WO2023077909A1 - 可滤血色素冻干球均相发光免疫检测试剂盒及其制备方法 - Google Patents

可滤血色素冻干球均相发光免疫检测试剂盒及其制备方法 Download PDF

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WO2023077909A1
WO2023077909A1 PCT/CN2022/113365 CN2022113365W WO2023077909A1 WO 2023077909 A1 WO2023077909 A1 WO 2023077909A1 CN 2022113365 W CN2022113365 W CN 2022113365W WO 2023077909 A1 WO2023077909 A1 WO 2023077909A1
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freeze
reagent
dried
sample
blood
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PCT/CN2022/113365
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English (en)
French (fr)
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陈东慧
张国锋
张伟
杨苏清
敖艳艳
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厦门宝太生物科技股份有限公司
深圳宝恒投资有限公司
上海宝太生物科技有限公司
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Publication of WO2023077909A1 publication Critical patent/WO2023077909A1/zh

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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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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
    • 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
    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin

Definitions

  • the invention belongs to the technical field of immunoassay kits, in particular to a filterable hemoglobin freeze-dried sphere homogeneous luminescent immunoassay kit and a preparation method thereof.
  • Light-excited homogeneous luminescence is to connect antigens, antibodies or gene probes to acceptor microspheres and donor microspheres respectively. When specific molecular binding occurs, the two microspheres are connected together. If there is a specific When the light source irradiates the reaction system, the photosensitizer contained in one of the donor microspheres will convert the ambient oxygen into excited singlet oxygen, and the singlet oxygen can oxidize the luminescent agent of another acceptor microsphere to emit light; if The two microspheres are not bound, and due to the distance, the shorter-lived singlet oxygen cannot reach the other acceptor microsphere and cause it to emit light. Therefore, the optically excited homogeneous luminescence technology shows great advantages, such as simple operation, fast test speed, good precision, and reduces the pipeline cleaning system, greatly reducing the size of the instrument and the cost of maintenance and consumables.
  • the labeling immunoassay technology is still based on the solid-phase method, that is, the immune complex is formed on the solid-phase carrier, and the unbound reactants and reaction substrates are removed by washing, while the light-excited homogeneous luminescence technology is characterized by no need for separation
  • solid-phase immunoassays have the advantages of high detection sensitivity and wide linear range, the disadvantages are that the reaction time is long, the operation steps are many, and the precision of detection results is not as good as that of homogeneous reactions.
  • Chinese patent CN101750487B discloses a dry photochemiluminescence immunoassay kit.
  • Trehalose needs to be added to the microparticle luminescent solution to support the setting effect during the drying process and prevent dehydration between reagent molecules, but thus increases the cost.
  • the process is complicated.
  • the kit needs to undergo two-step incubation before photon signal detection during use, which increases the error in the manual sample addition process.
  • Chinese patent CN113092767A discloses a limulus reagent freeze-dried microsphere and its preparation method and application. The freeze-drying process of the microsphere requires temperature control for pre-freezing, which increases the difficulty of operation.
  • the conventional light-excited homogeneous luminescent reagents on the market are mainly liquid-phase products, and the transportation and storage stability of liquid reagents are greatly limited.
  • the present invention provides a homogeneous luminescent immunoassay kit for filterable hemoglobin freeze-dried spheres and a preparation method thereof;
  • the donor microspheres coated with avidin and filled with phthalocyanine derivatives can generate singlet oxygen.
  • the donor microspheres are connected with biotin-labeled antibodies or antigens
  • the detection object is further connected with the receptor microspheres coated with antibodies or antigens
  • the singlet oxygen can be transferred to the receptor microspheres, and chemically react with the dimethylthiophene filled on the receptor microspheres to produce Ultraviolet light, ultraviolet light further excites the europium chelate filled on the acceptor microspheres to generate photons; if the acceptor and donor microspheres are not combined by the detection object, the singlet oxygen cannot reach the acceptor because the transmission distance is too far Microspheres make it glow.
  • the reagent detection card of the present invention includes a reaction part and a blood filtering part
  • the lower end of the filtering part is connected to the middle part of the reaction part through a channel;
  • the blood filtering part has a compound blood filtering device, and the compound blood filtering device includes a blood filtering membrane and a filter element;
  • the blood filtering device includes a blood filtering membrane and a filter element, wherein the filter element is a polyethylene sintered filter element with a lower pore diameter of about 2-10 ⁇ m, a diameter of 4.5-4.7 mm, and a thickness of 2-3 mm, and a filter element with a pore size of 1-2 ⁇ m is installed above the filter element.
  • Blood film, the position of the filter element and the blood filter membrane in the blood filtration device can be interchanged, and the device can directly add samples containing red blood cells to be tested, and the impurities and blood cells that do not participate in the reaction such as flocculent particles in the sample to filter.
  • the filterable hemoglobin freeze-dried sphere homogeneous luminescent immunoassay kit consists of a reagent detection card, lyophilized spheres and sample injection diluent;
  • sample injection diluent is in the form of adding preservatives to physiological saline
  • the preparation method of the filterable hemoglobin freeze-dried sphere homogeneous luminescence immunoassay kit is as follows:
  • marker reagents respectively mixing acceptor microspheres coated with antibodies or antigens, biotin-labeled antibodies or antigens, donor microspheres coated with avidin and buffer;
  • step 2) Freeze-drying: use the three marker reagents in step 1) of the bead dropping machine to drop into small droplets through the bead dropping machine. Transfer directly to an aluminum pan, stainless steel pan or vial that has been precooled with liquid nitrogen, and then transfer to a lyophilizer for freeze-drying;
  • Sorting Take one of the three kinds of freeze-dried pellets and pack them into the reagent test card, seal the reagent card after packing, put the reagent card into a sealed bag with desiccant and seal it.
  • the markers in step 1) are biotin-labeled antibody/antigen, acceptor microspheres coated with antibody/antigen, and donor microspheres coated with avidin.
  • the labeling process of the three markers is as follows :
  • Biotin-labeled antibody/antigen take a certain volume of 0.05M pH9.5 CB buffer, and put in a certain molar ratio of biological aminocaproyl-6-aminocaproic acid N-hydroxysuccinimide ester and antibody/ Antigen, after reacting at 25°C for 2 hours, add 1% glycine to block for 30 minutes, dialyze in 0.01M PBS buffer with pH7.4 until no residual biotin is removed, and use Thermo protein detector to detect protein concentration;
  • the buffer in the step 1) is one or a combination of PB, MES or TRIS systems containing sodium chloride, Tween20, bovine serum albumin, Proclin-300 and deionized water;
  • the concentration of NaCl affects the temperature and time of sublimation drying by affecting the freeze-drying eutectic point, further affects the shape of the freeze-dried pellets, and finally affects the detection results of light-excited chemiluminescence. Therefore, the present invention optimizes NaCl content;
  • the NaCl addition amount of the buffer solution in the step 1) is 0-0.9%;
  • the buffer in the step 1) can also be added with a protective agent of dextran 20000, ascorbic acid, casein, lysine, sorbitol, sucrose, trehalose, glucose, Tween80, Tween20, Triton X-405, Triton X One or more of -100, casein sodium salt, glycine, PEG20000, PRG8000, PEG6000, PEG2000, PEG200, PVP29000, PVP40, PVP10.
