WO2022104534A1 - 一种用于化学发光免疫法的试剂盒及其制备方法和应用 - Google Patents

一种用于化学发光免疫法的试剂盒及其制备方法和应用 Download PDF

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WO2022104534A1
WO2022104534A1 PCT/CN2020/129422 CN2020129422W WO2022104534A1 WO 2022104534 A1 WO2022104534 A1 WO 2022104534A1 CN 2020129422 W CN2020129422 W CN 2020129422W WO 2022104534 A1 WO2022104534 A1 WO 2022104534A1
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secondary antibody
antibody
preparation
kit
solution
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PCT/CN2020/129422
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English (en)
French (fr)
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陈小茹
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深圳上泰生物工程有限公司
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Publication of WO2022104534A1 publication Critical patent/WO2022104534A1/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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled 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
    • G01N33/531—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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the invention relates to the field of immunodetection, in particular to a kit for chemiluminescence immunoassay and a preparation method and application thereof.
  • Chemiluminescence immunoassay consists of two parts: immunoassay and chemiluminescence assay.
  • Immunoassay uses chemiluminescent substances or enzymes as markers, which are directly labeled on antigens or antibodies to form antigen-antibody immune complexes through the reaction of antigens and antibodies.
  • Chemiluminescence analysis is after the end of the immune reaction, adding an oxidant or a luminescent substrate of an enzyme. After the chemiluminescent substance is oxidized by the oxidant, an intermediate in an excited state is formed, which will emit photons to release energy to return to a stable ground state, and emit light. The intensity can be detected using a luminescence signal measuring instrument.
  • chemiluminescence immunoassay techniques can be divided into three categories: direct chemiluminescence marker immunoassay, chemiluminescence enzyme immunoassay and electrochemiluminescence immunoassay.
  • Chemiluminescence immunoassay technology has many applications in laboratory medicine, including the diagnosis of various disease markers and the monitoring of therapeutic drug concentrations. It has the advantages of high sensitivity, good specificity, wide linear range and short detection time. However, this technology has certain requirements on sample size, and it cannot accurately detect certain ultra-low levels of disease markers or drug concentrations.
  • some chemiluminescence detection methods such as chemiluminescence enzyme immunoassay are applied to signal amplification technology, which can amplify the luminescence enzyme signal.
  • signal amplification technology which can amplify the luminescence enzyme signal.
  • it also has defects, such as the need for repeated annealing and enzyme addition, and for direct chemiluminescence
  • marker immunoassays it is also not suitable for enhancing the detection effect by amplifying the luminescent enzyme signal. Therefore, the direct chemiluminescent marker immunoassay cannot be used in biological detection with extremely low analyte content.
  • the commonly used chemiluminescence system is direct chemiluminescence labeled with acridine ester or luminol luminescent groups, but the sensitivity of such methods often fails to meet clinical requirements, which limits the promotion of this method.
  • the invention provides a kit for chemiluminescence immunoassay, a preparation method and application thereof, and a signal amplification technology is used to enhance the signal in the chemiluminescence immunoassay.
  • the present application provides a kit for chemiluminescence immunoassay, the kit comprising an immunomagnetic bead complex coated with an antigen or antibody to be tested, and a polymer molecule conjugated with an immunomagnetic bead complex. Secondary antibody or secondary antibody, the polymer molecule is labeled with a plurality of light source groups.
  • kit in the first aspect of the present application can be applied to the chemiluminescence immunoassay method to detect the analyte in the sample to be tested by the competition method or the sandwich method.
  • the magnetic beads suitable for the present invention are also referred to as magnetic spheres, and can be magnetic microspheres commonly used in the art.
  • the magnetic beads used in the present invention are modified with active groups, so that the magnetic beads can specifically bind to the antigen or antibody of the analyte.
  • the magnetic beads in the immunomagnetic bead complex are modified with active groups, and the active groups include at least one of carboxyl group, amino group, aldehyde group, and avidin.
  • the polymer molecule should have multiple binding sites for simultaneously labeling multiple light source groups.
  • the chemiluminescence group in the light source group is based on the principle of chemical reaction.
  • the kit further includes a chemiluminescence initiator for inducing the chemiluminescence group to generate a light signal.
  • the fluorophore uses the principle of physical reaction, which can directly emit light signals, and does not need to add a chemiluminescence initiator when using it as a light source group.
  • the polymer molecule includes at least one of polystyrene, polymethacrylic acid, polymethyl methacrylate, biological macromolecules, and polyamino acids.
  • the biological macromolecule comprises at least one of BSA (referring to bovine serum albumin) and KLH (referring to hemocyanin), and the polyamino acid comprises polylysine.
  • the light source group includes a chemiluminescent group and a fluorescent group; preferably, the chemiluminescent group includes at least one of acridine esters and luminol derivatives ;
  • the fluorescent group includes at least one of fluorescein isothiocyanate, tetraethylrhodamine, phycoerythrin, 7-amino-4 methyl coumarin, and Eu3+ chelate.
  • the kit for chemiluminescence immunoassay may further include a calibrator, and the calibrator may be combined into the kit of the present application, or may be purchased commercially.
  • the calibrator consists of several samples with known concentrations, such as 6, 8, or 9, etc. can be configured. Its concentration range is determined according to the concentration range of the analyte in the human body. For example, for TXB 2 , its concentration in humans is in pg/mL, so a kit for detection of TXB 2 was prepared with a standard concentration range of 0-1000 pg/mL.
  • sST2 its concentration in the human body is in ng/mL, so a kit for the detection of sST2 is prepared, and the concentration range of its standard is 0-1000 ng/mL.
  • PD-1 monoclonal antibody its concentration in the human body is ⁇ g/mL, so a kit for detecting PD-1 monoclonal antibody is prepared, and the concentration range of its standard is 0-1000 ⁇ g/mL.
  • the sensitivity of the kit prepared in the present application can be increased by dozens of times compared to the kit without signal amplification technology.
  • the kit for detecting TXB 2 prepared in this application has a minimum detection limit of 0.05 pg/mL, which is especially suitable for detecting the analyte with extremely low content (0.05-0.1 pg/mL).
  • the present application provides a method for preparing the kit of the first aspect, comprising the following steps:
  • the coating solution containing the antigen or antibody of the analyte is added to the magnetic beads modified with active groups, and the antigen or antibody of the analyte is coupled and immobilized on the magnetic beads to obtain the antigen or antibody containing the analyte.
  • the immunomagnetic bead complex ;
  • Protect and activate polymer molecules couple light source groups to activated polymer molecules, and then couple with secondary antibodies or secondary antibodies to obtain labeled secondary antibodies or secondary antibodies that simultaneously label multiple light source groups Antibody.
  • the specific steps of preparing the immunomagnetic bead complex include:
  • the buffer used in the steps can be Tris-HCl buffer, PBS buffer or HEPES buffer, etc.; the coupling solution can be a mixture of EDC solution and NHS solution, and the washing solution can be conventional in PBS buffer. Add 0.05% Tween 20 to the PBST wash solution.
