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WO2024241780A1 - Procédé permettant de détecter la quantité d'une première structure et la quantité d'une seconde structure - Google Patents

Procédé permettant de détecter la quantité d'une première structure et la quantité d'une seconde structure Download PDF

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Publication number
WO2024241780A1
WO2024241780A1 PCT/JP2024/015380 JP2024015380W WO2024241780A1 WO 2024241780 A1 WO2024241780 A1 WO 2024241780A1 JP 2024015380 W JP2024015380 W JP 2024015380W WO 2024241780 A1 WO2024241780 A1 WO 2024241780A1
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Prior art keywords
label
amount
bound
binding
nucleic acid
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PCT/JP2024/015380
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English (en)
Japanese (ja)
Inventor
哲生 鳥巣
進 内山
ソシュ，セレイラス
未香子 ▲高▼倉
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国立大学法人大阪大学
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Publication of WO2024241780A1 publication Critical patent/WO2024241780A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention relates to a method for detecting the amount of a first structure and the amount of a second structure, a method for evaluating the ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid, and a kit for use in these methods.
  • Non-Patent Document 2 https://darkhorseconsultinggroup.com/wp-content/uploads/2022/2017DHC_Proposed-DRAFT-Guidance-for-FDA-Consideration.pdf.
  • This method has concerns such as errors in the complete particle/empty particle ratio due to the combination of two different mechanisms, and the need to quantify complete particles and empty particles using samples that are not strictly the same. In addition, performing two measurements requires time and effort.
  • the method of the present invention for detecting the amount of a first structure and the amount of a second structure includes the steps of binding a first label to the first structure, releasing the second structure from the first structure, binding a second label to the second structure, and jointly detecting the amount of the bound first label and the amount of the bound second label, where the second structure is contained within the first structure, and the first label and the second label are different but can be detected together.
  • the method for assessing the ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid includes the steps of immobilizing the viral capsid on a support, indirectly binding a first label to the viral capsid via an antibody that binds to the viral capsid, washing away the unbound first label, releasing nucleic acid from the viral capsid, binding a second label to the released nucleic acid, detecting the amount of the bound first label and the amount of the bound second label together, and calculating or determining the ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid based on the amount of the bound first label and the amount of the bound second label, wherein the nucleic acid is encapsulated in the viral capsid, and the first label and the second label can be detected together but are different.
  • the kit according to the present invention includes a first label for a first structure, a second label for a second structure, and instructions for detecting the amount of bound first label and the amount of bound second label together, where the second structure is contained within the first structure, and the first label and the second label are different but can be detected together.
  • the present invention provides the following: [1] A method for detecting an amount of a first structure and an amount of a second structure, the method comprising: Binding a first label to the first structure; Releasing the second structure from the first structure; binding a second label to the second structure and jointly detecting the amount of bound first label and the amount of bound second label; Including, a second structure is contained within the first structure, and the first label and the second label are detectable together but are different; method. [2] The method according to [1], further comprising the step of washing away the first label that has not bound to the first structure.
  • the first structure is selected from the group consisting of a virus particle, a virus capsid, a liposome, a lipid nanoparticle (LNP), and a polymer particle.
  • the virus is selected from the group consisting of adeno-associated virus (AAV), retrovirus, lentivirus, and adenovirus.
  • AAV adeno-associated virus
  • retrovirus retrovirus
  • lentivirus lentivirus
  • adenovirus adenovirus
  • step of binding the first label to the first structure comprises indirect binding via a molecule that specifically binds to one or more first structures, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide, or comprises indirect binding via the molecule that specifically binds to the first structure and a secondary antibody that specifically binds to the molecule.
  • step of jointly detecting the amount of bound first label and the amount of bound second label comprises detecting them together using one device or detector.
  • a method for evaluating a ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid comprising: Immobilizing the viral capsid on a support; Indirectly binding a first label to the viral capsid via an antibody that binds to the viral capsid; washing away unbound first label; Releasing nucleic acid from the viral capsid; binding a second label to the released nucleic acid; detecting the amount of bound first label and the amount of bound second label together, and calculating or determining the ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid based on the amount of bound first label and the amount of bound second label; Including, the nucleic acid is encapsulated in a viral capsid, and the first label and the second label are detectable together but are different, method.
  • a first marker for a first structure a first marker for a first structure; a second label on the second structure and instructions for jointly detecting the amount of bound first label and the amount of bound second label;
  • a detection kit comprising: a second structure is contained within the first structure, and the first label and the second label are detectable together but are different; Detection kit.
  • the method of the present invention includes a step of detecting the amount of the first label bound to the first structure and the amount of the second label bound to the second structure together.
  • the method of the present invention can, for example, reduce the time or effort required to detect the amount of the first structure and the amount of the second structure, reduce the cost of detecting the amount of the first structure and the amount of the second structure, reduce the detection error of the amount of the first structure and the amount of the second structure, eliminate the need to purify the first structure or the second structure from a sample containing impurities, or make it possible to detect the amount of the first structure and the amount of the second structure at low concentrations.
  • Example 1 is a simplified flow chart of one embodiment of the method of the present invention.