  • the liquid nitrogen frozen pellets are placed in an aluminum pan, a stainless steel pan or a vial, and then transferred to a freeze dryer, directly vacuumed and freeze-dried at -40 to -50°C, and finally pass through the eutectic point Determine the best process conditions.
  • the present invention has studied the influence of not using pigments on the detection kit, and selected the pigments that do not affect the detection kit to add the freeze-dried bead pigments.
  • the purpose is to distinguish the three components, so as to facilitate packaging, Preservation, the selected pigments in the present invention are warm-toned color systems: red, orange, and yellow, and the pigments added can be phenol red, phenol red sodium salt, sunset yellow or tartrazine.
  • the sorting environment in step 3) is as follows: the temperature is below 25° C., the humidity is below 30%, and the sorting time is not affected within 0-8 hours.
  • the filterable hemoglobin freeze-dried sphere homogeneous luminescence immunoassay kit its reaction mode is: 10-50 ⁇ L sample + 150 ⁇ L sample injection diluent, and three kinds of lyophilized spheres are reconstituted after suction filtration;
  • the reagent reaction mode after reconstitution of the freeze-dried beads is: 10-50 ⁇ L sample + 50 ⁇ L biotin-labeled antibody/antigen working solution + 50 ⁇ L receptor microspheres coated with antibody/antigen working solution + 50 ⁇ L bag Working solution of donor microspheres coated with avidin;
  • the coated protein can be antibody or antigen, which is mainly distinguished according to the method of project selection: 1 If the detected protein is a macromolecular antigen, the double-antibody sandwich method is used (See Figure 2), at this time, the receptor microspheres are coated with antibodies and biotin-labeled antibodies, and the reaction mode is 10-50 ⁇ L sample + 50 ⁇ L biotin-labeled antibody working solution + 50 ⁇ L receptor microspheres coated with antibody working solution + 50 ⁇ L of donor microsphere working solution coated with avidin; 2 If the detected protein is a small molecule antigen, use the competition method (see Figure 3).
  • the reaction mode is 10-50 ⁇ L sample + 50 ⁇ L biotin-labeled antibody working solution + 50 ⁇ L antigen-coated acceptor microsphere working solution + 50 ⁇ L avidin-coated donor microsphere working solution.
  • Receptor microspheres coated with antibodies, biotin-labeled antigens the reaction mode is 10-50 ⁇ L sample + 50 ⁇ L biotin-labeled antigen working solution + 50 ⁇ L receptor microspheres coated with antibody working solution + 50 ⁇ L coated with avidin Donor microsphere working solution.
  • the kit of the present invention uses 50 ⁇ L of three different working solutions containing microspheres and biotin labels to make dry reagents in the form of freeze-drying or drying. It has been verified that the freeze-drying form is superior In the form of drying, the reconstitution is relatively simple and the structure of the protein is not easily damaged; on the other hand, if the 50 ⁇ L working solution is directly freeze-dried into pellets, the volume is too large, resulting in poor surface morphology of the freeze-dried pellets, so , the present invention concentrates 50 ⁇ L of reagent components by 1.6-5 times, and after the concentrated working solution is frozen into small balls, each small ball is then diluted to 1X working concentration by adding 50 ⁇ L of injection diluent for further testing, and at the same time , in order to match the volume of three kinds of small balls that can be placed in the detection hole at the same time, so the volume of the freeze-dried small balls required by the present invention is 10
  • test kit of the present invention is:
  • the reagent detection card is provided with a handle, which is convenient for the operator to hold and operate, and a barcode is provided on one side of the reagent card, which is convenient for instrument identification and automation; the freeze-dried beads are loaded into the reaction detection hole of the reagent detection card, and the sample is tested. , remove the medical easy-to-tear film above the reagent card, add the sample to the filter hole of the reagent test card, then add an appropriate amount of sample injection diluent, put the reagent test card into the instrument, and the instrument draws negative pressure on the reaction test hole.
  • the detection pressure and time during the negative pressure pumping process should ensure that the blood filtration membrane will not be broken during the suction filtration process.
  • the instrument shakes the reagent card for a few seconds, and incubates in one step. After the incubation, read the photon signal, calculate the concentration of the sample to be tested according to the standard curve, and directly obtain the quantitative detection result of the sample.
  • the detection process See attached picture 1.
  • the sample and sample injection diluent are suction-filtered to the detection hole through the filter device, and the three concentrated beads in the detection hole are diluted into a working concentration liquid and react with the sample. Since the sample injection diluent is In order to dilute the three concentrated freeze-dried pellets, the different components of the injection diluent have a great influence on the reconstitution of the freeze-dried pellets and the detection performance of the sample.
  • the specific impact process is as follows:
  • the present invention optimizes the composition and content of the injection diluent.
  • PB system buffer-protectant composition is PB concentrate + 0-0.5% dextran 20000, 0-5% sucrose, 0-0.5% Triton X-405, 0-0.5% Triton X-100, 0- 0.5% PEG20000, 0-0.5% PEG8000, 0-0.5% PEG6000, 0-0.5% PEG2000, 0-5% trehalose, 0-4% glycine, 0-5% mannitol or 0-5% glucose;
  • MES system buffer-protectant composition is MES concentrate + 0-0.5% dextran 20000, 0-5% sucrose, 0-0.5% Triton X-405, 0-0.5% Triton X-100, 0- 0.5% PEG20000, 0-0.5% PEG8000, 0-0.5% PEG6000, 0-0.5% PEG2000, 0-5% trehalose, 0-4% glycine, 0-5% mannitol, 0-5% sorbitol or 0 -5% glucose;
  • TRIS system buffer-protectant composition is TRIS concentrate + 0-5% sucrose, 0-0.5% TX100;
  • each one needs to be taken and packed into the detection hole of the reagent test card.
  • the pellets placed in the environment will be affected by the environment. After research It was found that the freeze-dried pellets placed in an environment with a temperature ⁇ 25°C and a humidity ⁇ 30% for 8 hours had no effect on the performance of the pellets, while freeze-dried pellets placed in a humidity > 30% and a temperature > 25°C were easily deliquescent. , thereby affecting the storage stability of the pellets.
