WO2023087942A1 - Échantillon d'essai immuno-enzymatique numérique (delisa) capable d'augmenter la proportion de microbilles efficaces, et procédé de préparation et de détection associé - Google Patents

Échantillon d'essai immuno-enzymatique numérique (delisa) capable d'augmenter la proportion de microbilles efficaces, et procédé de préparation et de détection associé Download PDF

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WO2023087942A1
WO2023087942A1 PCT/CN2022/122482 CN2022122482W WO2023087942A1 WO 2023087942 A1 WO2023087942 A1 WO 2023087942A1 CN 2022122482 W CN2022122482 W CN 2022122482W WO 2023087942 A1 WO2023087942 A1 WO 2023087942A1
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mms
reporter
complex
microbeads
enzyme molecule
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PCT/CN2022/122482
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English (en)
Chinese (zh)
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崔玉峰
王志民
陈夏敏
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上海北昂医药科技股份有限公司
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Priority to KR1020247010847A priority Critical patent/KR20240048570A/ko
Publication of WO2023087942A1 publication Critical patent/WO2023087942A1/fr

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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
    • G01N33/543—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
    • 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
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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
    • G01N33/54326—Magnetic particles

Definitions

  • the invention belongs to the field of immunoanalysis, is an enzyme-linked immunoassay technology, and specifically relates to a digital enzyme-linked immunosorbent assay (Digital Enzyme-linked immunosorbent assay, dELISA) sample that increases the proportion of effective microbeads, and the preparation and processing of the sample. Detection method.
  • a digital enzyme-linked immunosorbent assay Digital Enzyme-linked immunosorbent assay, dELISA
  • ELISA is a high-efficiency analysis and detection method combined with enzyme reaction on the basis of specific immune response, and is widely used in the detection of tumors, infectious diseases and other diseases, as well as food and environment.
  • Rissin et al. proposed the digital ELISA (dELISA) method in 2010 (Rissin et al., Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations.
  • the specific capture antibody is attached to the surface of 2.7 micron microbeads, and after capturing the antigen (the specific use in the comparative literature is the prostate specific antigen PSA), the biotin-labeled detection antibody is bound to the antigen, and the report of further linking the avidin
  • the molecule enzyme molecule
  • the substrate is catalyzed by the enzyme molecule to generate a fluorescent product, which emits fluorescence under the excitation of the excitation light source, and is detected by the optical system.
  • the volume of the micro-pit is about tens of femtoliters, and the extremely small volume ensures a local high concentration of fluorescent molecules, which greatly reduces the interference of background light.
  • One microbead is only connected to one marker (PSA antigen in the comparative literature) molecule and forms an enzyme-linked immune complex is the premise of dELISA.
  • PSA antigen in the comparative literature PSA antigen in the comparative literature
  • dELISA enzyme-linked immune complex
  • the beads are bound to the marker such that for each bead there is either only one chance to bind the marker or no chance to bind. Therefore, only a small percentage (1.5%) of microbeads can form an ELISA complex, which belongs to effective microbeads, and most microbeads (98.5%) are not connected with markers, so ELISA cannot be formed.
  • Complex belonging to the void microbeads.
  • the effective microbeads cannot be separated from the ineffective microbeads, when the microbeads are loaded into the micropits array to form a detection chip, the effective microbeads and the ineffective microbeads must be loaded together at the same time, and the utilization rate of the chip is extremely low.
  • the object of the present invention is to provide a digital enzyme-linked immunosorbent assay (dELISA) sample with an increased effective bead ratio, as well as a preparation and detection method for the sample. That is to provide a method for enriching effective microbeads in the dELISA detection process, which can greatly improve the detection efficiency of the chip.
  • dELISA digital enzyme-linked immunosorbent assay
  • the method converts the markers to be tested into effective reporters, and quantitatively detects, and finally, converts the number of reporters into the number of markers.
  • Reporters can be detected in a variety of ways. If the converted reporter is the enzyme-linked immune complex carried by microbeads, these effective microbeads are loaded into the micropits array, and the detection efficiency of the chip will be greatly improved (if the equivalent microbead loading efficiency is calculated, the effective microbeads of the present invention It is about 100%, the effective microbead of the reference file is 1.5%, and the detection efficiency of the chip is about 100 ⁇ 1.5 ⁇ 66 times of the reference file).