  • the specific steps of preparing the labeled secondary antibody or the labeled secondary antibody include:
  • Protection and activation steps including placing the polymer molecules in a buffer, adding a protection reagent under stirring, and reacting at 0°C-37°C for 1-72 hours; after the reaction is completed, ultrasonication, centrifugation, and removal of the supernatant , and washed with washing solution; put the obtained polymer molecules in the buffer solution, add the activation reagent under stirring, and react at 0°C-37°C for 1-72h; after the reaction, ultrasonic, centrifuge and remove the supernatant, And wash with washing liquid to obtain activated polymer molecules;
  • the preparation step of the signal amplification marker including placing the activated polymer molecule in the buffer solution, adding the light source group under stirring, and reacting at 0°C-37°C for 1-72h; after the reaction, ultrasonication and centrifugation are completed. , remove the supernatant, and wash with washing solution; put the obtained activated polymer molecules in the deprotection reagent, and react at 0 °C-37 °C for 1-72 h; after the reaction is completed, ultrasonication, centrifugation, and removal of the supernatant, And wash with washing solution to obtain the signal amplification marker;
  • the step of labeling the secondary antibody or labeling the secondary antibody including placing the secondary antibody or secondary antibody in a buffer, adding a signal amplification marker under stirring, and reacting at 0°C-37°C for 1-72h; the reaction After ultrasonication, centrifugation, removal of the supernatant, and washing with washing solution, the secondary antibody or secondary antibody is purified to obtain a label that simultaneously labels multiple light source groups (polymer molecules coupled with light source groups) Secondary antibody or labeled secondary antibody.
  • the coupling solution in this application is mainly used to couple the antigen or antibody of the analyte to the magnetic beads, and to couple the polymer molecules labeled with multiple light source groups to the secondary antibody or on the secondary antibody.
  • the coupling preferably adopts EDC solution (Chinese: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution) and NHS solution (Chinese: N-hydroxysuccinimide solution) to improve the coupling efficiency.
  • the function of the coating solution is to coat the antigen or antibody of the object to be tested.
  • the coating solution includes a Na 2 CO 3 solution and/or a NaHCO 3 solution, which is the antigen or antibody to be tested.
  • Antibodies provide an alkaline environment.
  • the buffer is to provide a buffer environment for the preparation of the kit or the chemiluminescence immunoassay reaction, including Tris-HCl buffer, PBS buffer (ie, phosphate buffered saline) and HEPES buffer.
  • a phosphate buffered saline solution is preferably used as the buffer solution, and its pH value is adjusted to about 7 to provide neutral or weakly alkaline buffer conditions for the reaction.
  • the purpose of the protective reagent is to protect the amino group of the polymer molecule, and to avoid the direct reaction between the polymer molecule and the activated carboxyl group and the amino group in the activation step;
  • the purpose of the activation reagent is to activate the carboxyl group, so that the carboxyl group reacts with the chemiluminescence or fluorescent group;
  • the purpose of the deprotection reagent It is to dissociate the protected amino group in the polymer molecule, so that the amino group can be coupled with the carboxyl group activated by the secondary antibody or the second antibody.
  • the protection reagent preferably includes at least one of di-tert-butyl dicarbonate, p-toluenesulfonyl chloride, acetic anhydride and benzophenone; the deprotection reagent preferably includes at least one of acetic acid and hydrochloric acid.
  • the present application also provides a method for detecting a sample to be tested using the kit prepared in the first aspect or the kit prepared by the preparation method in the second aspect, comprising: adding the immunomagnetic bead complex to Mix with the sample to be tested to carry out an immune reaction; after the immune reaction is completed, obtain an immune complex by magnetic separation; add a secondary antibody or secondary antibody labeled with multiple light source groups to the immune complex to make the secondary antibody or secondary antibody
  • the antibody is combined with the immune complex to form a new immune complex; the new immune complex is magnetically separated, and the new immune complex is detected under the chemical reaction or excitation wavelength according to the labeled light source group
  • the generated light signal is used to detect the content of antigen or antibody in the sample to be tested by the intensity of the light signal.
  • the present application also provides an application of the kit prepared by the first aspect in detecting TXB 2 , PD-1 mAb or sST2.
  • PD-1 monoclonal antibody is used as a drug, and the detection of its blood concentration is to assist the adjustment of clinical drug regimens and reduce adverse drug reactions;
  • sST2 and TXB 2 are used as biomarkers, and the detection of its blood (or urine) content is to diagnose the progress of the disease happening.
  • the competition method was used for TXB2 as an organic small molecule, and the antibody sandwich method was used for PD-1 monoclonal antibody and sST2 as protein macromolecules. Since the concentrations of PD-1 mAb, sST2 and TXB2 in the human body are of different orders of magnitude, the corresponding preparation conditions and concentrations can be selected according to actual needs.
  • the minimum detection limit of the prepared kit is the detection limit of the TXB2 chemiluminescence immunoassay kit, which is 0.05 pg/mL, while the ELISA detection reagent for TXB2 produced by Shanghai Yubo Biological Co., Ltd. is on the market.
  • the minimum detection limit was 2.3 pg/mL, which was much higher than the detection limit of the present application. Experiments have proved that the sensitivity can be increased by dozens of times compared with the prior art by using the signal amplification technology applied in the present application.
  • the present application prepares a kit with multiple light source groups labeled on the secondary antibody or the secondary antibody by combining the signal amplification technology, which can be used in the chemiluminescence immunoassay to accurately and quantitatively detect ultra-low levels (such as In the detection of disease markers or drug concentrations of 0.05-0.10 pg/mL), the sensitivity of the detection is greatly improved.
  • 1 is a schematic diagram of the binding of labeled antibodies in the preparation process of the kit described in the application;
  • Fig. 2 is the concentration regression curve diagram of PD-1 monoclonal antibody test substance in Example 1;
  • Fig. 3 is the concentration regression curve diagram of sST2 test substance in embodiment two;
  • FIG. 4 is a graph of the concentration regression curve of the TXB 2 analyte in Example 3.
  • each secondary antibody or secondary antibody only binds to one light source group, and the intensity of the luminescent signal depends on the number of molecules in the analyte.
  • the generated light signal is also weak, and the content of the molecule in the analyte cannot be detected more accurately and sensitively.
  • the signal amplification technology is used to multiply the optical signal during detection.
  • the signal amplification technology is to couple multiple light source groups to the polymer molecules by dehydration condensation, that is, each obtained Each polymer molecule is coupled with multiple light source groups, and then the secondary antibody or secondary antibody is covalently coupled to the polymer molecule in a direct or indirect manner, so that the quantum yield of luminescence is doubled, thereby The detection sensitivity is greatly improved, and the immunological activity of the labeled substances before and after labeling will not be affected.
  • the covalent coupling methods include but are not limited to carbodiimide method, glutaraldehyde method, sodium periodate method, N-hydroxysuccinimide ester method, maleimide method and the like.
  • the principle of the kit using signal amplification technology is as follows: the corresponding antigen (or antibody) of the analyte is coupled on the surface of the magnetic beads, after the analyte and the antigen (or antibody) immunoreact, in the magnetic field Under the action, the immune complex is separated; then a second antibody labeled with multiple light source groups is added. After the immune complex reacts with the second antibody, the new immune complex is separated under the action of a magnetic field. Then, a light signal is generated, and the luminescence intensity of the light signal is proportional to the binding amount of the antibody to be tested, and the quantitative detection of the analyte in the sample to be tested can be realized by detecting the luminescence intensity of the light signal. Labeling multiple light source groups can achieve multiple amplification of the signal, so it is suitable for accurate quantitative measurement of analytes with very low content.
  • the principle of the kit using signal amplification technology is as follows: the antigen to be tested is coupled to the surface of the magnetic beads, and the antigen to be tested coupled with the magnetic beads competes with the antigen to be tested and the primary antibody in the sample. Under the action, the antigen-antibody immune complexes coupled with magnetic beads are separated; then secondary antibodies labeled with multiple light source groups are added. After the immune complexes react with the secondary antibodies, under the action of a magnetic field, new The immune complexes are separated; then a light signal is generated. The luminescence intensity of the light signal is inversely proportional to the binding amount of the antigen to be tested in the sample.
  • the quantitative detection of the analyte in the sample to be tested can be achieved. Labeling multiple light source groups can achieve multiple amplification of the signal, so it is suitable for accurate quantitative measurement of analytes with very low content.