  • a homogeneous luminescence immunoassay kit for leachable hemoglobin lyophilized spheres contains lyophilized spheres of three components, and the working solution containing the three components is concentrated by 1.6 to 5 times, and the volume is controlled In 10-30 ⁇ L, the CV of the obtained freeze-dried pellets can still reach below 6%;
  • freeze-dried pellets of the present invention are screened by freeze-drying boxes of different materials. Compared with stainless steel freeze-drying trays and vials, the freeze-dried pellets prepared by aluminum product freeze-drying boxes have a better shape;
  • the humidity of the sorting environment adopted is less than or equal to 30%, and the temperature is less than or equal to 25°C.
  • the sorting time can be extended to 8h;
  • sucrose can be added to the buffer in the freeze-drying process of the present invention as a protective agent or even the freeze-dried pellets prepared without the addition of a protective agent have smooth performance and shape, are not easy to produce debris, and have good high temperature stability. cost savings;
  • the present invention optimizes the NaCl concentration in the buffer system, wherein the freeze-drying eutectic point produced by NaCl with a buffer solution of 0 to 0.9% mass concentration is higher, which improves the temperature of sublimation drying in the vacuuming process, The freeze-drying time is shortened, the shape of the freeze-dried pellets is stable, and the cost is saved at the same time;
  • the present invention adopts the freeze-dried pellets that do not use pigments to distinguish the three components, so as to facilitate packaging and preservation;
  • the test method adopted by the kit prepared by the present invention is to inject samples on the left side of the reagent card, pump and filter on the right side and directly enter the right side detection cup to redissolve three small balls, and incubate and detect in one step, which shortens the time and also The addition steps of liquid reagents are reduced, the instrument does not need pipeline cleaning, and the requirements for instruments and equipment are reduced.
  • Figure 2 is the double-antibody sandwich detection mode
  • A, B, C, D, F are the donor microspheres coated with avidin, the biotin-labeled antibody, the antigen to be tested, and the acceptor microspheres coated with the antibody. spheres, immune complexes.
  • Figure 3 is the detection mode of the competition method
  • a, b, c, d, f1, and f2 are the donor microspheres coated with avidin, the antibody labeled with biotin, the antigen to be tested, and the acceptor coated with the antigen, respectively Microspheres, antigen competition binding immune complexes, antigen binding immune complexes to be tested.
  • Figure 4 is a blood filtration device.
  • NT-proBNP amino-terminal brain natriuretic peptide precursor
  • the working liquid drops in the form of small droplets through the bead dropping machine, and the dropped droplets are in the shape of small balls after being frozen by liquid nitrogen.
  • step 4 Transfer the liquid nitrogen frozen pellets obtained in step 4) to a freeze dryer that has been pre-frozen to -40°C for freeze-drying. After drying, sort the pellets in an environment with a humidity below 30% and a temperature below 25°C.
  • One of the three kinds of freeze-dried pellets was divided into the reaction detection hole of the reagent test card, and the reagent card was sealed after the division was completed, and the reagent card was put into a sealed bag containing a desiccant for sealing.
  • Table 1 show that the luminescence value of the pigment freeze-dried pellet reagent has a significant correlation with the concentration of NT-proBNP antigen. As the concentration of NT-proBNP antigen increases, the luminescence value gradually increases, and the intra-assay precision is less than 10 %, can be used for the detection of NT-proBNP antigen.
  • Reagent test card stability The stability of the freeze-dried pellets stored at 37°C for 28 days was measured, and the NT-proBNP antigen calibration products with concentrations of 20000pg/mL, 5000pg/mL and 300pg/mL were respectively, Add 10 ⁇ L to the filter holes of the reagent test card, add 150 ⁇ L of diluent to each sample in 3 wells, incubate at 37°C for 10 minutes, and read with Reader. The average luminescence values obtained are shown in Table 2;
  • the hemofiltration device of the reagent test card in the kit was optimized, in which the hemofiltration device adopted different filling forms, and the three different filling forms were compared for the effect of filtering red blood cells and the degree of difficulty of loading.
  • the results are shown in Table 3;
  • the filter element is a relatively hard material sintered with polyethylene and has a certain pore size.
  • the problem of large size and inhomogeneity can not play a good role in blood filtration, and the effect of blood filtration is poor; the pore size principle of blood filtration can intercept red blood cells, and the device using the blood filtration membrane alone needs to use a suitable suction pressure to obtain better filtration.
  • the blood filtration membrane itself is a soft and thin material, and it is difficult to fill the reagent hole evenly. It is difficult to load the blood filtration membrane alone, and after filling, the pressure of the suction filtration is too high.
  • the blood film ruptures or collapses inwardly; therefore, as can be seen from the results in Table 3, the filtered erythrocytes obtained in the composite form of filter element+blood filter membrane have a good effect, and the filtrate does not see redness.
  • the combined blood filter device is shown in Figure 4.
  • the base can be loaded with a filter element, and a layer of translucent blood filter membrane is covered on the filter element. There is a concentrated glue with good fluidity between the filter element and the blood filter membrane. After the membrane was pasted, it was cut into individual compound blood filtration devices by steel knife accessories. It was found that after compounding, it can filter red blood cells well and be easily filled into the blood filter holes of the reagent card.
  • This design solves the problem of time-consuming and laborious filling in production. The problem of a softer blood filter film can be solved, and the production automation can be directly facilitated through reasonable tooling.
  • the light value of the sample containing red blood cells filtered by the current reagent card filter device is about 15% lower than that of the sample without red blood cells, and has little effect on the gradient of the detection sample. Therefore, the present invention uses this method to filter red blood cells.
  • the working solutions containing microspheres and biotin-labeled were concentrated into working solutions of 10 ⁇ L, 12.5 ⁇ L, 15 ⁇ L, 15 ⁇ L, and 30 ⁇ L, which were further freeze-dried to form freeze-dried pellets, and different volumes of freeze-dried pellet samples Carry out performance measurement, and measurement result is shown in Table 5;
  • the volume of freeze-dried pellets is determined by the degree of concentration.
  • the original working solution is 50 ⁇ L/part.
  • the original 50 ⁇ L/part of the reagent components needs to be concentrated 5 times for preparation.
  • the corresponding 25 ⁇ L needs to be concentrated for 2 times, the greater the concentration, the greater the error of diluting back to the original working concentration after being prepared into pellets, and the 10-30 ⁇ L freeze-dried pellets prepared by the freeze-drying process of the present invention can meet the requirements of samples and processed samples.
  • the deviation of the final detected light value of the sample dilution is within ⁇ 10%, and the CV is kept within 6%.
