WO2020082720A1 - 一种基于磁棒法的单人份化学发光免疫检测方法及系统 - Google Patents

一种基于磁棒法的单人份化学发光免疫检测方法及系统 Download PDF

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WO2020082720A1
WO2020082720A1 PCT/CN2019/085653 CN2019085653W WO2020082720A1 WO 2020082720 A1 WO2020082720 A1 WO 2020082720A1 CN 2019085653 W CN2019085653 W CN 2019085653W WO 2020082720 A1 WO2020082720 A1 WO 2020082720A1
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magnetic
solution
module
mixed solution
detection
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PCT/CN2019/085653
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English (en)
French (fr)
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潘琴
张冠斌
田进
周晶
杨芳艳
高云
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成都博奥新景医学科技有限公司
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Publication of WO2020082720A1 publication Critical patent/WO2020082720A1/zh

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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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • 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

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  • the invention belongs to the technical field of biological detection, and in particular relates to a single-part chemiluminescence immunodetection method and system based on the magnetic rod method.
  • Chemiluminescence immunoassay technology has become the mainstream technology for clinical testing and analysis due to its high detection sensitivity, wide analysis range, and wide coverage of detection indicators.
  • the traditional chemiluminescence immunoassay technology still has many defects.
  • the packaging volume of the traditional chemiluminescence immunoassay kit is too large to meet the needs of primary hospitals.
  • the top three hospitals account for only a small part of all hospitals, and the vast majority of hospital groups are community, township, and county-level primary hospitals with a small sample size.
  • Traditional luminescence kits are usually packaged in 100 tests / box, that is, a box of reagents contains 100 servings, which will result in the 100 tests / box of reagents not being used within the validity period (1 month) of the bottle opening.
  • the traditional chemiluminescence immunoassay method also has the problems of more contamination and insufficient mixing of the reaction solution.
  • each test reaction, cleaning, detection and other steps are completed in a reaction tube.
  • a magnet is used to adsorb magnetic beads on the outer wall, and the washing liquid and the waste liquid are sucked (or dumped) into the tube.
  • the adsorption of the tube wall will cause various liquids to remain on the tube wall, such as samples, magnetic bead working fluid, enzyme working fluid, etc.
  • the cleaning step must be performed 3 to 6 times, which is cumbersome and complicated And it cannot be thoroughly cleaned, and it is inevitable to always carry pollution: carrying pollution, the most critical and core indicator for evaluating chemiluminescence systems, has always been a problem that traditional chemiluminescence immunological researchers cannot overcome.
  • the cleaning is not thorough enough, high-value samples, enzyme conjugates, and washing liquid will cause adsorption and residue on the tube wall, which will result in high detection background value, unsatisfactory signal-to-noise ratio, and poor sensitivity.
  • one of the objects of the present invention is to provide a single-part chemiluminescence immunodetection method based on the magnetic rod method. This method is to wash the magnetic beads thoroughly, carry less pollution, and mix well, suitable for single
  • the human chemiluminescence immunoassay kit is flexible in use and has low background noise.
  • the second object of the present invention is to provide a chemiluminescence immunodetection system capable of implementing the above method.
  • the instrument required by the system is small and compact, and can be carried into the diagnosis and treatment site. There are no special requirements for the size of the laboratory sample, the price is low, and there is basically no maintenance cost. It is suitable for primary medical institutions to promote the use of chemiluminescent immunoassay technology.
  • the certain frequency is selected from 1 time / min to 360 times / min.
  • the invention adopts the up-and-down mixing method from 1 time / minute to 360 times / minute, which overcomes the following defects of the traditional instrument eccentric force vortex mixing method: the mixing frequency is unstable, and the reaction solution is easy to splash, resulting in the cleaning solution cannot be washed. Severe jump points occur and false positive test results occur.
  • the salt content of the cleaning solution is easy to leak out and corrode the control motor, which results in the motor being easily damaged. This will cause frequent replacement of the motor and increase the cost of use.
  • the added volumes of the analysis buffer, magnetic bead working solution, enzyme working solution, cleaning solution 1, cleaning solution 2 and substrate solution are 1 to 1000 ⁇ l .
  • the reaction time of steps 2) and 4) is 30 seconds to 60 minutes.
  • the particle diameter of the magnetic beads is 0.01 ⁇ m to 50 ⁇ m.
  • the magnetic beads in one test can be magnetic beads of the same particle size or mixed magnetic beads of different particle sizes.
  • the principle of the luminescence signal detection includes but is not limited to alkaline phosphatase enzymatic luminescence, horseradish peroxidase luminescence, acridine The pyridyl ester emits light directly.
  • the substrate solution includes but is not limited to AMPPD, APS-5, CDP-Star, Luminol and its derivatives, dilute hydrogen peroxide Alkaline solution.
  • the enzyme working solution is a product labeled with alkaline phosphatase, horseradish peroxidase, or acridinium ester or other labeled products .
  • the alkaline phosphatase includes but is not limited to calf intestinal alkaline phosphatase, bacterial alkaline phosphatase or placental alkaline phosphatase.
  • the luminescence result is finally detected by a PMT or an analyzer containing PMT.
  • the above-mentioned detection method of the present invention can be operated manually or completed by an instrument system.
  • manual operation only need to carry out the fixed packaging single serving kit according to the above steps, there is no need to accurately draw 1 serving from the traditional serving kit of 100 servings for testing, so no longer rely on traditional large equipment, you can Flexible implementation in primary medical institutions.
  • the instrument detection system is used to complete the above detection method, thanks to the ingenious magnetic bead and liquid separation method of the present invention, the detection system no longer requires various cumbersome mixing and liquid suction mechanisms, only a magnetic rod is required After the completion of multiple steps, the magnetic beads and liquid are separated. And this separation method makes the cleaning more thorough, effectively reducing the number of cleanings and saving reagents.
  • the mixing method keeps the mixed solution in a uniformly suspended state, the magnetic beads will not sink, the reaction between the antigen and antibody molecules is free and smooth, and the binding is sufficient, the reaction platform period can be reached in a short time, which can shorten the sample detection Time can also improve detection sensitivity.
  • the present invention also provides a system for chemiluminescence immunoassay using a method as described above.
  • the system includes a reagent module, a magnetic suction module, an isolation and mixing module, and a control module; the reagent module, magnetic Both the suction module and the mixing module are connected to the control module.
  • the reagent module includes a reagent tube assembly 1 and a working platform 2 for fixing the reagent tube assembly.
  • the magnetic suction module includes a magnetic rod 3 that can move in a horizontal direction and a vertical direction. And a motor that drives the magnetic bar 3 to move.
  • the isolation and mixing module includes an isolation sleeve 7 that can move in the horizontal and vertical directions, and a motor that drives the isolation sleeve 7 to move.
