲和素连接起来，抗原或抗体标记生物素，最后稀土螯合物通过生物素与链酶亲和素反应，与抗原或抗体连接起来。
  5. 根据权利要求1所述的时间分辨荧光免疫检测方法，其特征在于，步骤(2)所述不带芳香基的双功能基团的聚氨基多羧基为异硫腈酸苯基-乙二胺四乙酸，异硫腈酸-二乙三胺基四乙酸、二乙三胺基五乙酸或N-(P-异硫氢基苄基)-二乙烯 三胺四乙酸；所述稀土离子为Eu 3+、Sm 3+，Tb 3+或Dy 3+。
  6. 根据权利要求1所述的时间分辨荧光免疫检测方法，其特征在于，步骤(2)所述稀土离子螯合物标记抗体或抗原制备过程为：抗原或抗体与稀土离子螯合物的质量比为1:1～10:1；在0.05M pH9.0的碳酸盐缓冲液下，加入稀土离子螯合物与已透析抗原或抗体，混合后溶于0.05M pH9.0的碳酸盐缓冲液，室温震荡过夜；用葡聚糖凝胶柱纯化后收集峰管；将上述制备好的荧光标记物稀释，备用。
  7. 根据权利要求1所述的时间分辨荧光免疫检测方法，其特征在于，步骤(2)所述免疫反应的温度为25℃～37℃，湿度为30～75％RH，在摇动、振荡下进行。
  8. 根据权利要求1所述的时间分辨荧光免疫检测方法，其特征在于，步骤(4)所述清洗液的配方为：0.8～1.5％Tween-20，0.05％Proclin300，pH7.2～7.5 0.025M Tris-Hcl缓冲液。
  9. 根据权利要求1所述的时间分辨荧光免疫检测方法，其特征在于，步骤(4)所述荧光增强液的配方为：0.0002～0.0009％β-NTA、0.0024％TOPO、0.08％醋酸、0.05％的Triton X-100组成，用适量邻苯二甲酸氢钾调整pH为3.0-3.2的水溶液。
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