  • This embodiment investigates the NaCl mass concentration in the buffer solution, wherein when the NaCl mass concentration is 0%, 0.9%, and 4.5%, the measured eutectic points are shown in Table 7;
  • the sorting environment parameters were investigated, respectively sorting at humidity>30% and temperature>25°C for 30 minutes, and sorting at humidity ⁇ 30% and temperature ⁇ 25°C for 8h. See Table 8 for the degree of difficulty in dissolution;

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Abstract

本发明涉及一种可滤血色素冻干球均相发光免疫检测试剂盒及其制备方法，该试剂盒包括试剂检测卡、冻干小球以及进样稀释液，其中所述试剂检测卡包括反应部和含有滤血膜和滤芯的滤血部，冻干小球为10～15μL包被有抗体或抗原的受体微球、生物素标记抗体或抗原、包被亲和素的供体浓缩微球试剂冻干形成的小球；本发明的试剂卡在孔抽负压下将样品经滤红细胞装置送至检测孔，与三种冻干小球复溶，一步法孵育，检测光信号，可对抗原或抗体进行定量检测，具有高精度、高稳定性以及好的滤红细胞效果。

Description

可滤血色素冻干球均相发光免疫检测试剂盒及其制备方法 技术领域
本发明属于免疫检测试剂盒技术领域，具体涉及一种可滤血色素冻干球均相发光免疫检测试剂盒及其制备方法。
技术背景
光激发均相发光是将抗原、抗体或者基因探针分别与受体微球和供体微球连接，当发生特异性的分子结合时，两种微球连接在一起，此时如果有特定的光源照射反应体系，则包含在其中的一个供体微球中的光敏剂会将环境氧转化为激发态的单线态氧，单线态氧可以氧化另一个受体微球的发光剂而发光；若是两种微球没有结合，由于距离较远，寿命较短的单线态氧无法到达另一个受体微球并使其发光。因此，光激发均相发光技术显示出巨大的优势，其操作简单，测试速度快，精密度好，并且减少管路清洗系统，很大程度减少仪器的体积大小及维护、耗材费用。
目前，标记免疫分析技术仍以固相方式为主，即免疫复合物在固相载体上形成，并通过洗涤去除未结合的反应物和反应基质，而光激发均相发光技术的特点是无需分离免疫复合物，虽然固相免疫具有检测灵敏度高、线性范围宽的优点，但不足之处是反应时间长，操作步骤多，检测结果精密度不及均相反应。中国专利CN101750487B公开了一种干法光激化学发光免疫检测试剂盒，其微粒发光溶液中需要添加海藻糖，用于干燥过程中起支撑定型效果，防止试剂分子之间脱水，但因此增加成本，工艺复杂化，另一方面，该试剂盒在使用过程中需要经两步温育后进行光子信号检测，增加了人工加样过程中的误差。中国专利CN113092767A公开了一种鲎试剂冻干微球及其制备方法和应用，该微球的冻干过程需要控制温度进行预冻，增加了操作难度。目前，市面常规的光激发均相发光试剂均以液相产品为主，液态试剂的运输和储存稳定性受到很大限制，供体和受体微球采取逐步加液体试剂的形式会影响检测的精密度，若出产产品需人工完成试剂加样，则造成偏差更大。因此，如何提高光激发均相发光试剂的运输、储存稳定性，简化试剂的制备工艺，同时在操作过程中减小人为因素的影响，提高检测的精密度，对光激发均相发光技术的应用提供广阔的前景。
发明内容
针对上述现有的技术问题，本发明提供了一种可滤血色素冻干球均相发光免疫检测试剂盒及其制备方法；
本发明的原理：在680nm左右激发光激发下，包被有亲和素的填充有酞菁衍生物的供体微球可以产生单线态氧，当供体微球连接生物素标记抗体或抗原，并且进一步连接检测对象 及包被有抗体或抗原的受体微球时，单线态氧可传递至受体微球上，并与填充在受体微球上的二甲基噻吩发生化学反应，产生紫外光，紫外光进一步激发填充于受体微球上的铕螯合物产生光子；若是受体和供体微球未通过检测对象结合，则单线态氧由于传输距离太远而无法到达受体微球使其发光。
本发明所述试剂检测卡包括反应部和滤血部；
所述滤血部下端和反应部中间部位通过通道连接；
所述的滤血部有复合滤血装置，所述复合滤血装置包括滤血膜、滤芯；
所述的滤血装置包含滤血膜和滤芯，其中滤芯为下方孔径2-10μm左右，直径4.5-4.7mm，厚度2-3mm的聚乙烯烧结滤芯，在滤芯上方加装1-2μm孔径的滤血膜，所述滤血装置中滤芯和滤血膜位置可以互换，该装置可直接添加含有红细胞的样品上样检测，对样品中絮状等粒径较大且不参与反应的杂质和血细胞进行过滤。
所述可滤血色素冻干球均相发光免疫检测试剂盒由试剂检测卡、冻干小球以及进样稀释液组成；
所述进样稀释液采用生理盐水添加防腐剂的形式；
所述可滤血色素冻干球均相发光免疫检测试剂盒的制备方法如下：
1)制备标记物试剂：分别将包被有抗体或抗原的受体微球、生物素标记抗体或抗原、包被有亲和素的供体微球与缓冲液混合得到；
2)冻干：采用滴珠机步骤1)中的三种标记物试剂经滴珠机呈小液滴滴落，滴落的液滴经液氮冷冻后呈小球状，将液氮冷冻小球直接转至液氮预冷过的铝制盘、不锈钢盘或西林瓶中，再进一步转移至冻干机进行冷冻干燥；
3)分拣：将三种冻干小球分别取1颗分装至试剂检测卡中，分装结束对试剂卡进行封口，并将试剂卡放入装有干燥剂的密封袋密封。
所述步骤1)中的标记物分别为生物素标记抗体/抗原、包被有抗体/抗原的受体微球、包被有亲和素的供体微球，三种标记物的标记过程如下：
1)生物素标记抗体/抗原：取一定体积的0.05M pH9.5 CB缓冲液，以此投入一定摩尔比的生物氨基己酰基-6-氨基己酸N-羟基琥珀酰亚胺酯和抗体/抗原，25℃反应2h后加入1％甘氨酸封闭30min，在0.01M pH7.4的PBS缓冲液透析至无残留生物素取出采用Thermo蛋白检测仪检测蛋白浓度；