  • 1 is a simplified flow chart of one embodiment of the method of the present invention.
  • Test results from Example 1 using AAV8. Test results from Example 2 using AAV2.
  • Test results from Example 3 using a mixture of AAV2 and AAV8.
  • Test results from Example 4 using Quantifluor.
  • Test results from Example 5 using AAV2 containing ssDNA and AAV2 containing scDNA.
  • Test results according to Example 6 using conventional wavelengths of 495 nm and 535 nm and wavelengths of 500 nm and 535 nm by the inventors.
  • A Quantification of capsid titer.
  • B Quantification of genome titer.
  • AAV8 content in unpurified samples was analyzed by three methods: dFLISA, ELISA, and dPCR.
  • dFLISA was used to quantify capsid and genome titers and to calculate the FP/EP ratio of AAV8 content in unpurified samples.
  • the present invention provides a method for detecting the amount (or concentration) of a first structure and the amount (or concentration) of a second structure, the method comprising the steps of binding a first label to the first structure, binding a second label to the second structure, and jointly detecting the amount of bound first label and the amount of bound second label, wherein the first label and the second label can be detected together but are different.
  • the present invention provides a method for detecting the amount (or concentration) of a first structure and the amount (or concentration) of a second structure, the method comprising the steps of binding a first label to the first structure, releasing the second structure from the first structure, binding a second label to the second structure, and jointly detecting the amount (or concentration) of the bound first label and the amount (or concentration) of the bound second label, wherein the second structure is contained within the first structure, and the first label and the second label are different but can be detected together.
  • the present invention provides a method for evaluating the ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid, the method comprising the steps of immobilizing the viral capsid on a support, indirectly binding a first label to the viral capsid via an antibody that binds to the viral capsid, washing away the unbound first label, releasing nucleic acid from the viral capsid, binding a second label to the released nucleic acid, detecting the amount (or concentration) of the bound first label and the amount (or concentration) of the bound second label together, and calculating or determining the ratio of viral capsids containing nucleic acid to viral capsids not containing nucleic acid based on the amount (or concentration) of the bound first label and the amount (or concentration) of the bound second label, wherein the nucleic acid is encapsulated in the viral capsid, and the first label and the second label can be detected together but are different.
  • the present invention provides a kit comprising a first label on a first structure, a second label on a second structure, and instructions for detecting the amount (or concentration) of the bound first label and the amount (or concentration) of the bound second label together, where the second structure is contained within the first structure, and the first label and the second label are different but can be detected together.
  • the "first structure” may be composed of any material or substance, for example, a protein, lipid, or polymer, or a combination thereof, for example, a virus particle, a virus capsid, a liposome, a lipid nanoparticle (LNP), and a polymer particle.
  • the “first structure” may be used as a tool for treating or diagnosing a disease or for in vivo gene transfer.
  • the "first structure” may include a "second structure”.
  • the “first structure” may not include a "second structure”.
  • the “first structure” may be a "first structure” that includes a "second structure” and/or a "first structure” that does not include a "second structure”.
  • the "first structure” may be bound to a "second structure”.
  • the “first structure” may not be bound to a “second structure”.
  • the “first structure” may be a "first structure” that is bound to a "second structure” and/or a “first structure” that is not bound to a "
  • Viruses include any virus, such as adeno-associated viruses (AAV), retroviruses, lentiviruses and adenoviruses, and those viruses with modified viral capsid proteins.
  • AAV includes natural adeno-associated viruses or recombinant adeno-associated viruses.
  • AAV includes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 and AAV-11.
  • Adeno-associated virus belongs to the Parvoviridae family.
  • the AAV genome consists of a linear single-stranded DNA molecule of approximately 4.7 kilobases (kb) and consists of two major open reading frames encoding the nonstructural Rep (replication) protein and the structural Cap (capsid) protein.
  • Recombinant vectors derived from AAV are used as tools for disease treatment or diagnosis, etc.
  • Adeno-associated virus (AAV) derived viral vectors are used as tools for in vivo gene transfer due to their superior in vivo efficiency, broad tissue tropism and excellent safety profile compared to other vectors.
  • Active (including DNA, etc.) adeno-associated virus (AAV) particles complete particles may be used as tools for disease treatment or diagnosis or in vivo gene transfer.
  • the "second structure” may be composed of any material or ingredient, for example, a nucleic acid, a protein, or a compound, or a combination thereof, for example, DNA, single-stranded DNA (ssDNA), self-complementary DNA (scDNA), RNA, miRNA, lncRNA, peptide, antibody, detection compound, and drug.
  • the "second structure” includes, for example, those that can be applied to the treatment, prevention, or diagnosis of a disease.
  • the disease may be cancer, an autoimmune disease, an infectious disease, etc.
  • the "second structure” may be an introduced gene, a transcription control gene, a gene therapy agent, a nucleic acid encoding a small molecule, a nucleic acid encoding an antibody, a nucleic acid encoding a protein, a small interfering RNA, an antisense RNA, a ribozyme, a nucleic acid encoding a therapeutic protein, a nucleic acid for genome editing, a nucleic acid for gene recombination, and a microRNA (miRNA), etc.