  • the magnetic attraction module further includes a magnetic rod fixing block 4, a magnetic rod horizontal arm 5, a magnetic rod vertical arm 6 and a horizontal arm fixing block 10, the magnetic The rod fixing block 4 clamps and fixes the magnetic rod 3, and can be moved horizontally along the magnetic rod horizontal arm 5 under the drive of the motor, and the magnetic rod horizontal arm 5 is fixed on the magnetic rod vertical arm 6 by the horizontal arm fixing block 10
  • the horizontal arm fixing block 10 can be moved in the vertical direction along the magnetic bar vertical arm 6 under the drive of another motor.
  • the manner of realizing the movement of the magnetic bar 3 in the horizontal direction and the vertical direction is not limited to this, this is just a manner provided by the embodiment of the present invention.
  • the isolation and mixing module further includes a horizontal telescopic rod 8, a vertical arm 9 of the isolation sleeve and a telescopic rod fixing block 11, and one end of the horizontal telescopic rod 8
  • the isolating sleeve 7 is movably connected, and the other end is fixed to the vertical arm 9 of the isolating sleeve by a telescopic rod fixing block 11. Driven by another motor, it moves in the vertical direction along the vertical arm 9 of the insulating sleeve.
  • the way to realize the movement of the isolation sleeve 7 in the horizontal direction and the vertical direction is not limited to this, but this is only a way provided by the embodiment of the present invention.
  • the above-mentioned system for performing single-part chemiluminescence immunodetection further includes a detection module connected to the control module, the detection module includes a detection gripper, a detection gripper motor, a detection disk with a hole position, and a PMT analyzer.
  • the single-part chemiluminescence immunodetection method based on the magnetic rod method achieves continuous mixing of the reaction solution. Whether the antibody on the magnetic particle reacts with the antigen in the sample, or the antigen-antibody complex on the magnetic particle reacts with the antibody in the enzyme conjugate to form a double antibody sandwich "sandwich" immune complex, the entire reaction process is at 37 °C (or other set temperature) continues to be mixed, at this time, the magnetic particles are always in a uniform suspension state, and will not sink, so that the reaction between the antigen and antibody molecules is free and smooth, and the antigen and antibody molecules are fully bound.
  • the reaction platform period can be reached in a short time, which can shorten the sample detection time and improve the detection sensitivity.
  • the single-part chemiluminescence immunodetection method based on the magnetic bar method adopts the single-person part, independent packaging, and the method of disassembly and use, which is tailor-made for the community, township and other grass-roots hospitals: the kit is single-person part, independent packaging There is no requirement for the average daily sample size of the hospital.
  • the kit is single-person part, independent packaging
  • the single-part chemiluminescence immunodetection system based on the magnetic bar method omits the traditional system of multiple transfer devices, cleaning devices, rotating devices and their corresponding motors. It is compact, simple to operate, and requires less timing, common Personnel can operate and use. And the instrument is a desktop computer, and there is no requirement for the storage place and venue at all.
  • FIG. 1 is a schematic structural diagram of an automatic chemiluminescence immunoassay instrument disclosed in the prior art CN107831322A;
  • FIG. 2 is a schematic structural diagram of a timing control system disclosed in the prior art CN108226549A for a chemiluminescence immunoanalyzer;
  • FIG. 3 is a schematic diagram of the main structure of a single-part chemiluminescence immunodetection system based on the magnetic rod method according to an embodiment of the present invention
  • 1-Reagent tube assembly 2-working platform, 3-magnetic rod, 4-magnetic rod fixing block, 5-magnetic rod horizontal arm, 6-magnetic rod vertical arm, 7-isolating sleeve, 8-horizontal telescopic rod, 9 -Isolated sleeve vertical arm, 10-horizontal arm fixing block, 11-telescopic pole fixing block.
  • the single-part chemiluminescence immunodetection method based on the magnetic rod method of the present invention can be used in a variety of chemiluminescence detection, and is not limited to the following HIV, HCV, and TP in the test examples; the system implementing the chemiluminescent immunodetection method of the present invention can combine some existing functional modules into the following embodiments according to actual needs. These are easily obtained by the basic invention and still belong to The scope of protection of the present invention.
  • a single-part chemiluminescence immunodetection system based on the magnetic rod method includes a reagent module, a magnetic attraction module, an isolation and mixing module, and a control module; the reagent module, magnetic attraction module, isolation and mixing module are all The control module is connected.
  • the reagent module includes a reagent tube assembly 1 and a working platform 2 for fixing the reagent tube assembly.
  • the magnetic suction module includes a magnetic rod 3 that can move in a horizontal direction and a vertical direction, and a motor that drives the magnetic rod 3 to move.
  • the isolation and mixing module includes an isolation sleeve 7 that can move in the horizontal and vertical directions, and a motor that drives the isolation sleeve 7 to move.
  • the magnetic rod 3 and the isolation sleeve 7 are shifted under the drive of the corresponding motor, first to directly above the magnetic bead working liquid reagent tube, after vertical movement, the magnetic rod 3 extends into Inside the isolation sleeve 7, it is further extended into the magnetic bead working fluid reagent tube to absorb the magnetic beads. Then the magnetic rod 3 and the isolation sleeve 7 are simultaneously lifted vertically upwards and moved into the analysis buffer reagent tube to which the sample is added. The magnetic rod 3 is lifted up, and the magnetic beads are left in the tube. The frequency oscillates up and down and mixes well. Then, the magnetic rod 3 extends into the tube, absorbs the magnetic beads, and then moves up together with the isolation sleeve 7 to transfer the magnetic beads to the next reagent tube until the reaction and cleaning are completed.
  • the magnetic attraction module further includes a magnetic bar fixing block 4, a magnetic bar horizontal arm 5, a magnetic bar vertical arm 6 and a horizontal arm fixing block 10
  • the magnetic bar fixing block 4 clamps and fixes the magnetic bar 3, and can be moved horizontally along the magnetic bar horizontal arm 5 under the drive of the motor.
  • the magnetic bar horizontal arm 5 is fixed on the magnetic bar by the horizontal arm fixing block 10
  • the horizontal arm fixing block 10 can be moved in the vertical direction along the magnetic bar vertical arm 6 under the drive of another motor.
  • the isolating and mixing module further includes a horizontal telescopic rod 8, an isolating sleeve vertical arm 9 and a telescopic rod fixing block 11, the horizontal telescopic One end of the rod 8 is movably connected to the isolation sleeve 7 and the other end is fixed to the vertical arm 9 of the isolation sleeve through a telescopic rod fixing block 11.
  • the rod fixing block 11 moves in the vertical direction along the vertical arm 9 of the insulating sleeve under the drive of another motor.
  • the movable connection isolating sleeve 7 is removed and replaced after each test, and is a disposable consumable.
  • the above system for performing chemiluminescence immunoassay for a single serving also includes a detection module connected to a control module, the detection module includes a detection gripper, a detection gripper motor, a detection disc with a hole position, and a PMT Analyzer.
  • the HIV 1 + 2 antibody single serving reagent is provided by Chengdu Boao Xinjing Medical Technology Co., Ltd.
  • the reagent components include: analysis buffer, magnetic bead working solution, enzyme working solution, cleaning solution 1, cleaning solution 2, and substrate solution.