  • One aspect of the present invention provides a method for preparing a dELISA sample that increases the effective bead ratio, comprising the following steps:
  • the avidin variants are bound to the surface of the biotin-coated magnetic microbeads, and then combined with the biotin-labeled detection antibody, so that the avidin-labeled reporter enzyme molecule and the biotin on the surface of the magnetic microbeads Binding to the avidin to form a (detection antibody n -magnetic microbead-reporter enzyme molecule m ) complex; wherein the biotin is combined with the avidin variant in a reversible manner;
  • the (detection antibody n -magnetic microbead-reporter enzyme molecule m ) complex is specifically bound to the capture-marker immune complex immobilized on the inner surface of the container in step (1), and no specificity occurs after washing away.
  • Sexually bound free state (detection antibody n -magnetic microbeads-reporter enzyme molecule m ) complex to obtain (capture-marker-detection antibody n -magnetic microbeads-reporter enzyme molecule m ) immune complex;
  • Dissociation (capture-marker-detection antibody n -magnetic microbeads-reporter enzyme molecule m ) in the immune complex, the avidin variant is connected to the detection antibody and/or magnetic microbeads with biotin , to obtain free state (magnetic microbeads-reporter enzyme molecule m ) and/or (detection antibody-magnetic microbead-reporter enzyme molecule m ) reporter complex;
  • step (4) Load the free state (magnetic microbead-reporter enzyme molecule m ) and/or (detection antibody-magnetic microbead-reporter enzyme molecule m ) reporter complex in the micropit array in step (4), so that Only one (magnetic microbead-reporter enzyme molecule m ) reporter complex is accommodated in each micron pit, and then a substrate is added, and then oil-sealed to obtain the digital ELISA sample;
  • the capture object is an antigen or an antibody
  • n and m are integers not less than 1.
  • Sample preparation of the dELISA detection chip fix the micro-pit array with a diameter of ⁇ in the micro-fluid chamber under the scanner lens and seal it, leaving only 1 sample inlet and 1 sample outlet to run the liquid The inflow and outflow; the free state (magnetic microbeads-reporter enzyme molecule m ) and/or (detection antibody-magnetic microbead-reporter enzyme molecule m ) reporter complex in step (4) is injected into the micrometer through the injection port In the pit array, the relationship between the diameter ⁇ of the micron pit and the diameter d of the magnetic microbead is: d ⁇ 2d, so that only one reporter complex can be accommodated in each micron pit, and with the assistance of an external magnet, the reporter complex Loading it into the micro-pit array to obtain the dELISA detection chip sample;
  • n and m are integers not less than 1.
  • n and m are on average > 1000 molecules.
  • Step (1) prepare the immune complex (Cp-Bm) of the capture object (Cp) and the marker (Bm):
  • Step (2) the preparation of the detection antibody (Ab2) n -magnetic microbead (MMS)-reporter enzyme molecule (EZ) m complex:
  • biotin-labeled Ab2 to the container and incubate fully to make it bind to the free site on the surface AM. Due to the large surface area of MMS, the biotin or avidin that can be immobilized on one MMS is about 5.8 ⁇ 10 4 / ⁇ m 2 , and the surface area of a magnetic bead with a diameter of about 3.5 ⁇ m is 38 ⁇ m 2 , and 2.2 ⁇ 10 6 biotin can be immobilized or avidin molecules, sufficient to link multiple Ab2s and EZs.
  • step (2) Add the (Ab2) n -MMS- EZm complex prepared in step (2) into the container (containing the Cp-Bm complex) in step (1), and fully incubate to make Ab2 specifically bind to Bm, "Cp-Bm-(Ab2) n -MMS-EZ m " immune complexes are formed.
  • the number of (Ab2) n -MMS-(EZ) m complexes should be greater than the number of Cp-Bm immune complexes to ensure that each Bm molecule binds to one (Ab2) n -MMS-(EZ) m complex substances, forming Cp-Bm-(Ab2) n -MMS-(EZ) m immune complexes.