2)包被有抗体/抗原的受体微球：取适量的200nm左右的羧基受体微球用0.05M pH5的MES清洗离心两遍后，按照一定的比例和顺序加入适量的抗体/抗原，m＝1/50m微球的EDC，加入10％Tween20至终浓度0.1％，30℃反应2h。偶联结束后，加入10％牛血清白蛋白浓度 至终浓度0.1％，加入0.6％乙醇胺，30℃封闭30min，封闭结束后根据缓冲液稀释至20-50mg/mL；
3)包被有亲和素的供体微球：取适量的200nm左右的羧基供体微球用0.05M pH5的MES清洗离心两遍后，按照一定的比例和顺序加入适量的亲和素，m＝1/50m微球的EDC，30℃反应2h，偶联结束后，加入0.1％牛血清白蛋白，加入0.6％乙醇胺，30℃封闭30min，封闭结束后根据缓冲液稀释至20-50mg/mL。
所述步骤1)中的缓冲液为含有氯化钠、Tween20、牛血清白蛋白、Proclin-300和去离子水的PB、MES或TRIS体系中的一种或组合；
由于本发明的试剂盒在制备过程中，NaCl浓度通过影响冻干共晶点，影响升华干燥的温度和时间，进一步影响冻干小球的成型形态，最终对光激发化学发光检测结果产生影响，因此，本发明对NaCl含量进行优化；
所述步骤1)中缓冲液的NaCl添加量为0～0.9％；
所述步骤1)中的缓冲液还可添加保护剂为葡聚糖20000、抗坏血酸、酪蛋白、赖氨酸、山梨醇、蔗糖、海藻糖、葡萄糖、Tween80、Tween20、Triton X-405、Triton X-100、酪蛋白钠盐、甘氨酸、PEG20000、PRG8000、PEG6000、PEG2000、PEG200、PVP29000、PVP40、PVP10中的一种或多种。
所述步骤2)中液氮冷冻小球放置于铝制盘、不锈钢盘或西林瓶中，进一步转移至冻干机中，在-40～-50℃直接抽真空冻干，最后通过共晶点判断最佳的工艺条件。
由于在不添加色素情况下，含有受体微球和生物素标记物的浓缩工作液小球颜色均为白色，肉眼难以区分，在生产分装上很难发现是否同时装载了同一种受体珠或者生物素珠，故本发明研究了不用色素对检测试剂盒的影响，挑选出不影响检测试剂盒的色素进行冻干小球色素添加，目的是为了区分三种组分，以便于分装、保存，本发明选取的色素为暖色调色系：红色、橙色、黄色，所述色素添加可为苯酚红、苯酚红钠盐、和日落黄或柠檬黄。
所述步骤3)的分拣环境为：温度为25℃以下，湿度为30％以下，分拣时间在0～8h不受影响。
本发明的可滤血色素冻干球均相发光免疫检测试剂盒在使用过程中，其反应模式为：10-50μL样品+150μL进样稀释液，经抽滤后复溶3种冻干小球；
进一步地，所述冻干小球复溶后的试剂反应模式为：10-50μL样品+50μL生物素标记抗体/抗原工作液+50μL包被有抗体/抗原的受体微球工作液+50μL包被有亲和素的供体微球工作液；
所述的受体微球和生物素标记中提到包被的蛋白可为抗体或抗原，其主要根据项目选取 的方法进行区分：①若检测的蛋白为大分子抗原，则采用双抗体夹心法(见附图2)，此时受体微球包被抗体，生物素标记抗体，反应模式是10-50μL样品+50μL生物素标记抗体工作液+50μL包被有抗体的受体微球工作液+50μL包被有亲和素的供体微球工作液；②若检测的蛋白为小分子抗原，则采用竞争法(见附图3)，此时受体微球包被抗原，则生物素标记抗体，反应模式是10-50μL样品+50μL生物素标记抗体工作液+50μL包被有抗原的受体微球工作液+50μL包被有亲和素的供体微球工作液。受体微球包被抗体，则生物素标记抗原，反应模式是10-50μL样品+50μL生物素标记抗原工作液+50μL包被有抗体的受体微球工作液+50μL包被有亲和素的供体微球工作液。
本发明的试剂盒在上述反应模式基础上，将50μL的三种含有微球和生物素标记的不同工作液采用冻干或者烘干形式做成干法试剂，经验证表明，冻干的形式优于烘干的形式，表现为复溶较简单且蛋白的结构不易破坏；另一方面，若是50μL的工作液直接冻干成小球，体积太大，导致冻干小球表面形态较差，因此，本发明对50μL的试剂成分进行1.6-5倍的浓缩，浓缩后的工作液冻成小球后，每颗小球再加入50μL的进样稀释液稀释至1X工作浓度，进一步进行测试，同时，为了配合检测孔可同时放置三种小球的体积大小，故本发明所要求冻干小球的体积大小为10-30μL，优选为10～15μL。
本发明所述试剂盒的使用方法为：
所述的试剂检测卡设有手柄，方便操作人员手持操作，在试剂卡的一侧设置有条码，方便仪器识别及自动化；将冻干小球装载至试剂检测卡的反应检测孔中，样品测试时，去掉试剂卡上方的医用易撕膜，将样品加入至试剂检测卡的过滤孔，后加入适量进样稀释液，将试剂检测卡放入仪器，仪器对反应检测孔抽负压，所述抽负压过程中的检测压力和时间应保证滤血膜在抽滤过程中不会破碎，样品及进样稀释液随压力经过滤血材料从通道进入至反应检测孔，进入检测孔的液体复溶3种冻干小球，仪器对试剂卡进行震动数秒，进行一步法孵育，孵育结束后，读取光子信号，根据标准曲线计算待测样品浓度，直接得到样品的定量检测结果，该检测过程见附图1。
本发明的试剂盒在使用过程中，样品和进样稀释液经过滤装置抽滤至检测孔，检测孔的三颗浓缩小球被稀释成工作浓度液体并与样品反应，由于进样稀释液是为了稀释3颗浓缩的冻干小球，因此进样稀释液的组分不同对冻干小球的复溶情况以及样品的检出性能均有有很大程度的影响，其具体影响过程如下：
①由于过滤装置选取的滤芯和滤血膜的孔径较小，若是进样稀释液含有表面活性剂或者是蛋白，则容易引起泡沫的产生，妨碍检测孔的检测；
②另一方面，由于进样稀释液是与样品一同加入，若是加入的样品中含有细胞，则稀释 液的环境应当满足与细胞渗透压接近，则容易引起细胞破碎；
综上所述，本发明对进样稀释液成分和含量进行了优化。
本发明所述含有微球和生物素标记的三种缓冲液体系的冻干工艺如下：
(1)PB体系冻干工艺如下：
a：PB体系缓冲液-保护剂组成为PB浓缩液+0-0.5％葡聚糖20000、0-5％蔗糖、0-0.5％Triton X-405、0-0.5％Triton X-100、0-0.5％PEG20000、0-0.5％PEG8000、0-0.5％PEG6000、0-0.5％PEG2000、0-5％海藻糖、0-4％甘氨酸、0-5％甘露醇或0-5％葡萄糖；