  • the detection method can be implemented by the detection system of Example 1, or can also be achieved by manual operation. When using the detection system, the analysis buffer, enzyme working solution, cleaning solution 1, cleaning solution 2, and substrate solution are placed in the reagent tube assembly in advance. The method specifically includes the following steps:
  • Step 1 Add the sample to be tested to the analysis buffer to obtain mixed solution A. (Aspirate the sample into the analysis buffer tube of the reagent tube assembly, and then fix the reagent tube assembly on the working platform 2.)
  • Step 2 Remove the magnetic beads from the magnetic bead working solution of HIV 1 + 2 antibody reagent by magnetic attraction, and add them to the mixed solution A. Then mix them up and down, shaking at a frequency of 120 times / minute And react for 5 minutes to obtain mixed solution B.
  • the magnetic bar 3 and the isolating sleeve 7 move to the top of the magnetic bead working fluid tube under the drive of the motor, and extend downward into the tube. Then the magnetic bar 3 absorbs the magnetic beads and transfers it to the analysis buffer tube. The magnetic bar 3 is lifted vertically From now on, the magnetic beads fall, and the isolation sleeve 7 is driven by the motor at a frequency of 120 vibrations per minute for 5 minutes.
  • Step 3 Remove the magnetic beads from the reaction mixture B by magnetic attraction, add it to the cleaning solution 1 to obtain the mixture C, and then shake up and down at 180 times / min to mix and wash.
  • the magnetic rod extends downward into the isolation sleeve 7, draws the magnetic beads in the tube, and transfers the magnetic beads together with the isolation sleeve 7 to the tube of cleaning solution 1, the magnetic rod 3 is lifted vertically, the magnetic beads fall, and the isolation sleeve 7 is driven by the motor Washing up and down at a frequency of 180 times / minute.
  • Step 4 Remove the magnetic beads from the mixed solution C by magnetic attraction, and add it to the working solution of the HIV 1 + 2 antibody reagent enzyme, and then mix it up and down by shaking at a frequency of 120 times / min. Mix well and react for 15 minutes to obtain mixed solution D.
  • the magnetic rod extends downward into the isolation sleeve 7, draws the magnetic beads in the tube, and transfers the magnetic beads to the enzyme working fluid tube together with the isolation sleeve 7, the magnetic rod 3 is lifted vertically, the magnetic beads fall, and the isolation sleeve 7 is driven by the motor Shake up and down at a frequency of 120 times / minute to mix and react for 15 minutes.
  • Step 5 Remove the magnetic beads from the reaction mixture D by magnetic attraction, add it to the cleaning solution 2 to obtain the mixture E, and then shake up and down at 180 times / min to mix and wash.
  • the magnetic rod extends downward into the isolation sleeve 7, sucks the magnetic beads in the tube, and transfers the magnetic beads together with the isolation sleeve 7 to the cleaning solution 2 tube, the magnetic rod 3 is lifted up vertically, the magnetic beads fall, and the isolation sleeve 7 is driven by the motor Shake up and down at 180 times / min to mix and wash.
  • Step 6 Remove the magnetic beads from the mixed solution E by magnetic attraction, move it to the substrate solution, and mix by shaking up and down at a frequency of 120 times / minute to obtain the mixed solution F, and then emit light Signal Detection.
  • the magnetic rod extends downward into the isolation sleeve 7, sucks the magnetic beads in the tube, and transfers the magnetic beads into the liquid tube with the isolation sleeve 7, the magnetic rod 3 is lifted up vertically, the magnetic beads fall, and the isolation sleeve 7 is driven by the motor 120 times / minute frequency mixing.
  • Step 7 Perform luminescence signal detection on the mixed liquid.
  • the detection gripper grabs the substrate liquid tube and puts it into the hole of the detection tray, and uses the PMT analyzer to perform signal detection.
  • HIV-1 p24 antigen reagent was provided by Chengdu Boao Xinjing Medical Technology Co., Ltd.
  • the reagent components include: analysis buffer, magnetic bead working solution, enzyme working solution, cleaning solution 1, cleaning solution 2, and substrate solution. The specific steps are as follows. When the system is completed, it is similar to Test Example 1:
  • Step 1 Add the sample to be tested to the analysis buffer to obtain mixed solution A.
  • Step 2 Remove the magnetic beads from the working solution of HIV-1 p24 antigen reagent magnetic beads by magnetic attraction, and add them to the mixed solution A. Then mix them up and down, shaking at a frequency of 120 times / min. After 5 minutes of reaction, a mixed liquid B was obtained.
  • Step 3 Remove the magnetic beads from the reaction mixture B by magnetic attraction, add it to the cleaning solution 1 to obtain the mixture C, and then shake up and down at 180 times / min to mix and wash.
  • Step 4 Remove the magnetic beads from the mixed solution C by magnetic attraction, and add it to the working solution of HIV-1 p24 antigen reagent enzyme, and then mix it up and down by shaking at a frequency of 120 times / min. And react for 15 minutes to obtain mixed solution D.
  • Step 5 Remove the magnetic beads from the reaction mixture D by magnetic attraction, add it to the cleaning solution 2 to obtain the mixture E, and then shake up and down at 180 times / min to mix and wash.
  • Step 6 Remove the magnetic beads from the mixed solution E by magnetic attraction, move it to the substrate solution, and mix by shaking up and down at a frequency of 120 times / minute to obtain the mixed solution F, and then emit light Signal Detection.
  • Step 7 Perform luminescence signal detection on the mixed liquid.
  • test results of HIV-1 p24 antigen national reference product (purchased from China Food and Drug Administration, Catalog No. 22015-20150505) were used as a reference to investigate the accuracy of the detection method of the present invention.
  • Anti-HCV single serving chemiluminescence detection method based on magnetic bar method alkaline phosphatase method
  • Anti-HCV reagent was provided by Chengdu Boao Xinjing Medical Technology Co., Ltd.
  • the reagent components include: analysis buffer, magnetic bead working solution, enzyme working solution, cleaning solution 1, cleaning solution 2, and substrate solution.
  • the operation is similar to Test Example 1.
  • the specific steps are as follows. When the system is completed, the test example 1 Similar:
  • Step 1 Add the sample to be tested to the analysis buffer to obtain mixed solution A.
  • Step 2 Remove the magnetic beads from the magnetic bead working solution of the anti-HCV reagent by magnetic attraction, and add them to the mixed solution A. Then mix them up and down, shaking at a frequency of 120 times / minute, reaction 5 Minutes to obtain mixed solution B.
  • Step 3 Remove the magnetic beads from the reaction mixture B by magnetic attraction, add it to the cleaning solution 1 to obtain the mixture C, and then shake up and down at 180 times / min to mix and wash.
  • Step 4 Remove the magnetic beads from the mixed solution C by magnetic attraction, and add the anti-HCV reagent enzyme working solution, and then mix up and down, shake and mix at a frequency of 120 times / minute, and react After 15 minutes, mixed solution D was obtained.
  • Step 5 Remove the magnetic beads from the reaction mixture D by magnetic attraction, add it to the cleaning solution 2 to obtain the mixture E, and then shake up and down at 180 times / min to mix and wash.