  • the “second structure” may be included in the “first structure”.
  • the “second structure” may be bound to the "first structure”.
  • the term "encompassing" in the context of "a second structure is contained in a first structure,” "a first structure containing a second structure,” or “a first structure not containing a second structure” means a structure or state in which a first structure contains a second structure.
  • a second structure is contained in a first structure means that a second structure is present inside a first structure.
  • the second structure may or may not be attached to the inner surface of the first structure.
  • a first structure not containing a second structure means that a second structure is not present inside the first structure.
  • label includes any detectable signal source, such as fluorescent, visible, optical, colorimetric, dyes, enzymes, GCMS tags, avidin, biotin, and radioactive substances (radioactive labels and radiopaque, etc.).
  • label includes various molecules such as dyes (fluorescent dyes) that emit fluorescent signals, radioactive labels, molecules that emit MRI signals, and molecules that emit ultrasound signals, as well as various microparticles such as iron oxide microparticles, gold microparticles, gold nanorods, and microparticles of metals and metal oxides such as platinum and silver.
  • the "label” can be detected by an apparatus or detector based on the properties, activity, strength, or amount of the label.
  • label may be only a detectable signal source, or may be a protein, antibody, peptide, probe, etc. that has a detectable signal source.
  • the fluorescent dye may be any dye that emits a fluorescent signal, such as commercially available fluorescent dyes, known fluorescent dyes, Alexa Fluor (registered trademark) 647, SYBR (registered trademark) Gold, SYBR (registered trademark) green, QuantiFluor (registered trademark) ssDNA Dye Systems, fluorescein, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, SYBR Gold, SYBR Green I, SYBR Green II, SYBR Safe, or QuantiFluor ssDNA Dye Systems (QuantiFluor dsDNA, RNA or ssDNA System).
  • fluorescent dyes such as commercially available fluorescent dyes, known fluorescent dyes
  • the fluorescent dye can be detected by a light sensor, a light detector, a microscope, a device or a detector, etc., based on the wavelength, characteristic, activity, intensity or amount of the fluorescent signal or fluorescent dye.
  • a light sensor a light detector, a microscope, a device or a detector, etc.
  • Those skilled in the art can appropriately select an optical sensor, optical detector, microscope, device, detector, etc. based on the wavelength, characteristics, activity, intensity, or amount of the fluorescent signal or fluorescent dye.
  • Radioactive labels include any label that emits radioactive substances, for example, commercially available radioactive labels, known radioactive labels, 18F, 32P, 35S, 129I, 131I, 64Cu, or 67Cu. Radioactive labels can be detected by a radiation or radioactivity sensor, radiation or radioactivity detector, device, or detector, etc., based on the characteristics, activity, strength, or amount of the radioactive label. Those skilled in the art can appropriately select a radiation or radioactivity sensor, radiation or radioactivity detector, device, or detector, based on the characteristics, activity, strength, or amount of the radioactive label.
  • a "first label for a first structure” or a “first label” is directly or indirectly bound to a first structure.
  • a "first label for a first structure” or a “first label” may be indirectly bound to a first structure via a molecule that specifically binds to one or more first structures, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide.
  • a "first label for a first structure” or a “first label” may be indirectly bound to a first structure via an antibody that specifically binds to the first structure and a secondary antibody that specifically binds to the antibody.
  • a "first label for a first structure” or a “first label” may be a label that is not destroyed or malfunctioned, or is not easily destroyed or malfunctioned, in a method that includes a step of releasing a second structure from a first structure.
  • the "first label for the first structure” or the “first label” may be a label that is not destroyed or disabled, or is not easily destroyed or disabled, in the step of washing away the first label that is not bound to the first structure when the method includes the step of washing away the first label that is not bound to the first structure.
  • the first structure may have one or more additional labels attached to it in addition to the "first label for the first structure" or the "first label".
  • the "first label for the first structure” or the “first label” may be a fluorescent dye or a radioactive label.
  • the "first label for the first structure” or the “first label” may be, for example, a commercially available fluorescent dye, a known fluorescent dye, Alexa Fluor (registered trademark) 647, fluorescein, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 5, or a similar fluorescent dye.
  • the "second label for the second structure” or “second label” is directly or indirectly bound to the second structure.
  • the "second label for the second structure” or “second label” may be indirectly bound to the second structure via one or more molecules that specifically bind to the second structure, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide.
  • the "second label for the second structure” or “second label” may be indirectly bound to the second structure via an antibody that specifically binds to the second structure and a secondary antibody that specifically binds to the antibody.
  • the "second label for the second structure” or “second label” may be a label that generates a different detectable signal when bound to the second structure compared to when not bound to the second structure.
  • the "second label for the second structure” or “second label” may be a label that generates a detectable signal when bound to the second structure, but does not generate a detectable signal when not bound to the second structure.
  • a “second label for a second structure” or a “second label” may be a label that generates a greater or lesser or different detectable signal when bound to the second structure than when not bound to the second structure.
  • the second structure may have one or more additional labels bound to it in addition to the "second label for a second structure" or the "second label”.