  • Step (4) the elution of effective microbeads:
  • step (3) After step (3), apply favorable elution conditions to the reaction system to dissociate between AM and biotin on Ab2 and/or MMS, MMS is released into the solution, and a free state (MMS-EZ m ) reporter complex is obtained , referred to as the MMS reporter complex, realizes the equimolar conversion of Bm and MMS.
  • the conditions that are beneficial to elution include but are not limited to measures such as changing the pH value of the reaction system and adding excess biotin.
  • the conditions that are beneficial to elution are to adjust the pH value of the reaction system to 8-12 , preferably, the pH value is 10; for the AM of Tamavidin 2-REV, the conditions that are beneficial for elution are the addition of biotin, and the number of molecules of biotin ⁇ 4 times the number of molecules of Tamavidin 2-REV, the added biotin, Competes for the binding site with biotin bound to Tamavidin 2-REV to achieve the purpose of elution.
  • the MMS amount is smaller than the Bm amount of the sample, also known as a near equimolar transition. It needs to be corrected by the standard curve to truly reflect the actual Bm concentration of the sample.
  • the above-mentioned full incubation is to make the reaction sufficient, there is no specific condition referred to, it can be incubated at 37°C for 2 hours, or incubated at 25°C for 10 hours; proper shaking can also further shorten the incubation time.
  • the quantity of the captured substance in step (1) is not less than 100 times the quantity of the marker.
  • AM includes but not limited to CaptAvidin, Tamavidin 2-REV; the ratio of the number of the avidin variant to the detection antibody ranges from 2.2 ⁇ 10 ⁇ 6 to 2.2 ⁇ 10 6 .
  • Avidin variant is a special kind of avidin, that is, the amino acid at a specific position of avidin has been changed to form an avidin variant, which is a characteristic that avidin does not have; step five uses AM Elution is performed by reversibility of binding to biotin.
  • the avidin variant (AM) is CaptAvidin, which is conducive to binding biotin under low pH conditions.
  • the ratio of the number of (detection antibody n -magnetic microbeads-reporter enzyme molecule m ) complexes to the capture-marker immune complexes described in step (3) is not less than 1.
  • the material of the container includes but not limited to polystyrene, polyethylene, polypropylene, glass, etc.; the passivating agent includes but not limited to bovine serum albumin.
  • the reporter enzyme molecule includes but is not limited to one of horseradish peroxidase (HRP), ⁇ -galactosidase ( ⁇ -Gal), and alkaline phosphatase (ALP). species; the immobilization sequence of MMS, EZ and Ab2 described in step (2), can first fix biotin-labeled Ab2 to MMS via AM, and then fix avidin-labeled EZ to magnetic microbeads. It is also possible to reverse the order, fix EZ first, and then fix Ab2.
  • HRP horseradish peroxidase
  • ⁇ -Gal ⁇ -galactosidase
  • ALP alkaline phosphatase
  • the detection of the MMS reporter complex adopts an optical system.
  • the enzyme molecule, HRP, ⁇ -Gal, ALP or other enzymes can effectively catalyze the substrate to generate another product with a different color from the substrate, or a product that can emit fluorescence with a different wavelength from the substrate when excited, Or self-luminous products.
  • the ratio of the number of (Ab2) n -MMS-EZ m complexes to the Cp-Bm immune complexes in step (3) is not less than 1.
  • Another aspect of the present invention also provides a dELISA sample prepared by the above method.
  • Another aspect of the present invention also provides a method for using the above-mentioned sample to perform ELISA detection, comprising the following steps:
  • the specific detection method is:
  • dELISA detection In bright field, the scanner scans and records the number of effective reporter complexes in the chip; in dark field, the substrate of the reporter enzyme molecule is added to the chip through the injection port and oil-sealed, and after sufficient enzymatic reaction, optical scanning is used The system collects and records the emission wavelength of the enzymatic product in each micron pit in the detection chip;
  • the free state reporter complex obtained above was subjected to optical detection for quantification. Load the free-state MMS reporter complex in the container into the pre-prepared array of micropits.