  • Step 6 Remove the magnetic beads from the mixed solution E by magnetic attraction, move it to the substrate solution, and mix by shaking up and down at a frequency of 120 times / minute to obtain the mixed solution F, and then emit light Signal Detection.
  • Step 7 Results testing. The results were detected by semi-automatic chemiluminescence immunoassay analyzer.
  • Anti-TP chemiluminescence detection method based on magnetic bar method (alkaline phosphatase method)
  • the anti-TP reagent comes from Chengdu Boao Xinjing Medical Technology Co., Ltd.
  • the reagent components include: enzyme working solution 1, magnetic bead working solution, enzyme working solution 2, cleaning solution 1, cleaning solution 2, and substrate solution.
  • the operation is similar to Test Example 1.
  • the specific steps are as follows. Test Example 1 is similar:
  • Step 1 Add the sample to be tested into enzyme working solution 1 to obtain mixed solution A.
  • the second step remove the magnetic beads from the magnetic bead working solution of anti-TP reagent by magnetic attraction, and add them to the mixed solution A, and then mix them up and down, shaking at a frequency of 120 times / minute, reaction 5 Minutes to obtain mixed solution B.
  • Step 3 Remove the magnetic beads from the reaction mixture B by magnetic attraction, add it to the cleaning solution 1 to obtain the mixture C, and then shake up and down at 180 times / min to mix and wash.
  • Step 4 Remove the magnetic beads from the mixed solution C by magnetic attraction, and add the anti-TP reagent enzyme working solution, and then mix it up and down by shaking at a frequency of 120 times / min. After 15 minutes, mixed solution D was obtained.
  • Step 5 Remove the magnetic beads from the reaction mixture D by magnetic attraction, add it to the cleaning solution 2 to obtain the mixture E, and then shake up and down at 180 times / min to mix and wash.
  • Step 6 Remove the magnetic beads from the mixed solution E by magnetic attraction, move it to the substrate solution, and mix by shaking up and down at a frequency of 120 times / min to obtain the mixed solution F, and then emit light Signal Detection.
  • Step 7 Results testing. The results were detected by semi-automatic chemiluminescence immunoassay analyzer.
  • test results of the Treponema pallidum antibody diagnostic kit (chemiluminescence method) and CHEMCLIN 1500 automatic chemiluminescence immunoassay analyzer of Beijing Kemei Biotechnology Co., Ltd. were used as references to investigate the accuracy of the detection method of the present invention.
  • test results are 20+ days after opening, and the test results show higher detection results than the present invention. Noise background.
  • the reasons include different magnetic bead absorption and transfer methods and changes in reagents due to time.
  • the method of the present invention has higher sensitivity.