  • a “second label for a second structure” or a “second label” may be a fluorescent dye or a radioactive label.
  • the "second label for the second structure” or the “second label” may be, for example, a commercially available fluorescent dye, a known fluorescent dye, SYBR (registered trademark) Gold, SYBR (registered trademark) green, QuantiFluor (registered trademark) ssDNA Dye Systems, fluorescein, SYBR Gold, SYBR Green I, SYBR Green II, SYBR Safe, QuantiFluor ssDNA Dye Systems (QuantiFluor dsDNA, RNA, etc.) or ssDNA System), SYPRO orange, Thioflavin T, SYPRO orange, SYPRO Tangerine, SYPRO Red, SYPRO Ruby, SYTOX Blue, SYTOX Green, SYTOX Orange, SYTOX Red, SYTOX AADvanced, or Hoechst, for example Hoechst
  • the "first label for the first structure” or the "first label” and the “second label for the second structure” or the “second label” are different labels that can be detected together.
  • the "first label” and the “second label” are labels that can be detected together using one or more devices or detectors, and a detection result of each label of the first structure and the second structure can be obtained.
  • the "first label” and the “second label” are fluorescent dyes that emit different fluorescent signals and can be detected together using one or more microscopes, devices or detectors, and a detection result of the fluorescent signal of each fluorescent dye of the first label and the second label can be obtained based on, for example, each wavelength.
  • the "first label” and the "second label” can be detected together using one device or detector.
  • a person skilled in the art can appropriately select the "first label” and the "second label” based on the characteristics of the first structure, the second structure, the first label, the second label, the device, the detector, or the detection result.
  • detecting together means detecting two or more substances to be detected in one space or location that contains them (e.g., one sample, one container, or one well in a plate).
  • detecting the amount of bound first label and the amount of bound second label together may mean detecting the amounts of each of the first label and the second label in one space or location that contains the bound first label and the bound second label (e.g., one sample, one container, or one well in a plate).
  • detecting the amount of bound first label and the amount of bound second label together may mean detecting the amount of each of the first label and the second label simultaneously, or sequentially or sequentially (e.g., detecting the amount of each of the first label and the second label simultaneously, detecting the amount of the first label first and then the amount of the second label, or detecting the amount of the second label first and then the amount of the first label).
  • a “kit” includes a first label for a first structure, a second label for a second structure, and instructions for detecting the amount of bound first label and the amount of bound second label together.
  • the “kit” may be a kit for use in any method of detecting the amount of a first structure and the amount of a second structure described herein.
  • the “kit” may further include any peptide, protein, antibody, nucleic acid, additional label, enzyme, substrate, reagent, buffer, salt, plate, or container, etc.
  • the method of the invention includes a step of immobilizing or binding the first structure and/or the second structure to a support before or after the step of binding the first label and/or the step of binding the second label.
  • the first structure and/or the second structure may be indirectly bound to the support via one or more substances, such as a molecule that specifically binds to the first structure and/or the second structure, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide.
  • the step of indirectly immobilizing the first structure and/or the second structure on the support may include (i) attaching to the support a substance capable of binding to the support and capable of binding to the first structure and/or the second structure, such as a molecule that specifically binds to the first structure and/or the second structure, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide, (ii) washing the support by using a washing buffer, (iii) coating the support by using a blocking buffer, (iv) washing the support by using a washing buffer, (v) binding the first structure and/or the second structure to the substance, such as a molecule, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide, and/or (vi) washing the support by using a washing buffer.
  • the step of immobilizing the first structure and/or the second structure on the support may hold or maintain the first structure and/or the second structure on the support.
  • the process of immobilizing the first structure and/or the second structure on the support allows unwanted substances, including buffer and substances that are not bound to the support, such as molecules, e.g., antibodies, antibody fragments, lectins, aptamers, ligands, peptides, or cyclic peptides, unbound first structure and/or unbound second structure, to be washed from the support.
  • the support includes any plate or vessel, e.g., a petri dish, microplate, or vessel for culture, measurement, or ELISA.
  • the support may be of any color, e.g., transparent, translucent, white, or black.
  • the support may be white for chemiluminescence detection or black for fluorescence or chemiluminescence detection.
  • the support may be a black microplate for fluorescent dye detection.
  • the method of the present invention includes a step of binding a first label to a first structure and a step of binding a second label to a second structure.
  • the steps of binding the first label to the first structure and the second label to the second structure may be performed in any order or simultaneously.
  • the steps of binding the first label to the first structure and the second label to the second structure may be performed by first binding the first label to the first structure and then binding the second label to the second structure.
  • the steps of binding the first label to the first structure and the second label to the second structure may be performed by first binding the second label to the second structure and then binding the first label to the first structure.
  • the step of binding the first label to the first structure includes directly or indirectly binding the first label to the first structure.
  • the step of binding the first label to the first structure may include indirectly binding the first label to the first structure via one or more substances, such as a molecule that specifically binds to the first structure, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide.
  • the step of binding the first label to the first structure may include indirectly binding the first label to the first structure via an antibody that specifically binds to the first structure.