b：1.6-5倍浓缩液经液氮冷冻后形成液氮小球，转至经液氮预冷过的铝制盘、不锈钢盘或西林瓶中，再根据表1中的冻干程序进行抽真空冷冻干燥；
表1：PB体系冻干程序
-40℃ 2h
-30℃ 2h
-25℃ 6h
-10℃ 2h
0℃ 2h
10℃ 2h
20℃ 2h
30℃ 3h
(2)MES体系冻干工艺如下：
a：MES体系缓冲液-保护剂组成为MES浓缩液+0-0.5％葡聚糖20000、0-5％蔗糖、0-0.5％Triton X-405、0-0.5％Triton X-100、0-0.5％PEG20000、0-0.5％PEG8000、0-0.5％PEG6000、0-0.5％PEG2000、0-5％海藻糖、0-4％甘氨酸、0-5％甘露醇、0-5％山梨醇或0-5％葡萄糖；
b：1.6-5倍浓缩液经液氮冷冻后形成液氮小球，转至经液氮预冷过的铝制盘、不锈钢盘或西林瓶中，再根据表2中的冻干程序进行抽真空冷冻干燥；
表2：MES体系冻干程序
-40℃ 2h
-30℃ 6h
-20℃ 2h
-10℃ 2h
0℃ 2h
10℃ 4h
20℃ 4h
30℃ 3h
(3)TRIS体系冻干工艺如下：
a：TRIS体系缓冲液-保护剂组成为TRIS浓缩液+0-5％蔗糖、0-0.5％TX100；
b：1.6-5倍浓缩液经液氮冷冻后形成液氮小球，转至经液氮预冷过的铝制盘、不锈钢盘或西林瓶中，再根据表3中的冻干程序进行抽真空冷冻干燥；
表3：TRIS体系冻干程序
-50℃ 2h
-45℃ 1.5h
-40℃ 0.5h
-35℃ 10h
-30℃ 6h
-20℃ 2h
-10℃ 2h
0℃ 2h
10℃ 2h
20℃ 2h
30℃ 3h
对上述冻干工艺制备得到的冻干小球进行分拣，对其分拣环境进行测定：
三种不同组分的冻干小球制备后，需要各取1颗分装至试剂检测卡的检测孔中，分拣的过程中，放置于环境中的小球会受环境的影响，经研究发现，冻干小球放置于温度≤25℃、湿度≤30％环境8h，对小球的性能不产生影响，而放置于湿度>30％、温度>25℃下的冻干小球溶易潮解，从而影响小球的存储稳定性。
相应的与现有技术相比，本发明的有益效果如下：
(1)本发明提供的一种可滤血色素冻干球均相发光免疫检测试剂盒包含三种成分的冻干小球，将含有三种成分的工作液进行浓缩1.6～5倍，体积大小控制在10-30μL，得到的冻干小球的CV仍可达到6％以下；
(2)本发明的冻干小球在制备过程中无需将经液氮冷冻后的小球置于冻干机中进行预冻，液氮小球可直接转移至冻干机中抽真空冻干，优化了工艺步骤，降低了实际过程中的操作难度，减少误差，有利于放大生产；
(3)本发明的冻干小球对不同材质的冻干箱进行筛选，相比不锈钢冻干盘和西林瓶，铝制品冻干箱制备的冻干小球形态更好；
(4)本发明在分拣封装过程中为了避免吸湿变形等问题，采用的分拣环境湿度≤30％以下，温度≤25℃，在此环境下，分拣时间可延长至8h；
(5)本发明冻干过程中的缓冲液中可添加5％蔗糖的蔗糖作为保护剂或者甚至无需添加保护剂制备的冻干小球性能形态光滑，不易产生碎屑，高温稳定性良好，同时可节约成本；
(6)本发明对缓冲液体系中的NaCl浓度进行了优化，其中NaCl以0～0.9％质量浓度的缓冲液产生的冻干共晶点较高，提高了抽真空过程中升华干燥的温度，缩短了冻干时间，冻干的小球形态稳定良好，同时节约了成本；
(7)本发明采用了不用色素区分三种组分的冻干小球，以便于分装、保存；
(8)本发明制备的试剂盒采用的测试方式为试剂卡左侧进样，右侧泵抽过滤后直接进入右侧检测杯复溶三个小球，一步温育检测，缩短了时间，也减少了液态试剂的添加步骤，仪器无需管路清洗，降低对仪器设备的要求。
附图说明
图1中(A)左边的滤芯和滤血膜位置可以互换，右边检测孔上方黑色部分为泵抽的吸头，检测孔中的三颗球分别为生物素标记物小球、受体微球标记小球、供体微球标记小球；(B)为通过负压抽滤后，样本+稀释液通过过滤装置过滤至右边的检测孔中，迅速复溶三颗小球，随后孵育结束后检测。
图2为双抗体夹心发检测模式，A、B、C、D、F分别为包被有亲和素的供体微球、生物素标记抗体、待测抗原、包被有抗体的受体微球、免疫复合物。
图3为竞争法检测模式，a、b、c、d、f1、f2分别为包被有亲和素的供体微球、生物素标记的抗体、待测抗原、包被有抗原的受体微球、抗原竞争结合免疫复合物、待测抗原结合免疫复合物。
图4为滤血装置。
具体实施方式
本发明下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。
本发明的术语“包括”和“具有”以及它们任何变形，意图在于覆盖不排他的包含。例如包含了一系列步骤的过程、方法、装置、产品或设备没有限定于已列出的步骤或模块，而是可选地还包括没有列出的步骤，或可选地还包括对于这些过程、方法、产品或设备固有的其它步骤。
为使本发明的目的、技术方案和优点更加清楚明了，下面结合具体实施方式，对本发明进一步详细说明。应该理解，这些描述只是示例性的，而并非要限制本发明的范围。此外，在以下说明中，省略了对公知结构和技术的描述，以避免不必要地混淆本发明的概念。
以下实施例对本发明做进一步的描述，但该实施例并非用于限制本发明的保护范围。
实施例1 NT-proBNP(氨基末端脑钠肽前体)冻干小球诊断试剂的制备
(1)抗NT-proBNP抗体包被的受体微球制备及工作液的配制：
受体微球的制备：(a)将100μL，浓度为10mg/mL受体微球混悬液放入试管内，于14000rpm的转速下离心15min，去除上清液，加入两倍体积的MES溶液(pH＝5)清洗两遍，同样于14000rpm的转速下离心15min，去除上清，加入200μL的MES溶液(pH＝5)，超声分散，制成初级微球混悬液I；(b)向步骤(a)制得的初级微球混悬液I中分别加入50μg抗NT-proBNP抗体、2μL 10mg/mL的EDC-MES溶液、2μL 10％的Tween20-去离子水，混匀，在30℃摇床下震荡2h，反应结束后加入1mg牛血清白蛋白和1.2μL乙醇胺，混匀，置于30℃摇床上封闭30min，于14000rpm转速下离心15min，去除上清液，加入两倍体积的缓冲液清洗两遍，加入50μL的缓冲液贮存；