  • the detection method of the present invention has low carry pollution and fewer cleaning times, which simplifies the procedure and reduces the difficulty of software control and staff operation. Due to the cleaning method of the method of the present invention and the characteristics of single serving detection, the noise signal generated by the contamination and reagent changes is effectively reduced, which is more helpful for the improvement of sensitivity.
  • the detection system adopting the detection method of the present invention saves multiple transfer mechanisms, mixing mechanisms and corresponding motors, reduces the volume of the instrument, and makes the traditional detection instruments like giants miniaturized, which can be applied to more limited areas In primary medical institutions, and the detection time is shorter.

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Abstract

一种基于磁棒法的单人份化学发光免疫检测方法及系统，利用磁棒(3)吸取磁珠加入到试剂内进行反应或清洗，采用一定频率持续震荡混合的方式进行反应，使磁珠处于均匀悬浮状态。系统包括试剂模块、磁吸模块、隔离和混匀模块以及控制模块。

Description

一种基于磁棒法的单人份化学发光免疫检测方法及系统 技术领域
本发明属于生物检测技术领域，具体涉及一种基于磁棒法的单人份化学发光免疫检测方法及系统。
背景技术
化学发光免疫分析技术以其检测灵敏度高、分析范围宽、检测指标覆盖面广等特点而成为临床检验分析的主流技术。但根据我们的观察研究和临床使用情况的反馈，传统化学发光免疫分析技术仍然存在多个缺陷。
首先，传统化学发光免疫试剂盒包装量过大，不能满足基层医院的使用需求。大三甲医院在所有医院中只占很小部分，绝大多数医院群体是样本量偏小的社区、乡镇、县级等基层医院。传统发光试剂盒通常采用以100测试/盒进行包装，即一盒试剂含100人份的量，这就会导致100测试/盒的试剂在开瓶有效期(1个月)内无法用完。而试剂在长时间存放过程中，由于需反复开瓶使用，且辗转于2℃～8℃冰箱和仪器试剂仓，会引起试剂各组份液体的严重挥发。另，由于试剂盒重复使用次数过多，由不同人甚至是同一人操作，都可能会出现封口或盖盖儿顺序颠倒、混乱的情况，这样会使试剂各组份之间产生交叉污染，导致组份变质失效，无法使用。以上种种情况均会导致医院和厂家资源的浪费，以及由于成分挥发和试剂污染增大检测背景噪声对检测灵敏度的干扰。
其次，传统化学发光免疫分析系统庞大，结构复杂，操作繁琐。传统化学发光免疫分析仪如专利CN107831322A(图1)和专利CN108226549A(图2)中所述，具有试剂盘、取样针、针头、清洗机构及其配套装置、多个移动臂及其电机、抽液泵及其电机等结构，导致系统体积庞大，占地面积大，对医院检验科的规模和场地都提出较高要求，而且增加了厂家生产难度。传统化学发光免疫分析仪的操作模块必须考虑非常复杂的时序逻辑如专利CN108226549A所述，导致操作系统使用复杂，每次装机验收时，都需要企业技术支持人员对医院专业人员进行详细的书面授课和实操培训。
此外，传统化学发光免疫分析方法还存在携带污染多和反应液混匀不充分的问题。
传统化学发光免疫分析方法每个测试的反应、清洗、检测等步骤均在一支反应管中完成，清洗时用磁铁在外壁吸附磁珠，向管内注入洗液和吸走(或倾倒)废液交替进行，但由于始终在一个管内，管壁的吸附作用会导致管壁残留各种液体，如样本、磁珠工作液、酶工作液等等，清洗步骤必须进行3～6遍，繁琐、复杂，而且无法做到彻底洗净，不可避免地始 终携带污染：而携带污染这一考核化学发光系统最关键、最核心的指标，一直是传统化学发光免疫研究人员无法攻克的难题。另由于清洗不够彻底，高值样本、酶结合物、洗液等在管壁会产生吸附和残留，进而产生检测背景值高，信噪比不理想，灵敏度不佳的结果。
传统化学发光免疫分析方法，无论是磁微粒上的抗体与样本中的抗原反应，还是磁微粒上的抗原抗体复合物与酶结合物中的抗体反应形成双抗体夹心“三明治”结构的免疫复合物，反应过程均处于37℃(或其它设定温度)静置状态，此时磁微粒会因为重力影响逐渐下沉，无法一直处于均匀悬浮状态，这就导致抗原、抗体分子之间的反应空间受阻，抗原、抗体分子之间结合不充分，不能在短时间内达到反应平台期，这既会影响样本检测时间，还会影响检测灵敏度。
虽然在CN106546732A、CN104090101A、CN106290864A等专利申请中公开了一些灵敏度高、准确性好的化学发光免疫试剂盒及其检测方法，但是都未能解决上述的问题。
发明内容
针对现有技术存在的不足，本发明的目的之一在于提供一种基于磁棒法的单人份化学发光免疫检测方法，该方法磁珠清洗彻底、携带污染少，混匀充分，适用于单人份化学发光免疫试剂盒，使用灵活、背景噪声低。
本发明的目的之二在于提供一种能实现上述方法的化学发光免疫检测系统。该系统所需仪器体积小巧，可随身携带进入诊疗现场，对实验室样本规模无特殊要求，价格低廉，基本无维护成本，适合基层医疗机构推广化学发光免疫分析技术的使用。
本发明的上述目的采用如下的技术方案实现：
一种基于磁棒法的单人份化学发光免疫检测方法，所用试剂包括：分析缓冲液、磁珠工作液、酶工作液、清洗液1、清洗液2、底物液，该方法包括以下具体步骤：
1)将待测样本加入分析缓冲液，得到混合液A；
2)通过磁吸的方式从磁珠工作液中取出磁珠，加入混合液A中，然后以上下混匀的方式以某种频率震荡，反应一定时间，得到混合液B；
3)通过磁吸的方式从混合液B中移出磁珠，加入清洗液1中，得到混合液C，然后又以某种频率上下震荡混匀清洗；
4)通过磁吸的方式从混合液C中取出磁珠，并加入酶工作液中，然后又以上下混匀的方式以某种频率震荡，反应一定时间，得到混合液D；
5)通过磁吸的方式从混合液D中移出磁珠，加入清洗液2中，得到混合液E，然后又 以某种频率上下震荡混匀清洗；
6)通过磁吸的方式从混合液E中取出磁珠，加入底物液中，以某种频率上下混匀，得到混合液F；
7)将混合液进行发光信号检测。
进一步地，前述的基于磁棒法的单人份化学发光免疫检测方法，所述某种频率选自1次/分钟～360次/分钟。
本发明采用1次/分钟～360次/分钟的上下混匀法，克服了传统仪器偏心力旋涡混匀法存在的以下缺陷：混匀频率不稳定，反应液易飞溅，导致清洗液无法洗涤而出现严重跳点，出现假阳性检测结果；另清洗液的盐成分易渗出腐蚀控制电机，导致电机极易损坏，这会导致电机频繁更换，增加使用成本。