  • 1% refers to the mass concentration of 1%
  • 0.05% refers to the mass concentration of 0.05%
  • Preparation steps of the immunomagnetic bead complex Take 10 mL of the stock solution containing carboxyl magnetic beads and mix with a vortex mixer, place it on the magnetic separator and let it stand for magnetic separation for 2 min, remove the supernatant; add 100 mL of coupling solution, set it to React on a rotary mixer for 30 min at room temperature, magnetically separate for 2 min, and remove the supernatant; add 100 mL of coating solution to resuspend, put it on a rotary reactor for 2 h, let stand for magnetic separation for 2 min, and remove the supernatant; wash with 10 mL Wash the magnetic beads with liquid, let stand for magnetic separation for 2 min, and remove the supernatant; add blocking solution, vortex and mix evenly, place it in a constant temperature incubation shaking box to react for 2 h, let stand for magnetic separation for 2 min, and remove the supernatant; add 10 mL of washing solution Wash the magnetic beads, stand for magnetic separation for 2 min, and remove the supern
  • the preparation steps of the labeled secondary antibody dilute the polymer molecule with buffer to 1.0% (w/w), add a protective reagent under stirring, react at room temperature for 2 hours, sonicate, centrifuge, and remove the supernatant;
  • the polymer molecules were diluted to 1.0% (w/w), the activation reagent was added under stirring, the reaction was carried out at room temperature for 2 hours, the supernatant was removed by ultrasonication, centrifugation, and the activated polymer molecules were obtained;
  • the activated polymer molecules were diluted to 1.0% ( w/w), add molecules containing light source groups for labeling under stirring, react at room temperature for 2 hours; ultrasonicate, centrifuge, remove supernatant; and place in deprotection reagent, react at room temperature for 2 hours;
  • the supernatant was obtained to obtain the signal-amplified labeled polymer molecule;
  • the secondary antibody was placed in the buffer, diluted to 1.0% (w/w), the signal-
  • Detection of PD-1 antibody plasma concentration by chemiluminescence immunoassay based on magnetic particles Take 30 ⁇ L of the immunomagnetic beads coated with PD-1 antigen obtained above and add it to a 2 mL centrifuge tube, and add 50 ⁇ L of calibration to the corresponding centrifuge tube in turn.
  • the above PD-1 mAb calibrator was dissolved in human blank serum to prepare 1.5625 ⁇ g/mL, 3.125 ⁇ g/mL, 6.25 ⁇ g/mL, 12.5 ⁇ g/mL, 25.0 ⁇ g/mL, 50.0 ⁇ g/mL, 100.0 ⁇ g /mL, 8 concentration gradients of 200.0 ⁇ g/mL, and detected according to the above detection method.
  • the measurement results are shown in Table 1.1, and the regression curve obtained by cubic curve fitting is shown in Figure 2.
  • the relative luminescence intensity of the zero-concentration calibrator was measured in parallel for 20 times, and the mean (M) and standard deviation (SD) of the relative luminescence intensity were calculated, and calibrated according to the zero-concentration calibrator (0 ⁇ g/mL) and the adjacent concentration (1.5625 ⁇ g/mL)
  • M mean
  • SD standard deviation
  • concentration-luminescence intensity results between the products were subjected to two-point regression fitting to obtain a linear equation, and the luminescence intensity corresponding to M+2SD was substituted into the above equation, and the obtained concentration was the minimum detection limit.
  • the lowest detection limit of the kit provided by the present invention is 0.0028 ⁇ g/mL.
  • the PD-1 mAb detection kit prepared above was used to test the intra-assay precision of quality control product L (3.125 ⁇ g/mL) and quality control product H (200.0 ⁇ g/mL), respectively. Take the same batch of kits prepared in Example 1, measure quality control substance L and quality control substance H in parallel 20 times, and calculate the coefficient of variation of all results. The result is as follows:
  • Quality control substance L ( ⁇ g/mL) Quality Control H ( ⁇ g/mL) mean 3.143 201.322 standard deviation 0.04 2.10 Coefficient of Variation % 1.21 1.04
  • the above-prepared kit was placed at room temperature, and the quality control substance L and the quality control substance H were tested on the 0th, 3rd, 5th, and 7th days, and the relative deviation between the 7th day and the 0th day was calculated.
  • the relative deviation between the 7th day and the 0th day of the quality control product L was 0.14%, and the relative deviation of the quality control product H was 0.05% between the 7th day and the 0th day, and the sample stability was good.
  • Example 1 Please refer to Example 1 for the preparation steps of the immunomagnetic bead complex.
  • Example 1 Please refer to Example 1 for the preparation steps of the labeled secondary antibody.
  • Detection of sST2 content by chemiluminescence immunoassay based on magnetic particles Take 30 ⁇ L of the immunomagnetic beads coated with sST2 antibody obtained above and add it to a centrifuge tube, and add 50 ⁇ L of calibrator/quality control product/to be tested to the corresponding centrifuge tube in turn Then, 50 ⁇ L of the light source group-labeled secondary antibody prepared above was added to the sample, and after 20 min of reaction at room temperature, the buffer was washed 3 times, and then placed in a constant temperature shaker at a speed of 180 rpm, and the reaction was performed at room temperature for 20 min. Wash the buffer 5 times, then add 5 ⁇ L of luminescence initiator, shake and mix, react for 5 minutes, detect the luminescence signal, and record the maximum luminescence intensity value.
  • the above sST2 calibrator was dissolved in human blank serum to prepare 0.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL, 20.0ng/mL, 40.0ng/mL, 80.0ng/mL, 160.0ng/mL 9 concentration gradients of ng/mL and 320.0ng/mL.
  • the detection was carried out according to the above-mentioned detection method, and the measurement results are shown in Table 1.
  • the regression curve obtained by the cubic curve fitting is shown in Figure 3.
  • the relative luminescence intensity of the zero-concentration calibrator was measured 20 times in parallel, and the mean (M) and standard deviation (SD) of the relative luminescence intensity were calculated. According to the concentration between the zero-concentration calibrator and the adjacent concentration (2ng/mL) calibrator- The luminescence intensity results were fitted by two-point regression to obtain a first-order equation, and the luminescence intensity corresponding to M+2SD was substituted into the above equation, and the obtained concentration was the minimum detection limit.
  • the minimum detection limit of the kit provided by the present invention is 0.0036ng/mL.
  • the sST2 quantitative detection kit prepared above was used to test the intra-assay precision of quality control product L and quality control product H, respectively. Take the same batch of kits prepared in Example 1, measure quality control substance L (10.0 ng/mL) and quality control substance H (160.0 ng/mL) 20 times in parallel, and calculate the coefficient of variation of all results. The result is as follows:
  • Quality control material L (ng/mL) Quality control material H (ng/mL) mean 9.99 161.67 standard deviation 0.19 1.18
  • the sST2 quantitative detection kit prepared above was used to test the inter-assay precision of quality control product L (10.0 ng/mL) and quality control product H (160.0 ng/mL), respectively. Two batches of kits prepared in Example 2 were taken, and each batch of kits was tested in parallel for quality control product L and quality control product H 20 times, and the coefficient of variation of all results was calculated. The result is as follows:
  • Quality control material L (ng/mL) Quality control material H (ng/mL) mean 9.98 161.67 standard deviation 0.18 1.29 Coefficient of Variation % 1.78 0.80
  • the above-prepared kit was placed at room temperature, and the quality control substance L and the quality control substance H were tested on the 0th, 3rd, 5th, and 7th days, and the relative deviation between the 7th day and the 0th day was calculated.
  • the relative deviation between the 7th day and the 0th day of the quality control product L was -0.62%, and the relative deviation of the quality control product H was 0.11% between the 7th day and the 0th day. 2-8°C stable for one year.