按质量百分数计，所述缓冲液组成为：0.25M MES、0.9％氯化钠、7.5％牛血清白蛋白、0.5％Tween20、0.5％Proclin-300，余量为去离子水，pH＝6.0。
工作液的配制：将包被有NT-proBNP抗体的受体微球贮存液用缓冲液+0.01％苯酚红钠盐稀释至工作浓度为1.0mg/mL；
(2)生物素标记抗NT-proBNP抗体制备及工作液的配制：
抗体的制备：50μg的NT-proBNP抗体和1.89μg的sigma长链生物素加入到50μL，0.01M的PBS缓冲液(pH＝7.4)中，于25℃下震荡2h，反应后加入1mg甘氨酸，于25℃下震荡封闭30min，生物素标记抗NT-proBNP抗体溶液在0.01M的PBS缓冲液(pH＝7.4)中透析24h。
工作液的配制：生物素标记抗NT-proBNP抗体标记液用缓冲液+0.01％日落黄稀释至工作浓度为6.25μg/mL；按质量百分数计，所述缓冲液组成为：0.25M MES、0.9％氯化钠、7.5％牛血清白蛋白、0.5％Tween20、0.5％Proclin-300，余量为去离子水，pH＝6.0。
(3)亲和素包被的供体微球制备及工作液的配制
供体微球的制备：(a)将100μL，浓度为10mg/mL供体微球混悬液放入试管内，于14000rpm的转速下离心15min，去除上清液，加入两倍体积的MES溶液(pH＝5)清洗两遍，同样于14000rpm的转速下离心15min，去除上清，加入200μL的MES溶液(pH＝5)，超声分散，制成初级微球混悬液I；(b)向步骤(a)制得的初级微球混悬液I中分别加入50μg亲和素、2μL 10mg/mL的EDC-MES溶液，混匀，在30℃摇床下震荡2h，反应结束后1mg牛血清白蛋白和1.2μL乙醇胺，混匀，置于30℃摇床上封闭30min，于14000rpm转速下离心15min，去除上清液，加入两倍体积的缓冲液清洗两遍，加入50μL的缓冲液贮存；
按质量百分数计，所述缓冲液组成为：0.25M MES、0.9％氯化钠、7.5％牛血清白蛋白、 0.5％Tween20、0.5％Proclin-300，余量为去离子水，pH＝6。
工作液的配制：包被有亲和素的供体微球贮存液用缓冲液稀释至工作浓度为0.25mg/mL。
(4)液氮冷冻小球制备
将滴珠机出液体积大小调整至10μL，包被有抗NT-proBNP抗体的受体微球工作液、生物素标记的NT-proBNP抗体工作液、包被有亲和素的供体微球工作液经滴珠机呈小液滴滴落，滴落的液滴经液氮冷冻后呈小球状。
(5)干燥、分拣
将步骤4)得到的液氮冷冻小球转移至已预冻至-40℃的冻干机后进行冷冻干燥，干燥结束后，于湿度30％以下，温度25℃以下环境进行小球分拣，3种冻干小球分别取1颗分装至试剂检测卡的反应检测孔中，分装结束对试剂卡进行封口，并将试剂卡放入装有干燥剂的密封袋密封。
(6)检测结果
(a)精密度：将已知浓度的NT-proBNP抗原定标品5个，分别取10μL加入试剂检测卡的过滤孔中，每个样品20孔，各添加150μL的进样稀释液，37℃孵育10min后，采用Reader读数，结过见表1；
表1：精密度测定结果
Figure PCTCN2022113365-appb-000001
由表1结果显示，色素冻干小球试剂的发光值与NT-proBNP抗原浓度具有明显的相关性，随着NT-proBNP抗原浓度的升高，发光值逐渐升高，批内精密度小于10％，可用于NT-proBNP抗原的检测。
(b)试剂检测卡稳定性：对37℃下储存28天的冻干小球稳定性进行测定，将浓度分别为20000pg/mL、5000pg/mL、300pg/mL的NT-proBNP抗原定标品，分别取10μL加入试剂检测卡的过滤孔中，每个样品3孔，各添加150μL的稀释液，37℃孵育10min后，采用Reader读数，得到的发光均值如表2所示；
表2：稳定性测定结果
Figure PCTCN2022113365-appb-000002
Figure PCTCN2022113365-appb-000003
由稳定性数据可知，冻干小球试剂37℃高温28天内稳定性良好，表明试剂检测卡也具有良好的稳定性。
实施例2滤血装置的优化
(1)滤血装置装的优化
对试剂盒中的试剂检测卡滤血装置进行优化，其中滤血装置采用不同的填充形式，三种不同的填充形式对滤过红细胞效果以及装在难以程度进行对比，结果如表3所示；
表3：不同滤血装置填充形式的优化结果
Figure PCTCN2022113365-appb-000004
由表3结果可知，单独使用滤芯填充的滤血装置虽然装载容易，但红细胞大多数可通过滤芯，滤芯是聚乙烯烧结的较硬有一定孔径的材料，可以起到支撑的结构，由于孔径较大和不均一的问题并不能起到良好的滤血作用，滤血效果较差；滤血的孔径原理可以截留红细胞，单独采用滤血膜的装置需要采用合适的抽滤压力才能得到较好的滤过效果，但滤血膜本身是属于软和薄的材质，在试剂卡孔较难平整填充，单独采用滤血膜的装载难度较大，且填塞后，抽滤的压力太大可能会导致滤血膜破裂或者向内坍塌；因此，由表3结果看出，以滤芯+滤血膜的复合形式得到的滤过红细胞效果好，且滤液不见红，本发明设计将滤血膜与其滤芯复合在一起的滤血装置见附图4，底座可装载滤芯，在滤芯上覆盖的一层偏透明色的滤血膜，滤芯和滤血膜之间有流动性较好的浓缩胶水，滤芯和滤血膜胶贴后通过钢刀配件裁剪至一个个的复合滤血装置，发现复合后能良好的过滤红细胞并且容易填装至试剂卡的滤血孔中，该设计解决了生产上费时费力填装单层较软滤血膜的问题，并且可以通过合理的工装直接便于生产自动化。
(2)滤血效果检测
取不含红细胞的CRP样品，向其添加红细胞，混合成含有40％红细胞压积比的混合液， 将不含红细胞样品和含有红细胞均通过上述滤芯+滤血膜的复合滤血装置，对比不含红细胞样品和含有红细胞样品的测试结果，见表4；
表4：过滤装置的滤过红细胞效果
Figure PCTCN2022113365-appb-000005
含有红细胞样品采用当前试剂卡过滤装置过滤后的光值相比不含红细胞样品平均偏低15％左右，并且对检测样品的梯度影响不大，因此，本发明采用该种方式进行红细胞过滤。