进一步地，前述的基于磁棒法的单人份化学发光免疫检测方法，分析缓冲液、磁珠工作液、酶工作液、清洗液1、清洗液2和底物液的加入体积为1～1000μl。
进一步地，前述的基于磁棒法的单人份化学发光免疫检测方法，步骤2)和4)的反应时间为30秒～60分钟。
进一步地，前述的基于磁棒法的单人份化学发光免疫检测方法，所述磁珠的粒径为0.01μm～50μm。在一次测试中的磁珠可以是同一粒径的磁珠，也可以是不同粒径的混合磁珠。
进一步地，前述的基于磁棒法的单人份化学发光免疫检测方法，步骤7)所述发光信号检测的原理包括但不限于碱性磷酸酶促发光、辣根过氧化物酶促发光、吖啶酯直接发光。
进一步地，前述的基于磁棒法的单人份化学发光免疫检测方法，所述底物液包括但不限于AMPPD、APS-5、CDP-Star、鲁米诺及其衍生物、过氧化氢稀碱溶液。
进一步地，前述的基于磁棒法的单人份化学发光免疫检测方法，所述酶工作液是通过碱性磷酸酶标记、辣根过氧化物酶标记或吖啶酯标记的产物或者其他标记产物。
进一步地，前述的基于磁棒法的单人份化学发光免疫检测方法，所述碱性磷酸酶包括但不限于小牛肠碱性磷酸酶、细菌碱性磷酸酶或胎盘碱性磷酸酶。
本发明的检测方法，最后通过PMT或者包含PMT的分析仪进行发光结果检测。
本发明上述的检测方法可以人工操作也可以通过仪器系统完成。人工操作时，只需要将固定包装的单人份试剂盒按以上步骤进行，不需要从100人份的传统试剂盒中精密吸取1人份剂量进行试验，因此不再依赖传统的大型设备，可以在基层医疗机构灵活实现。当采用仪器检测系统完成上述检测方法时，得益于本发明巧妙的磁珠和液体分离方式，检测系统不再 需要各种繁琐的混匀机构和液体吸取机构，只需要一根磁棒即可完成多个步骤结束后的磁珠和液体分离。而且这种分离方式使得清洗更为彻底，有效地减少清洗次数，节约试剂。其混匀方式使混合液一直处于均匀悬浮状态，磁珠不会下沉，抗原、抗体分子之间的反应自由、畅通，结合充分，短时间内就能达到反应平台期，既可以缩短样本检测时间，还可以提高检测灵敏度。
根据上述，本发明还提供了一种利用上述方法进行单人份化学发光免疫检测的系统，该系统包括试剂模块、磁吸模块、隔离和混匀模块、以及控制模块；所述试剂模块、磁吸模块和混匀模块均与控制模块相连，所述试剂模块包括试剂管组件1和固定试剂管组件的工作平台2，所述磁吸模块包括能够沿水平方向和竖直方向移动的磁棒3、以及驱动磁棒3移动的电机，所述隔离和混匀模块包括能够沿水平方向和竖直方向移动的隔离套7、以及驱动隔离套7移动的电机。
进一步地，上述进行单人份化学发光免疫检测的系统，所述磁吸模块还包括磁棒固定块4、磁棒水平臂5、磁棒竖向臂6和水平臂固定块10，所述磁棒固定块4夹持固定磁棒3，且在电机的驱动下能够沿磁棒水平臂5进行水平移动，所述磁棒水平臂5由水平臂固定块10固定在磁棒竖向臂6上，水平臂固定块10在另一电机的驱动下能够沿磁棒竖向臂6在竖直方向上移动。实现磁棒3沿水平方向和竖直方向移动的方式不限于此，这只是本发明实施例提供的一种方式。
进一步地，上述进行单人份化学发光免疫检测的系统，所述隔离和混匀模块还包括水平伸缩杆8、隔离套竖向臂9和伸缩杆固定块11，所述水平伸缩杆8的一端活动连接隔离套7，另一端通过伸缩杆固定块11固定在隔离套竖向臂9上，水平伸缩杆8在电机驱动下进行水平伸缩使隔离套7在水平方向上移动，伸缩杆固定块11在另一电机驱动下沿隔离套竖向臂9在竖直方向上移动。实现隔离套7沿水平方向和竖直方向移动的方式不限于此，这只是本发明实施例提供的一种方式。
进一步地，上述进行单人份化学发光免疫检测的系统，还包括与控制模块相连的检测模块，所述检测模块包括检测抓手、检测抓手电机、具有孔位的检测盘和PMT分析仪。
本发明的技术方案具有以下优点：
1.基于磁棒法的单人份化学发光免疫检测方法彻底解决了传统检测方法携带污染的问题。由于检测背景值低，仅需较低的信号值，即能实现较高信噪比，提高检测灵敏度：每个测试的反应、清洗、检测等步骤均在不同反应管里进行，清洗步骤只需要1～2遍，清洗方式为通过磁棒把磁珠往分别装有样本、洗液、酶工作液等溶液的不同反应管里移动、放入和吸 出，这样就完全不存在管壁吸附和液体残留的问题，磁珠清洗效果更彻底，且均采用一次性耗材，这就完全避免了携带污染的存在。正是由于清洗彻底、干净，检测背景值低，仅需较低的信号值，即能实现较高信噪比，提高检测灵敏度。
2.基于磁棒法的单人份化学发光免疫检测方法实现了对反应液的持续混匀。无论是磁微粒上的抗体与样本中的抗原反应，还是磁微粒上的抗原抗体复合物与酶结合物中的抗体反应形成双抗体夹心“三明治”结构的免疫复合物，整个反应过程均处于37℃(或其它设定温度)持续混匀状态，此时磁微粒一直处于均匀悬浮状态，不会下沉，使抗原、抗体分子之间的反应自由、畅通，抗原、抗体分子之间结合充分，在短时间内就能达到反应平台期，既可以缩短样本检测时间，还可以提高检测灵敏度。
3.基于磁棒法的单人份化学发光免疫检测方法采用单人份、独立包装、随拆随用的方式，为社区、乡镇等基层医院量身定做：试剂盒采用单人份、独立包装的方式，对医院日均样本量无任何要求，病人随到随测，检测一位病人就开封1人份试剂，完全不存在一盒试剂反复使用造成的试剂组份挥发、试剂各组份之间交叉污染的问题，更不会造成医院和厂家的资源浪费。
4.基于磁棒法的单人份化学发光免疫检测系统省略了传统系统的多个转移装置、清洗装置、旋转装置和它们对应的电机，体积小巧，操作简单，对时序性的要求降低，普通人员即可操作和使用。且仪器为台式机，对存放地点和场地根本没任何要求。
附图说明
图1是现有技术CN107831322A公开的全自动化学发光免疫分析仪器结构示意图；
图2是现有技术CN108226549A公开的用于化学发光免疫分析仪的时序控制系统结构示意图；
图3是本发明的实施例的一种基于磁棒法的单人份化学发光免疫检测系统主要结构示意图；
1-试剂管组件，2-工作平台，3-磁棒，4-磁棒固定块，5-磁棒水平臂，6-磁棒竖向臂，7-隔离套，8-水平伸缩杆，9-隔离套竖向臂，10-水平臂固定块，11-伸缩杆固定块。
具体实施方式
以下通过具体实施例对本发明的发明内容做进一步的阐释，本领域技术人员应当知晓，本发明基于磁棒法的单人份化学发光免疫检测方法可用于多种化学发光检测中，而不限于下述试验例的HIV、HCV和TP；实现本发明化学发光免疫检测方法的系统可以根据实际需 要将已有的一些功能模块组合到下述实施例中，这些都是基本发明容易获得的，仍属于本发明保护的范畴。
实施例1
一种基于磁棒法的单人份化学发光免疫检测系统，包括试剂模块、磁吸模块、隔离和混匀模块、以及控制模块；所述试剂模块、磁吸模块、隔离和混匀模块均与控制模块相连。所述试剂模块包括试剂管组件1和固定试剂管组件的工作平台2，所述磁吸模块包括能够沿水平方向和竖直方向移动的磁棒3、以及驱动磁棒3移动的电机，所述隔离和混匀模块包括能够沿水平方向和竖直方向移动的隔离套7、以及驱动隔离套7移动的电机。
参考图3，在控制系统的控制下，磁棒3和隔离套7在对应电机的驱动下进行移位，首先到磁珠工作液试剂管的正上方，经过竖直移动，磁棒3伸入隔离套7内，再进一步伸入磁珠工作液试剂管内，吸取磁珠。然后磁棒3和隔离套7同时竖直向上抬起并移动到加了样本的分析缓冲液试剂管内，磁棒3抬起，磁珠留在管内，隔离套7在电机驱动下做一定幅度和频率的上下震荡，混匀。然后磁棒3伸入管内，吸附磁珠，再与隔离套7一起向上，将磁珠转移到下一个试剂管内，直至反应和清洗结束。