  • TXB 2 quantitative detection kit The preparation of TXB 2 quantitative detection kit, its specific proportion is as follows:
  • TXB2 antibody 1 ⁇ g/mL Carboxy Magnetic Bead Stock Solution 1.5mg/mL coating fluid Na 2 CO 3 1.59g, NaHCO 3 2.93g, add double distilled water to make up to 1L, adjust pH to 9.6 blocking solution 5% nonfat dry milk in PBS buffer
  • Magnetic particle-based chemiluminescence immunoassay to detect TXB 2 content Take 30 ⁇ L of the immunomagnetic beads coated with TXB 2 antigen obtained above and add it to a 2 mL centrifuge tube, and add 50 ⁇ L of calibrator/quality control substance to the corresponding centrifuge tube in turn / The sample to be tested and an equal volume of TXB 2 antibody were incubated at room temperature for 1 h, magnetically separated for 2 min, and the supernatant was removed; the magnetic beads were washed with the washing solution, the supernatant was removed, and 50 ⁇ L of the light source group-labeled secondary antibody prepared above was added.
  • TXB 2 calibrator was dissolved in human blank serum to prepare 0.0pg/mL, 5.0pg/mL, 10.0pg/mL, 20.0pg/mL, 40.0pg/mL, 80.0pg/mL, 160.0pg/mL, 9 concentration gradients of 320.0pg/mL, 640.0ng/mL.
  • the detection was carried out according to the above-mentioned detection method, and the measurement results are shown in Table 1.
  • the regression curve obtained by cubic curve fitting is shown in Figure 4.
  • the relative luminescence intensity of the zero-concentration calibrator was measured in parallel for 20 times, and the mean (M) and standard deviation (SD) of the relative luminescence intensity were calculated.
  • the concentration-luminescence intensity results between the calibrators were fitted by two-point regression to obtain a linear equation, and the luminescence intensity corresponding to M+2SD was substituted into the above equation, and the obtained concentration was the minimum detection limit.
  • the minimum detection limit of the kit provided by the present invention is 0.05pg/mL.
  • the TXB 2 quantitative detection kit prepared above was used to test the intra-assay precision of quality control product L (5.0 pg/mL) and quality control product H (320.0 pg/mL), respectively. Take the same batch of kits, measure quality control material L and quality control material H in parallel for 20 times, and calculate the coefficient of variation of all results. The result is as follows:
  • Quality control substance L (pg/mL) Quality Control H (pg/mL) mean 4.99 321.67 standard deviation 0.09 1.18 Coefficient of Variation % 1.81 0.37
  • the TXB 2 quantitative detection kit prepared above was used to test the inter-assay precision of quality control product L and quality control product H, respectively. Two batches of kits prepared in Example 3 were taken, and each batch of kits was tested in parallel for quality control product L and quality control product H 20 times, and the coefficient of variation of all results was calculated. The result is as follows:
  • Quality control substance L (pg/mL) Quality Control H (pg/mL) mean 4.99 321.67 standard deviation 0.09 1.29 Coefficient of Variation % 1.89 0.40
  • kits prepared above were placed at room temperature, and the quality controls L and H were tested on the 0th, 3rd, 5th, and 7th days, and the relative deviation between the 7th day and the 0th day was calculated.