实施例3冻干小球体积对试剂盒检测结果的影响
本实施例将分别含有微球和生物素标记的工作液浓缩成10μL、12.5μL、15μL、15μL、30μL的工作液，进一步冷冻干燥形成冻干小球，分别对不同体积的冻干小球样品进行性能测定，测定结果见表5；
表5：冻干小球体积对检测结果的影响
Figure PCTCN2022113365-appb-000006
冻干小球的体积根据浓缩的程度决定，原工作液为50μL/份，若要做成10μL的小球则需要将原来50μL/份的试剂成分浓缩5倍进行制备，相应的25μL需要浓缩2倍，浓缩的倍数 越大，制备成小球后稀释回原来的工作浓度的误差就越大，而本发明的冻干工艺制备出的10-30μL的冻干小球均可满足与样品和进样稀释液最终的检测光值偏差在±10％以内，并且CV保持在6％以内。
实施例4保护剂对冻干小球的影响
本实施例制备10μL的含有不同保护剂的工作液，进一步制备成冻干小球，小球的检测结果如表6所示；
表6：保护剂对冻干小球形态的影响
Figure PCTCN2022113365-appb-000007
Figure PCTCN2022113365-appb-000008
由表6结果所示，针对目前研究的工艺，发现并不需要加多余的保护剂即可做成形态较优不掉碎屑的小球，且发现45℃7天可保持小球良好的稳定性，另外，添加5％蔗糖也可得到较好的冻干小球。
实施例5缓冲液中NaCl质量浓度对冻干共晶点的影响
本实施例对缓冲液中的NaCl质量浓度进行考察，其中NaCl质量浓度分别为0％、0.9％、4.5％时，测得的共晶点见表7；
表7：NaCl质量浓度对共晶点的影响
Figure PCTCN2022113365-appb-000009
由表7结果看出缓冲液中NaCl质量浓度对共晶点的影响是很大的，当缓冲液中NaCl质量浓度较低时，共晶点较高，冻干时间缩短。
实施例6分拣环境对冻干小球的影响
本实施例对分拣环境参数进行考察，分别在湿度>30％、温度>25℃下分拣30min，湿度≤30％、温度≤25℃下分拣8h，得到的冻干小球形态以及复溶难以程度见表8；
表8：分拣环境对冻干小球的影响
Figure PCTCN2022113365-appb-000010
由表8结果看出分拣环境对小球的影响较大，在湿度>30％、温度>25℃下30min小球已经吸水，复溶困难，应严格控制分拣小球的环境，否则将导致检测样本的结果差异太大。
需要说明的是，本说明书中描述的具体特征、结构、材料或者特点可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例以及不同实施例的特征进行结合和组合。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种试剂检测卡，其特征在于，所述试剂检测卡包括反应部和滤血部；所述滤血部下端和反应部的中间部位通过通道连接；所述滤血部有复合滤血装置，所述复合滤血装置包括滤血膜、滤芯。
  2. 根据权利要求1所述的试剂检测卡，其特征在于，所述滤血膜孔径为1-2μm，滤芯为孔径2-10μm，直径4.5-4.7mm，厚度2-3mm的聚乙烯烧结滤芯；所述复合滤血装置中滤芯和滤血膜的位置可以互换。
  3. 一种可滤血色素冻干球均相发光免疫检测试剂盒，其特征在于，所述试剂盒由权利要求1所述的试剂检测卡、冻干小球以及进样稀释液组成，所述进样稀释液由生理盐水和防腐剂组成。
  4. 根据权利要求3所述的一种可滤血色素冻干球均相发光免疫检测试剂盒的制备方法，其特征在于，包括以下步骤：
    1)制备标记物试剂：分别将包被有抗体或抗原的受体微球、生物素标记抗体或抗原、包被有亲和素的供体微球与缓冲液混合制备工作浓度；
    2)冻干：采用滴珠机将步骤1)中的三种标记物试剂经滴珠机呈小液滴滴落，滴落的液滴经液氮冷冻后呈小球状，将液氮冷冻小球直接转至含有液氮的铝制盘中并进一步转移至冻干机进行冷冻干燥；
    3)分拣：将三种冻干小球分别取1颗分装至试剂检测卡中，分装结束对试剂检测卡进行封口，并将试剂检测卡放入装有干燥剂的密封袋密封。
  5. 根据权利要求4所述的一种可滤血色素冻干球均相发光免疫检测试剂盒的制备方法，其特征在于，所述步骤1)中的缓冲液为含有氯化钠、Tween20、牛血清白蛋白、色素、Proclin-300和去离子水的PB、MES或TRIS体系中的一种或其组合，所述体系中氯化钠质量浓度为0-0.9wt％。
  6. 根据权利要求4所述的一种可滤血色素冻干球均相发光免疫检测试剂盒的制备方法，其特征在于，所述的步骤2)中的小球体积为10-30μL，优选为10-15μL。
  7. 根据权利要求4所述的一种可滤血色素冻干球均相发光免疫检测试剂盒的制备方法，其特征在于，所述步骤3)中预冷过的铝制盘温度为-40～-50℃。
  8. 根据权利要求4所述的一种可滤血色素冻干球均相发光免疫检测试剂盒的制备方法，其特征在于，所述冻干小球的颜色为红色、橙色、黄色，所述色素添加分别为苯酚红、苯酚红钠盐、日落黄或柠檬黄。
  9. 根据权利要求4所述的一种可滤血色素冻干球均相发光免疫检测试剂盒的制备方法，其特征在于，所述分拣环境为：温度在25℃以下，湿度在30％以下，时间为0～8h。
  10. 根据权利要求4所述方法制备的试剂盒的使用方法，其特征在于，步骤如下：
    将冻干小球装载至试剂检测卡的反应检测孔中，样品测试时，去掉试剂卡上方的医用易撕膜，加入适量进样稀释液至试剂检测卡的滤血部中，然后加入样品，将试剂检测卡放入仪器，仪器对反应检测孔抽负压，样品及进样稀释液随压力经过复合滤血装置从通道进入至反应检测孔，进入检测孔的液体复溶3种冻干小球，仪器对试剂卡进行震动数秒，进行一步法孵育，孵育结束后，读取光子信号，根据标准曲线计算待测样品浓度，直接得到样品的定量检测结果。
PCT/CN2022/113365 2021-11-03 2022-08-18 可滤血色素冻干球均相发光免疫检测试剂盒及其制备方法 WO2023077909A1 (zh)

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