  • the specific method add the MMS reporter complex from the sample inlet of the microfluidic chamber containing the micro-pit array, use the magnet to shuttle back and forth under the micro-pit substrate several times, and load the MMS reporter complex into the micro-pit by magnetic force.
  • the diameter of the micropits can only accommodate one MMS reporter complex, and the number of micropits is much larger than the number of MMS reporter complexes. Therefore, it is basically guaranteed that all MMS reporter complexes can be loaded into the micropits.
  • each bright micro-pit is a positive molecule. Theoretically, the sum of all positive bright spots is close to the number of Bm to be tested, that is, nearly equimolar.
  • the above-mentioned ELISA method is suitable for marker detection of human and animal diseases, food hygiene and environmental pollution.
  • a dELISA sample preparation method that increases the ratio of effective microbeads, converting the marker to be tested (Bm) into microbeads loaded with reporters (enzyme molecules), through the detection of optically detectable products produced by enzyme-catalyzed substrates, to achieve For the accurate quantification of Bm to be measured, compared with traditional methods, this method has the following advantages:
  • the surface of the inner wall with a sufficiently large surface area in step (1) is used as a solid support carrier to immobilize the capture antibody (Cp), and in step (2), avidin variants with significant differences in binding ability under different pH and temperature conditions are used (AM) replaces streptavidin or avidin to ensure the desorption of AM in step (4) and the separation of microbeads from the reaction vessel; Bead separation, that is, the enrichment of effective microbeads, improves the loading efficiency of the micro-pit array during the detection process. Theoretically, the loading efficiency is equivalent to 66 times that of the reference file (100 ⁇ 1.5 ⁇ 66).
  • the traditional method combines avidin-labeled EZ with a biotin-labeled detection antibody (Ab2).
  • Ab2 forms an immune complex with Bm and Cp, and Ab2 only binds to one EZ molecule.
  • EZ is directly bound to the surface of biotin-coated magnetic microbeads. Biotin-coated magnetic microbeads provide more binding sites, and the number of EZ bound under optimal conditions can be increased by 1000 times. Above, this can further increase the sensitivity of the dELISA method.
  • Fig. 1 is the specific operation flowchart of ELISA detection method of the present invention
  • Fig. 2 is a schematic diagram of the preparation of the reporter complex in the free state (magnetic microbeads-reporter enzyme molecule m ) in the present invention.
  • Raw materials and equipment used in this application are commonly used raw materials and equipment in this field, all from commercially available products.
  • the methods used in this application, unless otherwise specified, are conventional methods in the art.
  • compositions eg, media
  • methods include the listed elements, but do not exclude other elements.
  • Consisting essentially of when used to define compositions and methods means excluding other elements of any significance to the combination for the stated purpose. Accordingly, a composition consisting essentially of the elements defined herein does not exclude other materials or steps which do not materially affect the basic and novel characteristics of the claimed application.
  • Consisting of means trace elements and substantial method steps excluding other constituents. Embodiments defined by each of these transitional terms are within the scope of this application.
  • This embodiment provides a method for converting a human CD40 antigen marker to a ⁇ -galactosidase ( ⁇ -Gal) reporter, as follows.
  • Fig. 1 The specific operation flow chart is shown in Fig. 1, and Fig. 2 is the illustration of the preparation of the MMS reporter complex.
  • Step 1 prepare the immune complex (Cp-Bm) of the capture antibody (Cp) and the antigen (Bm):
  • Activation treatment of the inner surface of the container made of polystyrene wash the inner wall of the container twice with PBST (0.01M phosphate buffer containing 0.1% Tween 20) solution; wash the 25% glutaraldehyde solution with PBS (phosphoric acid Salt buffer solution) was diluted to 8% (v/v) glutaraldehyde solution, 350 ⁇ L of 8% glutaraldehyde solution was added to the container, shaken at room temperature for 5 hours, and the inner surface of the container was repeatedly washed twice with PBST.
  • PBST phosphate buffer containing 0.1% Tween 20
  • Cp specifically binds to Bm: add Bm (human CD40 antigen) into the container, shake and react at room temperature for 5 hours, and specifically combine with Cp to form Cp-Bm.