  • the step of binding the first label to the first structure may or may not include washing the support after directly or indirectly binding the first label to the first structure.
  • the step of binding the first label to the first structure may include (i) attaching an antibody capable of specifically binding to the first structure to the first structure, (ii) washing the support by using a wash buffer, (iii) binding the first label to the antibody capable of specifically binding to said first structure, and/or (iv) washing the support by using a wash buffer.
  • the step of binding the first label to the first structure may include indirectly binding the first label to the first structure via an antibody that specifically binds to the first structure and a secondary antibody that specifically binds to said antibody.
  • the step of binding the first label to the first structure may include (i) attaching an antibody capable of specifically binding to the first structure (e.g., an antibody that specifically binds to a virus capsid derived from a mouse) to the first structure, (ii) washing the support by using a washing buffer, (iii) binding a secondary antibody that specifically binds to the antibody having the first label (e.g., an anti-mouse IgG antibody that specifically binds to the antibody derived from a mouse having Alexa Fluor (registered trademark) 647) to the antibody, and/or (iv) washing the support by using a washing buffer.
  • an antibody capable of specifically binding to the first structure e.g., an antibody that specifically binds to a virus capsid derived from a mouse
  • washing buffer e.g., an anti-mouse IgG antibody that specifically binds to the antibody derived from a mouse having Alexa Fluor (registered trademark) 647) to the antibody
  • a washing buffer
  • the step of binding the second label to the second structure includes directly or indirectly binding the second label to the second structure.
  • the step of binding the second label to the second structure may include indirectly binding the second label to the second structure via one or more substances, such as a molecule that specifically binds to the second structure, such as an antibody, an antibody fragment, a lectin, an aptamer, a ligand, a peptide, or a cyclic peptide.
  • the step of binding the second label to the second structure may include indirectly binding the second label to the second structure via a peptide that specifically binds to the second structure.
  • the step of binding the second label to the second structure may or may not include washing the support after directly or indirectly binding the second label to the second structure.
  • the step of binding the second label to the second structure may include binding a peptide (e.g., SYBR® Gold) that specifically binds to the second structure having the second label to the second structure, and/or (ii) not washing the unbound peptide from the sample.
  • the step of binding the second label to the second structure may include (i) binding a peptide (e.g., SYBR® Gold) that specifically binds to the second structure having the second label to the second structure, and/or (ii) washing the unbound peptide from the sample by using a wash buffer.
  • the method of the present invention includes a step of releasing the second structure from the first structure when the second structure is contained in the first structure.
  • the step of releasing the second structure from the first structure is performed, for example, by using a temperature change, heating, cooling, stirring, pipetting, a chemical, a pH change, an acid or an alkali.
  • a person skilled in the art can appropriately select a step of releasing the second structure from the first structure based on the properties, activity, strength or amount of the "first structure" and the "second structure".
  • the heating may be performed at about 75°C to about 95°C, about 80°C to about 90°C, or about 85°C.
  • the heating may be performed for about 5 minutes to about 25 minutes, about 10 minutes to about 20 minutes, or about 15 minutes.
  • the heating may be performed at about 85°C for about 15 minutes.
  • the step of releasing the second structure from the first structure may be performed before the step of binding the first label to the first structure.
  • the step of releasing the second structure from the first structure may be after the step of binding the first label to the first structure.
  • the step of releasing the second structure from the first structure may be before the step of binding the second label to the second structure.
  • the step of releasing the second structure from the first structure may be after the step of binding the first label to the first structure and before the step of binding the second label to the second structure.
  • the step of releasing the second structure from the first structure makes it easier for the second label to bind to the second structure.
  • the step of releasing the second structure from the first structure allows the second label to bind to the second structure that was included in the first structure.
  • the step of releasing the second structure from the first structure allows the second label to bind to the second structure more accurately or with greater precision.
  • the method of the present invention includes a step of detecting the amount of the bound first label and the amount of the bound second label together.
  • the step of detecting the amount of the bound first label and the amount of the bound second label together includes detecting them together, for example, using one or more devices or detectors.
  • the step of detecting the amount of the bound first label and the amount of the bound second label together includes detecting them together, for example, using one device or detector.
  • the step of detecting the amount of the bound first label and the amount of the bound second label together includes obtaining a detection result of each of the labels, the "first label" and the "second label".
  • the step of detecting the amount of the bound first label and the amount of the bound second label together includes obtaining a detection result of each of the fluorescent signals based on excitation and emission, when the "first label” and the "second label” are fluorescent dyes that emit different fluorescent signals.
  • the detection result of the fluorescent signal may be obtained based on a wavelength of 652 nm (excitation) and/or 680 nm (emission).
  • the detection result of the fluorescent signal may be obtained based on a wavelength of 495 nm (excitation) and/or 535 nm (emission), or based on a wavelength of 500 nm (excitation) and/or 535 nm (emission).
  • one optical sensor, photodetector, microscope, device or detector may be used to obtain detection results of fluorescent signals based on wavelengths of 652 nm (excitation) and/or 680 nm (emission) and detection results of fluorescent signals based on wavelengths of 495 nm (excitation) and/or 535 nm (emission) and/or 500 nm (excitation) and/or 535 nm (emission).