  • the relative deviation between day 7 and day 0 of quality control L was -1.22%, and the relative deviation between day 7 and day 0 of quality control H was 0.16%.
  • the accelerated destruction of the reagent at 37°C was stable for 7 days, equivalent to 2-8°C stable for one year.

Abstract

一种用于化学发光免疫法的试剂盒及其制备方法和应用。试剂盒包括包被有待测物的抗原或抗体的免疫磁珠复合物，以及偶联有聚合物分子的二抗或第二抗体，聚合物分子上标记有多个光源基团。由于采用信号放大技术，将多个发光基团共价偶联到聚合物分子上，再将聚合物分子共价偶联到二抗或第二抗体上，使发光基团成倍增加，从而使发光强度成倍增强，可用于检测含量极低的生物指标。

Description

一种用于化学发光免疫法的试剂盒及其制备方法和应用 技术领域
本发明涉及免疫检测领域，具体涉及一种用于化学发光免疫法的试剂盒及其制备方法和应用。
背景技术
化学发光免疫分析包含免疫分析和化学发光分析两部分，其中免疫分析是将化学发光物质或酶作为标记物，直接标记在抗原或抗体上，经过抗原与抗体反应形成抗原-抗体免疫复合物，而化学发光分析是在免疫反应结束后，加入氧化剂或酶的发光底物，化学发光物质经氧化剂的氧化后，形成一个处于激发态的中间体，会发射光子释放能量以回到稳定的基态，发光强度可以利用发光信号测量仪器进行检测。根据化学发光标记物与发光强度的关系，可利用标准曲线计算出被测物的含量。根据标记物的不同可将化学发光免疫分析技术分为三大类：即直接化学发光标记物免疫分析、化学发光酶免疫分析和电化学发光免疫分析。
化学发光免疫分析技术在检验医学中有多方面的应用，包括各类疾病标志物的诊断以及治疗药物浓度监测等，其具有灵敏度高、特异性好、线性范围宽和检测时间短等优点。然而该技术对样本量有一定要求，其对于某些超低含量的疾病标志物或药物浓度而言，无法做到精确检测。目前部分化学发光检测法如化学发光酶免疫分析法应用到信号放大技术，可对其发光酶信号进行放大，然而其也存在着缺陷，如需要反复退火和加酶等，并且，对于直接化学发光标记物免疫分析法而言，其也不适用通过对发光酶信号进行放大来增强检测效果，因此，直接化学发光标记物免疫法无法应用在待测物含量极低的生物检测中。
目前常用的化学发光体系为以吖啶酯或鲁米诺类发光基团标记的直接化学发光，但是此类方法灵敏度常常达不到临床要求，使得该方法的推广受到限制。
发明内容
本发明提供一种用于化学发光免疫法的试剂盒及其制备方法和应用，采用信号放大技术对化学发光免疫法中的信号进行增强。
在第一方面，本申请提供一种用于化学发光免疫法的试剂盒，所述试剂盒包括包被有待测物的抗原或抗体的免疫磁珠复合物，以及偶联有聚合物分子的二抗或第二抗体，所述聚合物分子上标记有多个光源基团。
即在本申请第一方面中的试剂盒，可以应用在化学发光免疫检测法中，通过竞争法或者夹心法对待测样品中的待测物进行检测。
适用于本发明的磁珠也称为磁球，可以是本领域中常用的磁性微球。优选的是，本发明使用的磁球经过活性基团进行修饰，使得磁珠可以与待测物的抗原或抗体进行特异性结合。同时利用磁珠自身的磁性，在外磁场的作用下可以实现定向移动与富集，从而达到分离纯化目的。在本申请的一种实现方式中，免疫磁珠复合物中的磁珠经过活性基团修饰，所述活性基团包括羧基、氨基、醛基、亲和素中的至少一种。
聚合物分子上应具有多个结合位点，以用于同时标记多个光源基团，位于同个聚合物分子上的光源基团越多，其信号放大效果越好。因此可以通过控制两者的浓度配比，以将更多的光源基团标记到同一个聚合物分子上。
光源基团中的化学发光基团是利用化学反应原理，当采用化学发光基团作为光源基团时，所述试剂盒中还包括化学发光引发剂，用于诱导化学发光基团产生光信号。而荧光基团是利用物理反应原理，其可直接发出光信号，采用其作为光源基团时无需添加化学发光引发剂。在本申请的一种实现方式中，聚合物分子包括聚苯乙烯、聚甲基丙烯酸、聚甲基丙烯酸甲酯、生物大分子以及聚氨基酸中的至少一种。优选地，所述生物大分子包括BSA(指牛血清白蛋白)、KLH(指血蓝蛋白)中的至少一种，所述聚氨基酸包括聚赖氨基酸。在本申请的一种实现方式中，所述光源基团包括化学发光基团和荧光基团；优选地，所述化学发光基团包括吖啶酯、鲁米诺类衍生物中的至少一种；所述荧光基团包括异硫氰酸荧光素、四乙基罗丹明、藻红蛋白、7-氨基-4甲基香豆素、Eu3+螯合物中的至少一种。
在本实施例中，该用于化学发光免疫法的试剂盒还可以包括校准品，校准品可以组合到本申请的试剂盒中，也可以市售购买。校准品由若干个含已知浓度的样品组成，例如可以配置成6个、8个或9个等。其浓度范围根据待测物在人体内的浓度范围所决定。例如，对于TXB 2而言，其在人体内的浓度单位为pg/mL，因此制备用于检测TXB 2的试剂盒，其标准品的浓度范围为0-1000pg/mL。对于sST2而言，其在人体内的浓度单位为ng/mL，因此制备用于检测sST2的试剂盒，其标准品的浓度范 围为0-1000ng/mL。对于PD-1单抗而言，其在人体内的浓度单位为μg/mL，因此制备用于检测PD-1单抗的试剂盒，其标准品的浓度范围为0-1000μg/mL。
由于采用同时标记多个光源基团的聚合物分子进行偶联，使得本申请所制备出的试剂盒灵敏度相较于未采用信号放大技术的试剂盒而言，灵敏度能提高数十倍。尤其地，本申请所制备的用于检测TXB 2的试剂盒，其最低检测限度为0.05pg/mL，特别适用于对含量极低(0.05-0.1pg/mL)的待测物进行检测。
在第二方面，本申请提供一种第一方面所述试剂盒的制备方法，包括以下步骤：
将含有待测物的抗原或抗体的包被液加入经过活性基团修饰的磁珠中，将待测物的抗原或抗体偶联固定于磁珠上，得到包含有待测物的抗原或抗体的免疫磁珠复合物；
对聚合物分子进行保护和活化，并将光源基团偶联到活化的聚合物分子上，再与二抗或第二抗体偶联，得到同时标记多个光源基团的标记二抗或第二抗体。
即在本申请第二方面中，提供应用于竞争法或夹心法的试剂盒的制备方法，二者的制备方法上除了所添加的成分有所区别外，其它步骤均相同。
一种实施例中，所述制备免疫磁珠复合物的具体步骤包括：
对经过活性基团修饰的磁珠原液进行涡旋混匀后，静置磁性分离、去除上清液；加入偶联液并反应，磁性分离、去除上清液；加入含待测物的抗原或抗体的包被液重悬，涡旋混匀后磁性分离、去除上清液；用洗涤液洗涤磁珠，磁性分离、去除上清液；加入封闭液，涡旋混匀反应0-72h，磁性分离、去除上清液；加入洗涤液洗涤磁珠，磁性分离、去除上清液；再加入缓冲液，涡旋混匀既得免疫磁珠复合物。步骤中所采用的缓冲液可以是Tris-HCl缓冲液、PBS缓冲液或HEPES缓冲液等；偶联液可以是EDC溶液和NHS溶液的混合液，洗涤液为可以是常规的在PBS缓冲液中添加0.05％的吐温20形成的PBST洗涤液。
一种实施例中，所述制备标记二抗或标记第二抗体的具体步骤包括：
(1)保护与活化步骤：包括将聚合物分子置于缓冲液中，在搅拌下加入保护试剂，并在0℃-37℃下反应1-72h；反应完毕后超声、离心、去除上清液，并用洗涤液进行清洗；将所得聚合物分子置于缓冲液中， 在搅拌下加入活化试剂，并在0℃-37℃下反应1-72h；反应完毕后超声、离心、去除上清液，并用洗涤液进行清洗，得到活化的聚合物分子；
(2)信号放大标记物制备步骤：包括将活化的聚合物分子置于缓冲液中，在搅拌下加入光源基团，并在0℃-37℃下反应1-72h；反应完毕后超声、离心、去除上清液，并用洗涤液进行清洗；将所得活化的聚合物分子置于脱保护试剂中，在0℃-37℃下反应1-72h；反应完毕后超声、离心、去除上清液，并用洗涤液进行清洗，得到信号放大标记物；
(3)标记二抗或标记第二抗体步骤：包括将二抗或第二抗体置于缓冲液中，在搅拌下加入信号放大标记物，并在0℃-37℃下反应1-72h；反应完毕后超声、离心、去除上清液，并用洗涤液进行清洗，对二抗或第二抗体进行纯化，得到同时标记多个光源基团(偶联有带光源基团的聚合物分子)的标记二抗或标记第二抗体。