  • Bm human CD40 antigen
  • the amount of Bm should be less than the amount of Cp (1 ⁇ Bm The number of molecules ⁇ the number of molecules of Cp), to ensure that all of Bm is combined with Cp.
  • Step 2 immobilize the detection antibody (Ab2) and the enzyme molecule (EZ) as a reporter on the surface of biotin-coated magnetic microbeads (MMS):
  • step 2 Add the (Ab2) n -MMS-(EZ) m complex prepared in step 2 into the container (containing the Cp-Bm complex) in step 1, shake and mix at room temperature for 2 hours, and incubate fully to make the formation of Ab2 and Bm Specifically combined to form Cp-Bm-(Ab2) n -MMS-(EZ) m immune complex.
  • the number of (Ab2) n -MMS-(EZ) m complexes should be greater than that of Cp-Bm, so as to ensure that each Bm molecule combines with one (Ab2) n -MMS-(EZ) m complexes to form Cp-Bm-(Ab2) n -MMS-(EZ) m immune complex.
  • MMS MMS
  • the immobilized Cp-Bm-(Ab2) n -MMS-(EZ) m immune complex (effective microbeads)
  • the unbound free state (Ab2) n -MMS -(EZ) m complex
  • the solution in the container was decanted and washed 3 times with PBST.
  • the invalid microbeads are removed by washing, leaving only the effective microbeads in the container.
  • the specific method add the MMS reporter complex from the sample inlet of the microfluidic chamber containing the micro-pit array, use the magnet to shuttle back and forth several times under the micro-pit substrate, and load the MMS reporter complex into the micro-pit by magnetic force.
  • the diameter of the micropits can only accommodate one MMS reporter complex, and the number of micropits is much larger than the number of MMS reporter complexes. Therefore, it is basically guaranteed that all MMS reporter complexes can be loaded into the micropits.
  • Step seven establish a standard curve:
  • This embodiment provides a method for converting a new crown antigen marker to a horseradish peroxidase (HRP) reporter, as follows:
  • Step 1 prepare the immune complex (Cp-Bm) of capture antibody (Cp, recombinant mouse anti-SARS-CoV-2N monoclonal antibody) and antigen (Bm, new coronavirus N recombinant protein):
  • Activation treatment of the inner surface of the container made of polyethylene material clean the inner wall of the container twice with PBST (0.01M phosphate buffer saline containing 0.1% Tween 20) solution; wash 25% glutaraldehyde solution with PBS (phosphate buffered saline) buffer solution) was diluted to 8% (v/v) glutaraldehyde solution, 300 ⁇ L of 8% glutaraldehyde solution was added to the container, shaken at room temperature for 5 hours, and the surface of the solid phase carrier was repeatedly washed twice with PBST.
  • PBST phosphate buffer saline containing 0.1% Tween 20
  • Cp specifically binds to Bm: add Bm (new coronavirus N recombinant protein) into the container, shake and react at room temperature for 5 hours, and specifically combine with Cp to form Cp-Bm.
  • Bm new coronavirus N recombinant protein
  • the amount of Bm should be less than the amount of Cp (1 ⁇ The number of molecules of Bm ⁇ the number of molecules of Cp), to ensure that all of Bm is combined with Cp.
  • Step 2 biotin-coated magnetic microbeads (MMS) surface immobilization detection antibody (Ab2, rabbit anti-SARS-CoV-2N polyclonal antibody) and enzyme molecule (EZ) as reporter:
  • step 2 Add the (Ab2) n -MMS-(EZ) m complex prepared in step 2 into the container (containing the Cp-Bm complex) in step 1, shake and mix at room temperature for 2 hours, and incubate fully to make the formation of Ab2 and Bm Specifically combined to form Cp-Bm-(Ab2) n -MMS-(EZ) m immune complex.
  • the number of (Ab2) n -MMS-(EZ) m complexes should be greater than that of Cp-Bm, so as to ensure that each Bm molecule combines with one (Ab2) n -MMS-(EZ) m complexes to form Cp-Bm-(Ab2) n -MMS-(EZ) m immune complex.