作为实现磁棒3实现沿水平方向和竖直方向移动的一种方式，所述磁吸模块还包括磁棒固定块4、磁棒水平臂5、磁棒竖向臂6和水平臂固定块10，所述磁棒固定块4夹持固定磁棒3，且在电机的驱动下能够沿磁棒水平臂5进行水平移动，所述磁棒水平臂5由水平臂固定块10固定在磁棒竖向臂6上，水平臂固定块10在另一电机的驱动下能够沿磁棒竖向臂6在竖直方向上移动。
作为实现隔离套7实现沿水平方向和竖直方向移动的一种方式，所述隔离和混匀模块还包括水平伸缩杆8、隔离套竖向臂9和伸缩杆固定块11，所述水平伸缩杆8的一端活动连接隔离套7，另一端通过伸缩杆固定块11固定在隔离套竖向臂9上，水平伸缩杆8在电机驱动下进行水平伸缩使隔离套7在水平方向上移动，伸缩杆固定块11在另一电机驱动下沿隔离套竖向臂9在竖直方向上移动。活动连接的隔离套7在每次测试后取下更换，为一次性耗材。
作为优选的实施例方式，上述进行单人份化学发光免疫检测的系统还包括与控制模块相连的检测模块，所述检测模块包括检测抓手、检测抓手电机、具有孔位的检测盘和PMT分析仪。
试验例1
基于磁棒法的HIV 1+2型抗体单人份化学发光检测方法(碱性磷酸酶法)
HIV 1+2型抗体单人份试剂由成都博奥新景医学科技有限公司提供。试剂组份包含：分析缓冲液、磁珠工作液、酶工作液、清洗液1、清洗液2、底物液，检测方法可以通过实施例1的检测系统实现，也可以人工操作实现。用检测系统时，分析缓冲液、酶工作液、清洗液1、清洗液2、底物液事先放置在试剂管组件中。该方法具体包括以下步骤：
第一步：将待测样本加入分析缓冲液里，得到混合液A。(吸取样本放入试剂管组件的分析缓冲液管内，然后将试剂管组件固定在工作平台2上。)
第二步：通过磁吸的方式从HIV 1+2型抗体试剂的磁珠工作液中取出磁珠，并加入混合液A中，然后以上下混匀的方式，以120次/分钟的频率震荡，反应5分钟，得到混合液B。(磁棒3和隔离套7在电机驱动下移动到磁珠工作液管上方，向下伸入管内，然后磁棒3吸取磁珠，再转移到分析缓冲液管内，磁棒3竖直向上抬起，磁珠落下，隔离套7在电机驱动下以120次/分钟的频率上小震荡反应5分钟。)
第三步：再从反应混合液B中通过磁吸的方式移出磁珠，加入清洗液1中，得到混合液C，然后又以180次/分钟的频率进行上下震荡混匀清洗。(磁棒向下伸入隔离套7，吸取管内磁珠，与隔离套7一起转移磁珠到清洗液1管内，磁棒3竖直向上抬起，磁珠落下，隔离套7在电机驱动下以180次/分钟的频率进行上下震荡清洗。)
第四步：通过磁吸的方式从混合液C中取出磁珠，并加入HIV 1+2型抗体试剂酶工作液中，然后又以上下混匀的方式，以120次/分钟的频率震荡混匀，反应15分钟，得到混合液D。(磁棒向下伸入隔离套7，吸取管内磁珠，与隔离套7一起转移磁珠到酶工作液管内，磁棒3竖直向上抬起，磁珠落下，隔离套7在电机驱动下以120次/分钟的频率上下震荡混匀反应15分钟。)
第五步：再从反应混合液D中通过磁吸的方式移出磁珠，加入清洗液2中，得到混合液E，然后又以180次/分钟的频率进行上下震荡混匀清洗。(磁棒向下伸入隔离套7，吸取管内磁珠，与隔离套7一起转移磁珠到清洗液2管内，磁棒3竖直向上抬起，磁珠落下，隔离套7在电机驱动下以180次/分钟上下震荡混匀清洗。)
第六步：通过磁吸的方式从混合液E中取出磁珠，移到底物液中，以上下混匀的方式，以120次/分钟的频率震荡混匀，得到混合液F，然后进行发光信号检测。(磁棒向下伸入隔离套7，吸取管内磁珠，与隔离套7一起转移磁珠到底物液管内，磁棒3竖直向上抬起，磁珠落下，隔离套7在电机驱动下以120次/分钟的频率震荡混匀。)
第七步：将混合液进行发光信号检测。(检测抓手抓取底物液管放到检测盘的孔内，用 PMT分析仪进行信号检测。)
用北京科美生物技术有限公司的人类免疫缺陷病毒抗体诊断试剂盒(化学发光法)和CHEMCLIN 1500全自动化学发光免疫分析仪检测的结果作为参照，以考察本发明检测方法的准确性。
检测结果如下：
Figure PCTCN2019085653-appb-000001
结论：经磁棒法HIV 1+2型抗体单人份化学发光法检测，样本阴阳性检测结果与参照厂家检测结果完全一致。
试验例2
基于磁棒法的HIV-1 p24抗原单人份化学发光检测方法(碱性磷酸酶法)
HIV-1 p24抗原试剂由成都博奥新景医学科技有限公司提供。试剂组份包含：分析缓冲液、磁珠工作液、酶工作液、清洗液1、清洗液2、底物液，具体步骤如下，当采用系统完成时，与试验例1类似：
第一步：将待测样本加入分析缓冲液里，得到混合液A。
第二步：通过磁吸的方式从HIV-1 p24抗原试剂的磁珠工作液中取出磁珠，并加入混合 液A中，然后以上下混匀的方式，以120次/分钟的频率震荡，反应5分钟，得到混合液B。
第三步：再从反应混合液B中通过磁吸的方式移出磁珠，加入清洗液1中，得到混合液C，然后又以180次/分钟的频率进行上下震荡混匀清洗。
第四步：通过磁吸的方式从混合液C中取出磁珠，并加入HIV-1 p24抗原试剂酶工作液中，然后又以上下混匀的方式，以120次/分钟的频率震荡混匀，反应15分钟，得到混合液D。
第五步：再从反应混合液D中通过磁吸的方式移出磁珠，加入清洗液2中，得到混合液E，然后又以180次/分钟的频率进行上下震荡混匀清洗。
第六步：通过磁吸的方式从混合液E中取出磁珠，移到底物液中，以上下混匀的方式，以120次/分钟的频率震荡混匀，得到混合液F，然后进行发光信号检测。
第七步：将混合液进行发光信号检测。
用HIV-1 p24抗原国家参考品(购自中国食品药品检定研究院、货号：22015-20150505)的检测结果作为参照，以考察本发明检测方法的准确性。
检测结果如下：
Figure PCTCN2019085653-appb-000002
Figure PCTCN2019085653-appb-000003
结论：经磁棒法HIV-1 p24抗原单人份化学发光法检测，样本阴阳性结果与HIV-1 p24抗原国家参考品的要求完全一致。
试验例3
基于磁棒法的anti-HCV单人份化学发光检测方法(碱性磷酸酶法)
anti-HCV试剂由成都博奥新景医学科技有限公司提供。试剂组份包含：分析缓冲液、磁珠工作液、酶工作液、清洗液1、清洗液2、底物液，操作与试验例1类似，具体步骤如下，当采用系统完成时，与试验例1类似：
第一步：将待测样本加入分析缓冲液里，得到混合液A。
第二步：通过磁吸的方式从anti-HCV试剂的磁珠工作液中取出磁珠，并加入混合液A中，然后以上下混匀的方式，以120次/分钟的频率震荡，反应5分钟，得到混合液B。
第三步：再从反应混合液B中通过磁吸的方式移出磁珠，加入清洗液1中，得到混合液C，然后又以180次/分钟的频率进行上下震荡混匀清洗。
第四步：通过磁吸的方式从混合液C中取出磁珠，并加入anti-HCV试剂酶工作液中，然后又以上下混匀的方式，以120次/分钟的频率震荡混匀，反应15分钟，得到混合液D。
第五步：再从反应混合液D中通过磁吸的方式移出磁珠，加入清洗液2中，得到混合液E，然后又以180次/分钟的频率进行上下震荡混匀清洗。