需要说明的是，本申请中的偶联液，主要应用在将待测物的抗原或抗体偶联到磁珠上，以及将标记有多个光源基团的聚合物分子偶联到二抗或第二抗体上。本申请的一种实现方式中，在偶联优选采用EDC溶液(中文：1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶液)和NHS溶液(中文：N-羟基琥珀酰亚胺溶液)的混合溶液，以提高偶联效率。
包被液的作用在于包被待测物的抗原或抗体，在本申请的一种实现方式中，所述包被液包括Na 2CO 3溶液和/或NaHCO 3溶液，其为待测抗原或抗体提供碱性环境。
缓冲液是为试剂盒的制备或化学发光免疫分析法反应提供缓冲环境，包括Tris-HCl缓冲液、PBS缓冲液(即磷酸缓冲盐溶液)和HEPES缓冲液等。在本申请的一种实现方式中，优选采用磷酸缓冲盐溶液作为缓冲液，并将其pH值调节在7左右，为反应提供中性或弱碱性缓冲条件。
保护试剂目的在于保护聚合物分子氨基，避免在活化步骤中，聚合物分子与活化的羧基与氨基直接反应；活化试剂目的在于活化羧基，使羧基与化学发光或荧光基团反应；脱保护试剂目的在于解离出聚合分子中被保护的氨基，使氨基可以与二抗或第二抗体活化的羧基偶联。在本申请中，保护试剂优选包括二碳酸二叔丁酯、对甲苯磺酰氯、乙酸酐和二苯甲酮中的至少一种；脱保护试剂优选包括乙酸、盐酸中的至少一种。
在第三方面，本申请还提供一种采用第一方面所制备的试剂盒或第二方面制备方法所制备出的试剂盒对待测样品进行检测的方法，包括：将所述免疫磁珠复合物与待测样品混合，进行免疫反应；免疫反应完成 后，通过磁性分离获得免疫复合物；向免疫复合物中加入标记有多个光源基团的二抗或第二抗体，使二抗或第二抗体与所述免疫复合物结合，形成新的免疫复合物；磁性分离所述新的免疫复合物，根据所标记的光源基团，在化学反应或激发波长下，检测所述新的免疫复合物产生的光信号，通过光信号的强度检测待测样品中抗原或抗体的含量。
在第四方面，本申请还提供一种采用第一方面所制备的试剂盒在检测TXB 2、PD-1单抗或sST2中的应用。PD-1单抗作为药物，检测其血药浓度是为了协助临床用药方案调整，降低药物不良反应；sST2，TXB 2作为生物标志物，检测其血液(或尿液)含量是为了诊断疾病的进展情况。
其中，TXB2作为有机小分子使用的是竞争法，PD-1单抗、sST2作为蛋白质大分子使用的是抗体夹心法。由于PD-1单抗、sST2以及TXB2在人体中的浓度数量级不同，因此可根据实际需求选择相应的制备条件和浓度。在本申请中，所制备出的试剂盒最低检测限度为TXB2化学发光免疫检测试剂盒的检测限度，其为0.05pg/mL，而市面上如上海钰博生物有限公司生产的TXB2的ELISA检测试剂盒，其最低检测限度为2.3pg/mL，远高于本申请的检测限度。实验证明，使用本申请所应用得信号放大技术，可使灵敏度相较于现有技术而言提高数十倍。
有益效果：本申请通过结合信号放大技术，制备出在二抗或第二抗体上标记有多个光源基团的试剂盒，应用于化学发光免疫法中，可以准确定量检测出超低含量(如0.05-0.10pg/mL)的疾病标志物或药物浓度检测中，极大地提高检测的灵敏度。
附图说明
图1为本申请所述试剂盒的制备过程中标记抗体的结合示意图；
图2为实施例一中PD-1单抗待测物的浓度回归曲线图；
图3为实施例二中sST2待测物的浓度回归曲线图；
图4为实施例三中TXB 2待测物的浓度回归曲线图。
具体实施方式
目前直接化学发光标记物免疫法技术中，每个二抗或第二抗体只结合一个光源基团，其发光信号强弱取决于待测物内分子数量，当待测物 内分子数量过低时，所产生的光信号也较弱，无法更精准灵敏地检测出待测物中分子的含量。
而在本申请中，采用信号放大技术对进行检测时的光信号进行倍数型放大，该信号放大技术是将多个光源基团通过脱水缩合的方式偶联到聚合物分子上，即所得的每个聚合物分子上均偶联多个光源基团，然后再将二抗或第二抗体与该聚合物分子以直接或间接的方式共价偶联，使得发光的量子产率成倍增加，从而大大提高了检测灵敏度，且标记前后被标记物的免疫活性不会受到影响。所述的共价偶联方法包括但不限于碳二亚胺法、戊二醛法、过碘酸钠法、N-羟基琥珀酰亚胺酯法、马来酰亚胺法等。
该试剂盒采用信号放大技术(夹心法)的原理为：将待测物相应的抗原(或抗体)偶联在磁珠表面上，待测物与抗原(或抗体)发生免疫反应后，在磁场作用下，免疫复合物被分离出来；然后加入标记有多个光源基团的第二抗体，待免疫复合物与第二抗体发生免疫反应后，在磁场作用下，新的免疫复合物被分离出来；然后产生光信号，光信号的发光强度与待测抗体的结合量成正比，通过检测光信号的发光强度可以实现对待测样品中待测物的定量检测。标记多个光源基团可实现对信号的倍数型放大，因此可适用于对含量极低的待测物的精准定量测量。
该试剂盒采用信号放大技术(竞争法)的原理为：将待测抗原偶联在磁珠表面上，偶联有磁珠的待测抗原与样本中待测抗原与一抗竞争反应，在磁场作用下，偶联有磁珠的抗原抗体免疫复合物被分离出来；然后加入标记有多个光源基团的二抗，待免疫复合物与二抗发生免疫反应后，在磁场作用下，新的免疫复合物被分离出来；然后产生光信号，光信号的发光强度与样本中待测抗原的结合量成反比，通过检测光信号的发光强度可以实现对待测样品中待测物的定量检测。标记多个光源基团可实现对信号的倍数型放大，因此可适用于对含量极低的待测物的精准定量测量。
下面将通过具体实施例并结合附图对本发明作进一步说明。应当理解，实施例仅是示例性的，并不构成对本发明保护范围的限制。下述实施例中的实验操作，如无特殊说明，均为常规操作方法。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。任何与下文记载的方法和材料相似或等同的方法及材料，都可应用于本发明中。
如无特殊标明，下文中的1％均指的是质量浓度为1％，0.05％指的是质量浓度为0.05％。
实施例一
1.PD-1抗体检测试剂盒的制备，其具体配比如下所示：
包被PD-1抗原的免疫磁珠复合物配方
Figure PCTCN2020129422-appb-000001
免疫磁珠复合物的制备步骤：取10mL含有羧基磁珠的原液用涡旋混匀器混匀，置于磁分离器上静置磁性分离2min，去除上清液；加入100mL偶联液，置于旋转混匀仪上室温反应30min，磁性分离2min，去除上清液；加入100mL包被液重悬，置于旋转反应仪上反应2h，静置磁性分离2min，去除上清液；用10mL洗涤液洗涤磁珠，静置磁性分离2min，去除上清液；加入封闭液，涡旋混匀后置于恒温培养振荡箱中反应2h，静置磁性分离2min，去除上清液；加入10mL洗涤液洗涤磁珠，静置磁性分离2min，去除上清液；加入100mL缓冲液，涡旋混匀既得免疫磁珠复合物。
化学发光基团标记的配方
Figure PCTCN2020129422-appb-000002
标记第二抗体的制备步骤：将聚合物分子用缓冲液稀释至1.0％(w/w)，搅拌下加入保护试剂，室温下反应2h，超声、离心、去除上清液；将上一步所得聚合物分子稀释至1.0％(w/w)，搅拌下加入活化试剂， 室温下反应2h，超声、离心、去除上清液，得活化的聚合物分子；将活化的聚合物分子稀释至1.0％(w/w)，搅拌下加入含光源基团的分子进行标记，室温下反应2h；超声、离心、去除上清液；并置于脱保护试剂中，室温下反应2h；超声、离心、去除上清液，得信号放大标记聚合物分子；将第二抗体置于缓冲液中，稀释至1.0％(w/w)，搅拌下加入信号放大标记聚合物分子，室温下反应2h；对第二抗体进行纯化：将上一步得到的第二抗体反应液转移至透析袋中，并将透析袋浸泡于缓冲液中2～8℃静置2h得所需纯化的标记第二抗体。
基于磁微粒的化学发光免疫分析检测PD-1抗体血药浓度：取上述所得的包被有PD-1抗原的免疫磁珠30μL加入至2mL离心管中，在相应的离心管中依次加入50μL校准品/质控品/待测样品，然后加入发光基团-聚合物标记的第二抗体50μL，室温反应20min后，缓冲液洗涤3次；置于恒温摇床中，转速为180rpm，并室温反应20min；缓冲液洗涤5次，然后加入发光引发剂H 2O 2 5μL，轻微振荡混匀后反应5min，对发光信号进行检测，记录最大的发光强度值。