  • MMS MMS
  • the immobilized Cp-Bm-(Ab2) n -MMS-(EZ) m immune complex (effective microbeads)
  • the unbound free state (Ab2) n -MMS -(EZ) m complex
  • the solution in the container was decanted and washed 3 times with PBST.
  • the invalid microbeads are removed by washing, leaving only the effective microbeads in the container.
  • the diameter of the micropits can only accommodate one MMS reporter complex, and the number of micropits is much larger than the number of MMS reporter complexes. Therefore, it is basically guaranteed that all MMS reporter complexes can be loaded into the micropits.
  • Step seven establish a standard curve:
  • the present embodiment provides a method for converting human prostate-specific antigen (PSA) to horseradish peroxidase (HRP) reporter, specifically as follows:
  • Step 1 prepare the immune complex (Cp-Bm) of capture antibody (Cp, PSA monoclonal antibody, derived from mouse) and antigen (Bm, PSA):
  • Activation treatment of the inner surface of the container made of polypropylene wash the inner wall of the container twice with PBST (0.01M phosphate buffer containing 0.1% Tween 20) solution; wash 25% glutaraldehyde solution with PBS (phosphate buffered saline) Buffer) was diluted to 8% (v/v) glutaraldehyde solution, and 300 ⁇ L of 8% glutaraldehyde solution was added to the container, shaken at room temperature for 5 hours, and the inner surface of the container was repeatedly washed twice with PBST.
  • PBST phosphate buffer containing 0.1% Tween 20
  • PBS phosphate buffered saline
  • Cp specifically binds to Bm: add Bm (PSA) to the container, shake and react at room temperature for 5 hours, and specifically combine with Cp to form Cp-Bm, the amount of Bm should be less than the amount of Cp (1 ⁇ the number of molecules of Bm ⁇ the number of molecules of Cp), to ensure that all of Bm is combined with Cp.
  • PSA Bm
  • Cp-Bm the amount of Bm should be less than the amount of Cp (1 ⁇ the number of molecules of Bm ⁇ the number of molecules of Cp), to ensure that all of Bm is combined with Cp.
  • Step 2 immobilize the detection antibody (Ab2, PSA polyclonal antibody, derived from rabbit) and the enzyme molecule (EZ) as a reporter on the surface of biotin-coated magnetic microbeads (MMS):
  • Biotin-labeled Ab2 (PSA polyclonal antibody, derived from rabbit) was added to the test tube, shaken and reacted at room temperature for 5 hours, and fully incubated to bind Ab2 to the free site on the AM of the MMS surface. Due to the large surface area of MMS, multiple Ab2s and EZs can be attached to one MMS.
  • step 2 Add the (Ab2) n -MMS-(EZ) m complex prepared in step 2 into the container (containing the Cp-Bm complex) in step 1, shake and mix at room temperature for 2 hours, and incubate fully to make the formation of Ab2 and Bm Specifically combined to form Cp-Bm-(Ab2) n -MMS-(EZ) m immune complex.
  • the number of (Ab2) n -MMS-(EZ) m complexes should be greater than that of Cp-Bm, so as to ensure that each Bm molecule combines with one (Ab2) n -MMS-(EZ) m complexes to form Cp-Bm-(Ab2) n -MMS-(EZ) m immune complex.
  • MMS MMS
  • the immobilized Cp-Bm-(Ab2) n -MMS-(EZ) m immune complex (effective microbeads)
  • the unbound free state (Ab2) n -MMS -(EZ) m complex
  • the solution in the container was decanted and washed 3 times with PBST.
  • the invalid microbeads are removed by washing, leaving only the effective microbeads in the container.
  • the diameter of the micropits can only accommodate one MMS reporter complex, and the number of micropits is much larger than the number of MMS reporter complexes. Therefore, it is basically guaranteed that all MMS reporter complexes can be loaded into the micropits.