  • the process of detecting the amount of bound first label and the amount of bound second label together can, for example, reduce the time or effort required to detect the amount of the first structure and the amount of the second structure, reduce the cost of detecting the amount of the first structure and the amount of the second structure, reduce the detection error of the amount of the first structure and the amount of the second structure, eliminate the need to purify the first structure or the second structure from a sample containing contaminants, or make it possible to detect the amount of the first structure and the amount of the second structure at low concentrations.
  • the method of the present invention includes a step of calculating or determining the amount (or concentration) of the first structure and the amount (or concentration) of the second structure based on the amount (or concentration) of the bound first label and the amount (or concentration) of the bound second label.
  • a person skilled in the art can appropriately calculate or determine the amount (or concentration) of the first structure and the amount (or concentration) of the second structure based on the characteristics of the first structure, the second structure, the label, the device, the detector, or the detection result.
  • the step of calculating or determining the amount (or concentration) of the first structure and the amount (or concentration) of the second structure may include creating a calibration curve of the amount (or concentration) of the first label and the amount (or concentration) of the first structure and/or a calibration curve of the amount (or concentration) of the second label and the amount (or concentration) of the second structure.
  • a calibration standard e.g., a commercially available standard, standard solution, and standard sample
  • Those skilled in the art can appropriately determine the standard for the calibration curve to be used based on the characteristics of the first structure, the second structure, the label, the device, the detector, or the detection result.
  • the standard for the calibration curve may be set for each of the first structure and/or the second structure to be calculated or determined.
  • the amount (or concentration) of one or more first structures and/or the amount (or concentration) of one or more second structures can be calculated or determined by performing a correction by calculation.
  • the amount (or concentration) of two or more second structures can be calculated or determined by using one type of standard for the calibration curve and performing a correction by calculation (for example, based on the correlation between DNA length and fluorescence intensity).
  • the amount (or concentration) of the first structure containing the second structure e.g., a viral capsid containing a nucleic acid
  • the amount (or concentration) of the first structure not containing the second structure e.g., a viral capsid not containing a nucleic acid
  • the amount (or concentration) of the first structure containing the second structure can be determined based on the calculated or determined amount (or concentration) of the second structure.
  • the amount (or concentration) of the first structure not containing the second structure can be determined based on the calculated or determined amount (or concentration) of the second structure from the calculated or determined amount (or concentration) of the first structure.
  • the ratio of the first structure containing the second structure e.g., viral capsids containing nucleic acid
  • the first structure not containing the second structure e.g., viral capsids not containing nucleic acid
  • the percentage (proportion) of the first structure containing the second structure e.g., viral capsids containing nucleic acid
  • the percentage (proportion) of the first structure not containing the second structure e.g., viral capsids not containing nucleic acid
  • the amount (or concentration) of the active viral vector (complete particle) and/or the amount (or concentration) of the inactive viral vector (empty particle) can be determined.
  • the ratio of full particles to empty particles can be evaluated or determined based on the calculated or determined amount (or concentration) of the first structure and the amount (or concentration) of the second structure.
  • the method of the present invention may use ELISA (Enzyme-Linked Immunosorbent Assay), which is an immunological assay.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • the method of the present invention is an application of ELISA, which is an immunological assay, and is called Dual Fluorescence-Linked Immunosorbent Assay (dFLISA).
  • the method of the present invention may include detecting the amount (or concentration) of one or more additional structures (e.g., a third structure and/or a fourth structure) in addition to detecting the amount (or concentration) of the first structure and the amount (or concentration) of the second structure.
  • additional structures e.g., a third structure and/or a fourth structure
  • a sample, a container, or a well in a plate may include one or more additional structures (e.g., a third structure and/or a fourth structure) in addition to the first structure and the second structure.
  • the step of detecting the amount (or concentration) of one or more additional structures may be the same as the method or step of detecting the amount (or concentration) of the first structure and the amount (or concentration) of the second structure described herein.
  • Coating of AAV VHH antibody onto plate 100 ⁇ L of diluted AAV VHH antibody was added to each well. An example is shown below. A sticker was placed on the top surface of the plate and pressed into place with a spatula. Incubated at 4°C for 16 hours.
  • Preparation of 1x PBS Weigh out the reagents and mix them in a 500 mL glass bottle. An example is shown below. The vial was shaken to mix well.
  • Preparation of 1% TWEEN20 in 1x PBS Weigh out the reagents into a 15 mL tube and mix them. An example is shown below. The tube was shaken and mixed well by vortexing.
  • wash buffer 0.05% TWEEN20 in 1x PBS, pH 7.4
  • the reagents were weighed and mixed in a 500 mL glass bottle. An example is shown below. The vial was shaken to mix well.
  • the plate was removed from the refrigerator (4°C).
  • the VHH solution in each well of the plate was removed with a multichannel pipette. After adding 200 ⁇ L of wash buffer to each well, the plate was turned over and the wash buffer was removed with a Kimtowel. This operation was repeated three times.
  • Blocking of the plate 200 ⁇ L of blocking buffer was added to each well.