2.方法学验证
2.1标准曲线
将上述PD-1单抗校准品溶于人空白血清中，配制成1.5625μg/mL、3.125μg/mL、6.25μg/mL、12.5μg/mL、25.0μg/mL、50.0μg/mL、100.0μg/mL、200.0μg/mL 8个浓度梯度，并按照上述检测方法进行检测。其测定结果如表1.1所示，利用三次曲线拟合得到回归曲线如图2所示。
表1.1校准品发光强度测定结果
Figure PCTCN2020129422-appb-000003
2.2最低检测限
平行测定20次零浓度校准品的相对发光强度，计算相对发光强度的平均值(M)和标准差(SD)，根据零浓度校准品(0μg/mL)与临近浓度 (1.5625μg/mL)校准品之间的浓度-发光强度结果进行两点回归拟合得出一次方程，将M+2SD所对应的发光强度代入上述方程，求得的浓度即为最低检测限。可得本发明提供的试剂盒最低检测限为0.0028μg/mL。2.3批内精密度
用上述制备的PD-1单抗检测试剂盒分别对质控品L(3.125μg/mL)和质控品H(200.0μg/mL)进行批内精密度测试。取实施例1制备的同一批试剂盒，平行测定质控品L和质控品H20次，计算所有结果的变异系数。结果如下：
表1.2批内精密度测定结果
  质控品L(μg/mL) 质控品H(μg/mL)
均值 3.143 201.322
标准偏差 0.04 2.10
变异系数％ 1.21 1.04
结果表明本发明的试剂盒质控品L和质控品H的批内精密度变异系数在分别为1.21％和1.04％，性能良好。
2.4批间精密度
用上述制备的PD-1单抗检测试剂盒分别对质控品L和质控品H进行批间精密度测试。取实施例1制备的两批试剂盒，每批试剂盒平行测定质控品L和质控品H 20次，计算所有结果的变异系数。结果如下：
表1.3批间精密度测定结果
Figure PCTCN2020129422-appb-000004
结果表明本发明的试剂盒质控品L和质控品H的批间精密度变异系数在分别为1.20％和1.03％，性能良好。
2.5样品稳定性
将上述制备的试剂盒放在室温下，于第0、3、5、7天对质控品L和质控品H进行测试，计算第7天与第0天的相对偏差。
表1.4试剂盒加速稳定性结果
Figure PCTCN2020129422-appb-000005
Figure PCTCN2020129422-appb-000006
质控品L第7天与第0天的相对偏差为0.14％，质控品H第7天与第0天的相对偏差为0.05％，样本稳定性良好。
实施例二
1.sST2定量检测试剂盒的制备
包被有sST2抗体的免疫磁珠复合物配方
Figure PCTCN2020129422-appb-000007
免疫磁珠复合物的制备步骤请参见实施例1。
化学发光基团标记的第二抗体配方
Figure PCTCN2020129422-appb-000008
标记第二抗体的制备步骤请参见实施例1。
基于磁微粒的化学发光免疫分析检测sST2含量：取上述所得的包被有sST2抗体的免疫磁珠30μL加入至离心管中，在相应的离心管中依次加入50μL校准品/质控品/待测样品，然后加入上述制备的光源基团标记的第二抗体50μL，室温反应20min后，缓冲液清洗3次，置于恒温摇床中，转速为180rpm，室温反应20min。洗涤缓冲液5次，然后加入发 光引发剂5μL轻微振荡混匀后反应5min，对发光信号进行检测，记录最大的发光强度值。
2.方法学验证
2.1标准曲线
将上述sST2校准品溶于人空白血清中，配制成0.0ng/mL、2.0ng/mL、5.0ng/mL、10.0ng/mL、20.0ng/mL、40.0ng/mL、80.0ng/mL、160.0ng/mL、320.0ng/mL 9个浓度梯度。按照上述检测方法进行检测，测定结果如表1所示。利用三次曲线拟合得到回归曲线如图3所示。
表2.1校准品发光强度测定结果
Figure PCTCN2020129422-appb-000009
2.2最低检测限
平行测定20次零浓度校准品的相对发光强度，计算相对发光强度的平均值(M)和标准差(SD),根据零浓度校准品与临近浓度(2ng/mL)校准品之间的浓度-发光强度结果进行两点回归拟合得出一次方程，将M+2SD所对应的发光强度代入上述方程，求得的浓度即为最低检测限。本发明提供的试剂盒最低检测限为0.0036ng/mL。
2.3批内精密度
用上述制备的sST2定量检测试剂盒分别对质控品L和质控品H进行批内精密度测试。取实施例1制备的同一批试剂盒，平行测定质控品L(10.0ng/mL)和质控品H(160.0ng/mL)20次，计算所有结果的变异系数。结果如下：
表2.2批内精密度测定结果
  质控品L(ng/mL) 质控品H(ng/mL)
均值 9.99 161.67
标准偏差 0.19 1.18
变异系数％ 1.89 0.73
结果表明本发明的试剂盒质控品L和质控品H的批内精密度变异系数在分别为1.89％和0.73％，性能良好。
2.4批间精密度
用上述制备的sST2定量检测试剂盒分别对质控品L(10.0ng/mL)和质控品H(160.0ng/mL)进行批间精密度测试。取实施例2制备的两批试剂盒，每批试剂盒平行测定质控品L和质控品H 20次，计算所有结果的变异系数。结果如下：
表2.3批间精密度测定结果
  质控品L(ng/mL) 质控品H(ng/mL)
均值 9.98 161.67
标准偏差 0.18 1.29
变异系数％ 1.78 0.80
结果表明本发明的试剂盒质控品L和质控品H的批间精密度变异系数在分别为1.78％和0.80％，性能良好。
2.5样品稳定性
将上述制备的试剂盒放在室温下，于第0、3、5、7天对质控品L和质控品H进行测试，计算第7天与第0天的相对偏差。
表2.4试剂盒加速稳定性结果
Figure PCTCN2020129422-appb-000010
质控品L第7天与第0天的相对偏差为-0.62％，质控品H第7天与第0天的相对偏差为0.11％,试剂的37℃加速破坏能稳定7天，相当于2-8℃稳定一年。
实施例三
1.TXB 2定量检测试剂盒的制备，其具体配比如下所示：
包被有TXB 2抗体的免疫磁珠复合物配方
TXB 2抗体 1μg/mL
羧基磁珠原液 1.5mg/mL
包被液 Na 2CO 3 1.59g，NaHCO 3 2.93g，加双蒸水定容1L，调节pH为9.6
封闭液 含5％脱脂奶粉的PBS缓冲液
缓冲液 为PBS缓冲液
洗涤液 为PBST洗涤液：在PBS缓冲液中加入0.05％的吐温20
偶联液 10mg/mL EDC溶液，75mg/mL NHS溶液
免疫磁珠复合物的制备步骤请参见实施例1。
化学发光基团标记的二抗配方
Figure PCTCN2020129422-appb-000011
标记二抗的制备步骤请参见实施例1中标记第二抗体的制备步骤(将其中的第二抗体替换成本申请中的二抗)。
基于磁微粒的化学发光免疫分析检测TXB 2含量：取上述所得的包被有TXB 2抗原的免疫磁珠30μL加入至2mL离心管中，在相应的离心管中依次加入50μL校准品/质控品/待测样品及等体积的TXB 2抗体，室温孵育1h后，磁性分离2min，去除上清液；洗涤液清洗磁珠，去除上清液，加入上述制备的光源基团标记的二抗50μL，置于恒温摇床中转动，转速为180rpm，室温反应20min；缓冲液洗涤5次，然后加入发光引发剂5μL轻微振荡混匀后反应5min，对发光信号进行检测，记录最大的发光强度值。
2.方法学验证
2.1标准曲线
将上述TXB 2校准品溶于人空白血清中，配制成0.0pg/mL、5.0pg/mL、10.0pg/mL、20.0pg/mL、40.0pg/mL、80.0pg/mL、160.0pg/mL、320.0pg/mL、640.0ng/mL 9个浓度梯度。按照上述检测方法进行检测，测定结果如表1所示。利用三次曲线拟合得到回归曲线如图4所示。
表3.1校准品发光强度测定结果