  • Step seven establish a standard curve:
  • This embodiment provides a method for switching the SARS-CoV-2 Spike protein IgM antibody marker of the new crown to an alkaline phosphatase (ALP) reporter, as follows:
  • Step 1 prepare the immune complex (Cp-Bm) of the capture object (Cp, SARS-CoV-2 Spike protein) and marker (Bm, SARS-CoV-2 Spike protein IgM antibody):
  • Activation treatment of the inner surface of the container made of glass wash the inner wall of the container twice with PBST (0.01M phosphate buffered saline containing 0.1% Tween 20) solution; wash 25% glutaraldehyde solution with PBS (phosphate buffered saline) solution) diluted to 8% (v/v) glutaraldehyde solution, add 300 ⁇ L of 8% glutaraldehyde solution to the container, shake at room temperature for 5 hours, and wash the inner surface of the container twice with PBST.
  • PBST phosphate buffered saline containing 0.1% Tween 20
  • Cp specifically binds to Bm: add Bm (SARS-CoV-2 Spike protein IgM antibody) to the container, shake and react at room temperature for 5 hours, and specifically combine with Cp to form Cp-Bm, the amount of Bm should be less than that of Cp amount (1 ⁇ number of molecules of Bm ⁇ number of molecules of Cp), to ensure that all of Bm is combined with Cp.
  • Bm SARS-CoV-2 Spike protein IgM antibody
  • Step 2 immobilize the detection antibody (Ab2, anti-IgM antibody) and the enzyme molecule (EZ) as a reporter on the surface of the biotin-coated magnetic microbeads (MMS):
  • step 2 Add the (Ab2) n -MMS-(EZ) m complex prepared in step 2 into the container (containing the Cp-Bm complex) in step 1, shake and mix at room temperature for 2 hours, and incubate fully to make Ab2 and Cp Specifically combined to form Cp-Bm-(Ab2) n -MMS-(EZ) m immune complex.
  • the quantity of (Ab2)n-MMS-(EZ)m complex will be greater than that of Cp-Bm, so as to ensure that each Cp molecule binds to an (Ab2)n-MMS-(EZ)m complex to form Cp-Bm-(Ab2)n-MMS-(EZ)m immune complex.
  • MMS MMS
  • the immobilized Cp-Bm-(Ab2) n -MMS-(EZ) m immune complex (effective microbeads)
  • the unbound free state (Ab2) n -MMS -(EZ) m complex
  • the solution in the container was decanted and washed 3 times with PBST.
  • the invalid microbeads are removed by washing, leaving only the effective microbeads in the container.
  • the diameter of the micropits can only accommodate one MMS reporter complex, and the number of micropits is much larger than the number of MMS reporter complexes. Therefore, it is basically guaranteed that all MMS reporter complexes can be loaded into the micropits.
  • Step seven establish a standard curve:

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Abstract

L'invention concerne un échantillon d'essai immuno-enzymatique numérique (dELISA) capable d'augmenter la proportion de microbilles efficaces, et un procédé de préparation et de détection associé. Le procédé de préparation et de détection comprend : l'immobilisation d'une capture (Cp) sur un site actif de la surface interne d'un conteneur, de sorte qu'un marqueur à détecter est lié à la capture pour former un complexe immunitaire capture-marqueur ; l'immobilisation d'un anticorps de détection (Ab2) et d'une molécule d'enzyme rapporteur (EZ) sur la surface d'une microbille magnétique recouverte de biotine (MMS) et liée à une variante d'avidine ; la fixation spécifique d'un complexe (anticorps de détection n-microbille magnétique-molécule d'enzyme rapporteur m) au complexe immunitaire capture-marqueur lier spécifiquement un complexe (anticorps de détection n-microbille magnétique-molécule d'enzyme rapporteur m) au complexe immunitaire de capture-marqueur ; le chargement d'un complexe rapporteur à l'état libre (microbille magnétique-molécule d'enzyme rapporteur m) et/ou (anticorps de détection n-microbille magnétique-molécule d'enzyme rapporteur m) obtenu après dissociation dans un réseau de micro-cuvettes ; et ajouter un substrat pour la détection optique. L'efficacité de détection de puce peut être considérablement améliorée.
PCT/CN2022/122482 2021-11-18 2022-09-29 Échantillon d'essai immuno-enzymatique numérique (delisa) capable d'augmenter la proportion de microbilles efficaces, et procédé de préparation et de détection associé WO2023087942A1 (fr)

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