  • a sticker was placed on the top surface of the plate and pressed into place with a spatula. Incubated at 37°C for 1 hour. After incubation, the blocking buffer in each well was removed with a multichannel pipette. After adding 200 ⁇ L of wash buffer to each well, the plate was turned over and the wash buffer was removed with a Kimtowel. This operation was repeated three times.
  • Preparation of AAV standard and sample solutions ⁇ Preparation of calibration curve solutions>
  • the AAV sample for the standard curve was diluted in a 1.5 mL microtube. An example is shown below.
  • the diluted AAV standard curve sample was serially diluted as follows to prepare standard curve samples (SD1 to SD7).
  • standard curve samples SD1 to SD7
  • sample AAV 100 ⁇ L of sample AAV was added to each well.
  • An example is shown below.
  • ADK8 anti-AAV8 antibody
  • ADK8 anti-AAV8 antibody
  • Goat Anti-Mouse IgG H&L (Alexa Fluor 647) 100 ⁇ L of Goat Anti-Mouse IgG H&L (Alexa Fluor 647) was added to each well.
  • a sticker was placed on the top surface of the plate and pressed into place with a spatula. The mixture was incubated at 37°C for 1 hour in the dark at 300 rpm. After incubation, the AAV solution in each well was removed with a multichannel pipette. After adding 200 ⁇ L of wash buffer to each well, the plate was turned over and the wash buffer was removed with a Kimtowel. This operation was repeated three times.
  • Preparation of SYBR Gold The reagents were weighed out and mixed in a 15 mL tube (1000-fold dilution). An example is shown below. The tube was gently shaken to mix well. The 15 mL tube was covered with aluminum foil.
  • Example 2 The test was carried out in the same manner as in Example 1, except that AAV8 and anti-AAV8 antibody (ADK8) were replaced with AAV2 and anti-AAV2 antibody (A20) shown in the table below. The results are shown in Figure 3.
  • Example 3 AAV8 and anti-AAV8 antibody (ADK8) were tested as in Example 1, using a mixture of AAV2 and AAV8 as shown in the table below, and anti-AAV2 antibody (A20) and anti-AAV8 antibody (ADK8). The results are shown in Figure 4. A dose-response curve was obtained for the total number of AAV8 capsids. Detection of antibodies against individual AAV serotypes (AAV8 & AAV2) was confirmed by the method of the present invention (dFLISA).
  • Example 4 The test was carried out in the same manner as in Example 1, except that SYBR Gold was replaced with QuantiFluor, and proteins were detected using antibodies labeled with HRP (Horse Radish Peroxidase) and absorbance measurements were performed when TMB (3,3',5,5'-tetramethylbenzidine) was added. The results are shown in Figure 5.
  • HRP Hase Radish Peroxidase
  • Example 5 A test was performed in the same manner as in Example 2 using AAV2 containing ssDNA and AAV2 containing scDNA. The results are shown in Figure 6.
  • the fluorescence intensity of AAV2 scDNA was about 1.4-1.6 times that of AAV2 ssDNA. Since scDNA is a long-chain DNA, it may be possible to perform quantification independent of DNA length by appropriately correcting for the DNA length.
  • Example 6 Measurement of DNA fluorescence intensity was performed in the same manner as in Example 2, using conventional wavelengths of 495 nm and 535 nm or wavelengths of 500 nm and 535 nm (optimized conditions) by the inventors. The results are shown in Figure 7. The signal background of the samples was equal or higher under the optimized conditions compared to the conventional wavelengths.
  • Example 7 Tests were performed in the same manner as in Example 2 using a transparent plate (Nunc MaxiSorpTM flat-bottom clear 96-well plate) and a black plate (The Nunc MaxiSorpTM flat-bottom black 96-well plate). The results are shown in FIG. 8. The fluorescence intensity of the sample showed a higher response in the black plate compared to the transparent plate. This result suggests that the black microplate is suitable for providing a signal suitable for the method of the present invention.
  • Example 8 To confirm whether the method of the present invention (dFLISA) can be used to analyze unpurified samples without purification, a spike recovery test was performed. The recovery rate was within ⁇ 25% of the predicted value, meeting the criteria. This indicates that the dFLISA results are not easily affected by contaminants contained in unpurified samples. This indicates that dFLISA is an excellent method for evaluating unpurified AAV samples.
  • the dFLISA method is a valuable and novel method that can be used to accurately quantify the titer of unpurified samples, and is unique in that it can directly quantify the full and empty capsid concentrations and FP/EP ratios of unpurified samples. The results are shown in Figure 9.
  • dFLISA can measure the capsid titer and genome titer of unpurified samples.
  • the capsid titer measured by dFLISA was comparable to that measured by ELISA.
  • the genome titer results by dFLISA were higher than those by dPCR. Considering the high recovery rate in the spike recovery experiment, the dFLISA results are considered reliable. Since previous studies have shown that dPCR can be affected by impurity interference, dFLISA may be a more accurate method to measure the concentration of AAV genome material in unpurified samples compared with the combination of ELISA and dPCR. The results are shown in Figure 10.