第六步：通过磁吸的方式从混合液E中取出磁珠，移到底物液中，以上下混匀的方式，以120次/分钟的频率震荡混匀，得到混合液F，然后进行发光信号检测。
第七步：结果检测。通过半自动化学发光免疫分析仪进行结果检测。
用雅培的丙型肝炎病毒抗体测定试剂盒(化学发光微粒子免疫检测法)、雅培ARCHITECT i2000 SR检测的结果作为参照，以考察本发明检测方法的准确性。
检测结果如下：
Figure PCTCN2019085653-appb-000004
Figure PCTCN2019085653-appb-000005
结论：经磁棒法anti-HCV单人份化学发光法检测，样本阴阳性结果与比对厂家检测结果完全一致。
试验例4
基于磁棒法的anti-TP单人份化学发光检测方法(碱性磷酸酶法)
anti-TP试剂来自成都博奥新景医学科技有限公司。试剂组份包含：酶工作液1、磁珠工作液、酶工作液2、清洗液1、清洗液2、底物液，操作与试验例1类似，具体步骤如下，当采用系统完成时，与试验例1类似：
第一步：将待测样本加入酶工作液1里，得到混合液A。
第二步：通过磁吸的方式从anti-TP试剂的磁珠工作液中取出磁珠，并加入混合液A中，然后以上下混匀的方式，以120次/分钟的频率震荡，反应5分钟，得到混合液B。
第三步：再从反应混合液B中通过磁吸的方式移出磁珠，加入清洗液1中，得到混合液C，然后又以180次/分钟的频率进行上下震荡混匀清洗。
第四步：通过磁吸的方式从混合液C中取出磁珠，并加入anti-TP试剂酶工作液中，然后又以上下混匀的方式，以120次/分钟的频率震荡混匀，反应15分钟，得到混合液D。
第五步：再从反应混合液D中通过磁吸的方式移出磁珠，加入清洗液2中，得到混合液E，然后又以180次/分钟的频率进行上下震荡混匀清洗。
第六步：通过磁吸的方式从混合液E中取出磁珠，移到底物液中，以上下混匀的方式， 以120次/分钟的频率震荡混匀，得到混合液F，然后进行发光信号检测。
第七步：结果检测。通过半自动化学发光免疫分析仪进行结果检测。
用北京科美生物技术有限公司的梅毒螺旋体抗体诊断试剂盒(化学发光法)、CHEMCLIN 1500全自动化学发光免疫分析仪检测的结果作为参照，以考察本发明检测方法的准确性。
检测结果如下：
Figure PCTCN2019085653-appb-000006
结论：经磁棒法anti-TP单人份化学发光法检测，样本阴阳性结果与比对厂家检测结果完全一致。
当用其他厂家方法检测样本时，因为厂家的试剂是100人份，多次使用均未用完，用开封后20+天的试剂进行检测，其检测结果表现出比本发明检测结果更高的噪声背景。其原因包括磁珠吸取转移方式不同以及试剂受时间影响发生变化。本发明方法具有更高的灵敏性。
以上试验例表明本发明的检测方法获得的检测结果是准确的，能够与多个厂家的检测结果保持高度一致。而且，本发明的检测方法携带污染低，清洗次数少，简化了程序，降低了软件控制和工作人员操作的难度。由于本发明方法的清洗方式和单人份检测的特点，有效降 低了污染物携带和试剂变化产生的噪声信号，更有助于灵敏度的提高。此外，采用本发明检测方法的检测系统，节约了多个转移机构、混匀机构和对应的电机，缩小了仪器的体积，使庞然大物般的传统检测仪器小型化，可以应用到面积有限的更多基层医疗机构中，而且检测时间更短。

Claims (10)

  1. 一种基于磁棒法的单人份化学发光免疫检测方法，所用试剂包括：分析缓冲液、磁珠工作液、酶工作液、清洗液1、清洗液2、底物液，其特征在于，该方法包括以下步骤：
    1)将待测样本加入分析缓冲液，得到混合液A；
    2)通过磁吸的方式从磁珠工作液中取出磁珠，加入混合液A中，然后以上下混匀的方式以某种频率震荡，反应一定时间，得到混合液B；
    3)通过磁吸的方式从混合液B中移出磁珠，加入清洗液1中，得到混合液C，然后又以某种频率上下震荡混匀清洗；
    4)通过磁吸的方式从混合液C中取出磁珠，并加入酶工作液中，然后又以上下混匀的方式以某种频率震荡，反应一定时间，得到混合液D；
    5)通过磁吸的方式从混合液D中移出磁珠，加入清洗液2中，得到混合液E，然后又以某种频率上下震荡混匀清洗；
    6)通过磁吸的方式从混合液E中取出磁珠，加入底物液中，以某种频率上下混匀，得到混合液F；
    7)将混合液F进行发光信号检测。
  2. 根据权利要求1所述的基于磁棒法的单人份化学发光免疫检测方法，其特征在于，所述某种频率为1次/分钟～360次/分钟；分析缓冲液、磁珠工作液、酶工作液、清洗液1、清洗液2和底物液的加入体积为1～1000μl。
  3. 根据权利要求1所述的基于磁棒法的单人份化学发光免疫检测方法，其特征在于，步骤2)和4)的反应时间为30秒～60分钟；所述磁珠的粒径为0.01μm～50μm。
  4. 根据权利要求1所述的基于磁棒法的单人份化学发光免疫检测方法，其特征在于，步骤7)所述发光信号检测的原理为碱性磷酸酶促发光、辣根过氧化物酶促发光或吖啶酯直接发光；所述酶工作液是通过碱性磷酸酶标记、辣根过氧化物酶标记或吖啶酯标记的产物或者其他标记产物。
  5. 根据权利要求1所述的基于磁棒法的单人份化学发光免疫检测方法，其特征在于，所述底物液为AMPPD、APS-5、CDP-Star、鲁米诺及其衍生物或过氧化氢稀碱溶液。
  6. 根据权利要求4所述的基于磁棒法的单人份化学发光免疫检测方法，其特征在于，所述碱性磷酸酶为小牛肠碱性磷酸酶、细菌碱性磷酸酶或胎盘碱性磷酸酶。
  7. 一种利用权利要求1～6任意一项所述的方法进行化学发光免疫检测的系统，其特征在于，该系统包括试剂模块、磁吸模块、隔离和混匀模块、以及控制模块；所述试剂模块、磁吸模块、隔离和混匀模块均与控制模块相连，所述试剂模块包括试剂管组件 (1)和固定试剂管组件(1)的工作平台(2)，所述磁吸模块包括能够沿水平方向和竖直方向移动的磁棒(3)、以及驱动磁棒(3)移动的电机，所述隔离和混匀模块包括能够沿水平方向和竖直方向移动的隔离套(7)、以及驱动隔离套(7)移动的电机。
  8. 根据权利要求7所述的进行化学发光免疫检测的系统，其特征在于，所述磁吸模块还包括磁棒固定块(4)、磁棒水平臂(5)、磁棒竖向臂(6)和水平臂固定块(10)，所述磁棒固定块(4)夹持固定磁棒(3)，且在电机的驱动下能够沿磁棒水平臂(5)进行水平移动，所述磁棒水平臂(5)由水平臂固定块(10)固定在磁棒竖向臂(6)上，水平臂固定块(10)在另一电机的驱动下能够沿磁棒竖向臂(6)在竖直方向上移动。
  9. 根据权利要求7所述的进行化学发光免疫检测的系统，其特征在于，所述隔离和混匀模块还包括水平伸缩杆(8)、隔离套竖向臂(9)和伸缩杆固定块(11)，所述水平伸缩杆(8)的一端活动连接隔离套(7)，另一端通过伸缩杆固定块(11)固定在隔离套竖向臂(9)上，水平伸缩杆(8)在电机驱动下进行水平伸缩使隔离套(7)在水平方向上移动，伸缩杆固定块(11)在另一电机驱动下沿隔离套竖向臂(9)在竖直方向上移动。
  10. 根据权利要求7～9任意一项所述的进行化学发光免疫检测的系统，其特征在于，该系统还包括与控制模块相连的检测模块，所述检测模块包括检测抓手、检测抓手电机、具有孔位的检测盘和PMT分析仪。
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