校准品浓度(pg/mL) RLU 拟合浓度(pg/mL) 拟合偏差(％)
0.0 42597103 -0.52 -
5.0 42344238 5.75 -
10.0 42151373 10.51 5.1
20.0 41735643 20.7 3.5
40.0 40994183 38.66 -3.35
80.0 39291263 78.96 -1.3
160.0 35685423 161.23 0.76
320.0 28523743 319.68 -0.10
640.0 15350383 640.03 0.0047
2.2最低检测限
平行测定20次零浓度校准品的相对发光强度，计算相对发光强度的平均值(M)和标准差(SD)，根据零浓度校准品(0.0pg/mL)与临近浓度(5.0pg/mL)校准品之间的浓度-发光强度结果进行两点回归拟合得出一次方程，将M+2SD所对应的发光强度代入上述方程，求得的浓度即为最低检测限。本发明提供的试剂盒最低检测限为0.05pg/mL。
2.3批内精密度
用上述制备的TXB 2定量检测试剂盒分别对质控品L(5.0pg/mL)和质控品H(320.0pg/mL)进行批内精密度测试。取同一批试剂盒，平行测定质控品L和质控品H20次，计算所有结果的变异系数。结果如下：
表3.2批内精密度测定结果
  质控品L(pg/mL) 质控品H(pg/mL)
均值 4.99 321.67
标准偏差 0.09 1.18
变异系数％ 1.81 0.37
结果表明本发明的试剂盒质控品L和质控品H的批内精密度变异系数在分别为1.81％和0.37％，性能良好。
2.4批间精密度
用上述制备的TXB 2定量检测试剂盒分别对质控品L和质控品H进行批间精密度测试。取实施例3制备的两批试剂盒，每批试剂盒平行测定质控品L和质控品H 20次，计算所有结果的变异系数。结果如下：
表3.3批间精密度测定结果
  质控品L(pg/mL) 质控品H(pg/mL)
均值 4.99 321.67
标准偏差 0.09 1.29
变异系数％ 1.89 0.40
结果表明本发明的试剂盒质控品L和质控品H的批间精密度变异系数在分别为1.89％和0.40％，性能良好。
2.5样品稳定性
将上述制备的试剂盒放在室温下，于第0、3、5、7天对质控品L和H进行测试，计算第7天与第0天的相对偏差。
表3.4试剂盒加速稳定性结果
Figure PCTCN2020129422-appb-000012
质控品L第7天与第0天的相对偏差为-1.22％，质控品H第7天与第0天的相对偏差为0.16％，试剂的37℃加速破坏能稳定7天，相当于2-8℃稳定一年。
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (10)

  1. 一种用于化学发光免疫法的试剂盒，其特征在于，所述试剂盒包括：包被有待测物的抗原或抗体的免疫磁珠复合物，以及偶联聚合物分子的标记二抗或标记第二抗体，所述聚合物分子上标记有多个光源基团。
  2. 如权利要求1所述用于化学发光免疫法的试剂盒，其特征在于，所述免疫磁珠复合物中的磁珠表面经过活性基团修饰，所述活性基团包括羧基、氨基、醛基、亲和素中的至少一种；
    优选地，所述聚合物分子包括聚苯乙烯、聚甲基丙烯酸、聚甲基丙烯酸甲酯、聚氨基酸和生物大分子中的至少一种；
    优选地，所述聚氨基酸为赖氨酸；
    优选地，所述生物大分子为牛血清白蛋白或血蓝蛋白；
    优选地，所述光源基团包括化学发光基团和荧光基团中的至少一种；
    优选地，所述化学发光基团包括吖啶酯、鲁米诺类衍生物中的至少一种；
    优选地，所述荧光基团包括异硫氰酸荧光素、四乙基罗丹明、藻红蛋白、7-氨基-4甲基香豆素、Eu3+螯合物中的至少一种。
  3. 如权利要求2所述用于化学发光免疫法的试剂盒，其特征在于，当采用化学发光基团作为光源基团时，所述试剂盒中还包括化学发光引发剂；
    优选地，所述化学发光引发剂包括H 2O 2。
  4. 一种如权利要求1-3任一项所述试剂盒的制备方法，其特征在于，包括：
    免疫磁珠复合物的制备：包括将含有待测物的抗原或抗体的包被液加入经过活性基团修饰的磁珠中，将待测物的抗原或抗体偶联固定于磁珠上，得到包被有待测物的抗原或抗体的免疫磁珠复合物；
    标记二抗或标记第二抗体的制备：对聚合物分子进行保护和活化，并将光源基团标记到活化的聚合物分子上，再与二抗或第二抗体偶联，得到标记二抗或标记第二抗体。
  5. 如权利要求4所述试剂盒的制备方法，其特征在于，所述免疫磁珠复合物的制备，具体步骤包括：
    将经过活性基团修饰的磁珠加入偶联液并反应后，加入含待测物的抗原或抗体的包被液重悬，涡旋混匀反应，磁性分离、去除上清液；用洗涤液洗涤磁珠，磁性分离、去除上清液；加入封闭液，涡旋混匀反应， 磁性分离、去除上清液；加入洗涤液洗涤磁珠，磁性分离、去除上清液；再加入缓冲液，涡旋混匀，既得包被有待测物的抗原或抗体的免疫磁珠复合物。
  6. 如权利要求5所述试剂盒的制备方法，其特征在于，所述标记二抗或标记第二抗体的制备，具体步骤包括：
    (1)保护与活化步骤：包括将聚合物分子置于缓冲液中，在搅拌下加入保护试剂，并在0℃-37℃下反应1-72h；反应完毕后超声、离心、去除上清液，并用洗涤液进行清洗；将所得聚合物分子置于缓冲液中，在搅拌下加入活化试剂，并在0℃-37℃下反应1-72h；反应完毕后超声、离心、去除上清液，并用洗涤液进行清洗，得到活化的聚合物分子；
    (2)信号放大标记物制备步骤：包括将活化的聚合物分子置于缓冲液中，在搅拌下加入光源基团，并在0℃-37℃下反应1-72h；反应完毕后超声、离心、去除上清液，并用洗涤液进行清洗；将所得活化的聚合物分子置于脱保护试剂中，在0℃-37℃下反应1-72h；反应完毕后超声、离心、去除上清液，并用洗涤液进行清洗，得到光源基团标记的聚合物分子，即信号放大标记物；
    (3)获得标记二抗或标记第二抗体步骤：包括将二抗或第二抗体置于缓冲液中，在搅拌下加入信号放大标记物，并在0℃-37℃下反应1-72h；反应完毕后超声、离心、去除上清液，并用洗涤液进行清洗，对二抗或第二抗体进行纯化，得到标记有多个光源基团的标记二抗或标记第二抗体。
  7. 如权利要求6所述试剂盒的制备方法，其特征在于，所述免疫磁珠复合物的制备中，待测物的抗原或抗体与磁珠偶联时所采用的偶联液包括EDC溶液和NHS溶液；
    优选地，所述包被液包括Na 2CO 3溶液、NaHCO 3溶液中的至少一种；
    优选地，所述标记二抗或第二抗体的制备中，所述保护试剂包括二碳酸二叔丁酯、对甲苯磺酰氯、乙酸酐、二苯甲酮中的至少一种；
    优选地，所述脱保护试剂包括乙酸、盐酸中的至少一种；
    优选地，所述活化试剂包括N,N'-二环己基碳二亚胺、1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐、N,N'-二异丙基碳二亚胺、N-羟基琥珀酰亚胺中的至少一种；
  8. 如权利要求6所述试剂盒的制备方法，其特征在于，所述免疫磁珠复合物的制备和所述标记二抗或第二抗体的制备中，所述缓冲液包括Tris-HCl缓冲液、PBS缓冲液、HEPES缓冲液中的至少一种。
  9. 一种采用如权利要求1-3任一项所述试剂盒或者如权利要求4-8任一项所述制备方法制备的试剂盒对待测样品进行检测的方法，其特征在于，包括将所述免疫磁珠复合物与待测样品混合，进行免疫反应；免疫反应完成后，通过磁性分离获得免疫复合物；向免疫复合物中加入标记有多个光源基团的二抗或第二抗体，使二抗或第二抗体与所述免疫复合物结合，形成新的免疫复合物；磁性分离所述新的免疫复合物，根据所标记的光源基团，在化学反应或激发波长下，检测所述新的免疫复合物产生的光信号，通过光信号的强度检测待测样品中抗原或抗体的含量。
  10. 一种如权利要求1-3任一项所述试剂盒或者如权利要求4-8任一项所述制备方法制备的试剂盒在检测TXB 2、sST2或PD-1单抗中的应用。
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