  • AAV8-Lot2 was concentrated by ultracentrifugation to final concentrations of 5.36 x 1013 cp/mL and 4.75 x 1013 vg/mL, and then diluted as follows in 1 x PBS (containing 0.05% Tween 20): High concentration (Spike H): 1.07 x 10 cp/mL and 9.50 x 10 vg/mL, medium concentration (Spike M): 7.15 x 10 cp/mL and 6.33 x 10 vg/mL, low concentration (Spike L): 5.36 x 10 cp/mL and 4.75 x 10 vg/mL.
  • Each spike sample solution was added to an unknown concentration unpurified sample (AAV8-Lot4) in a 1:1 volume ratio and mixed, and the prepared spike ratio sample solution was added to each well of the plate in 100 ⁇ L.
  • N 2 analyses were performed.
  • Mixed spike (HM) (1/2) (crude + spike H) - (1/2) (crude + spike L) (Equation 4)
  • the target recovery rate (%) was set within ⁇ 25%.
  • dFLISA Quantification of capsid and genome titers of unpurified samples.
  • dFLISA was performed as described above.
  • AAV8-Lot1 was diluted 60-fold in 1x PBS (containing 0.05% Tween 20) to a final concentration of 2.01 x 1011 cp/mL and 1.81 x 1011 vg/mL, and then serially diluted at a 1:2 ratio to serve as calibration curve standards.
  • Example 9 The fluorescence intensity in the quantification of genomes using fluorescent dyes varies depending on the length of the genome, which is also related to dFLISA.
  • dFLISA fluorescence intensity normalized by concentration
  • a correlation was observed between genome length and fluorescence intensity.
  • Figure 11 Furthermore, when the fluorescence intensity was compared between AAV vectors of different genome lengths (Table 22), the fluorescence intensity was high in samples with long genome lengths, and the ratio of genome length to fluorescence intensity was equivalent to that in Figure 11. From these results, the validity of dFLISA as a method for evaluating the genomes of AAV vectors with various nucleic acids was confirmed. In addition, even if the genome length is different from that of the standard AAV vector, the genome length can be corrected by calculation, and it was shown that it is not necessary to prepare a standard AAV for each AAV with a different genome length.
  • Fluorescence intensity of ssDNA from 1100 bp to 5100 bp was measured using ssDNA 7K ladder (PerkinElmer, Waltham, MA, USA).
  • the ssDNA ladder marker solution was appropriately diluted with Milli-Q and then loaded onto a 1% agarose gel.
  • 10 ⁇ L of sample was added to 2 ⁇ L of EzApplyDNA (6x loading buffer), mixed thoroughly by pipetting, and 10 ⁇ L of the mixture was loaded onto the gel. Electrophoresis was performed at 70 V for 45 min. After electrophoresis, the gel was stained and washed according to the manufacturer's instructions. SYBR Gold Nucleic Acid Gel Staining solution was used to stain the gel. Quantitative analysis of brightness density in the stained gel was performed.
  • MP mass photometry
  • AAV2-Lot1 containing ssDNA was diluted 100-fold in 1x PBS (containing 0.05% Tween 20) to final concentrations of 7.70 x 1010 cp/mL and 6.41 x 1010 vg/mL.
  • a standard curve was prepared by serial dilution in 1:2 ratio steps.
  • AAV2-Lot2 containing scDNA was diluted 100-fold to final concentrations of 2.11 x 1011 cp/mL and 1.93 x 1011 vg/mL.
  • a standard curve was prepared by serial dilution in 1:2 ratio steps.
  • the fluorescence intensity ratios of ssDNA AAV8 and scDNA AAV2 were measured by dFLISA and compared with the fluorescence intensity ratios calculated from the curves.

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Abstract

Le problème à résoudre dans le cadre de la présente invention consiste à fournir un procédé permettant de détecter la quantité d'une première structure et la quantité d'une seconde structure, le procédé comprenant une étape de détection de la quantité d'une première étiquette liée et de la quantité d'une seconde étiquette liée ensemble. La solution selon l'invention consiste en un procédé permettant de détecter la quantité d'une première structure et la quantité d'une seconde structure, le procédé comprenant : une étape de liaison d'une première étiquette à une première structure ; une étape de libération d'une seconde structure de la première structure ; une étape de liaison d'une seconde étiquette à la seconde structure ; et une étape de détection de la quantité de la première étiquette liée et de la quantité de la seconde étiquette liée ensemble, la seconde structure étant incluse dans la première structure et la première étiquette et la seconde étiquette pouvant être détectées ensemble, mais étant différentes.
PCT/JP2024/015380 2023-05-25 2024-04-18 Procédé permettant de détecter la quantité d'une première structure et la quantité d'une seconde structure WO2024241780A1 (fr)

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JP2017029151A (ja) * 2007-07-23 2017-02-09 クロンデイアグ・ゲーエムベーハー アッセイ
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JP2000189173A (ja) * 1998-11-17 2000-07-11 Cytos Biotechnology Ag 予め決定された特性を有するポリペプチドをコ―ドする遺伝子の、発見、特徴づけ、および単離のための発現クロ―ニングプロセス
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