[go: up one dir, main page]

WO2025196504A1 - Chromogènes bleus et leurs procédés d'utilisation - Google Patents

Chromogènes bleus et leurs procédés d'utilisation

Info

Publication number
WO2025196504A1
WO2025196504A1 PCT/IB2025/000104 IB2025000104W WO2025196504A1 WO 2025196504 A1 WO2025196504 A1 WO 2025196504A1 IB 2025000104 W IB2025000104 W IB 2025000104W WO 2025196504 A1 WO2025196504 A1 WO 2025196504A1
Authority
WO
WIPO (PCT)
Prior art keywords
solution
naphthol
phosphate
biological sample
blue
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IB2025/000104
Other languages
English (en)
Inventor
Stacey LINDSAY
Rachel C. JONES
Natalia LOS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leica Biosystems Newcastle Ltd
Original Assignee
Leica Biosystems Newcastle Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Leica Biosystems Newcastle Ltd filed Critical Leica Biosystems Newcastle Ltd
Publication of WO2025196504A1 publication Critical patent/WO2025196504A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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

Definitions

  • chromogens are chemical compounds that produce a visible color change upon reaction with specific enzymes or antigens. They are used for visualizing the location and distribution of target molecules within tissue samples. Chromogens are typically coupled with an enzyme-conjugated secondary antibody or linker molecule that binds to a primary antibody. The primary antibody, in turn, specifically binds to a target antigen. These interactions allow for detection and visualization of antigens, such as diagnostic markers, in a sample.
  • multiple chromogens of different colors is desirable, particularly with multiplex IHC assays.
  • multiplex assays the use of multiple chromogens having different colors allows for detection and visualization of multiple targets simultaneously within the same tissue section. This allows for reduced sample consumption, as more information can be extracted from a fewer tissue samples.
  • Different colors can be assigned to different target molecules or cellular structures, providing enhanced specificity in detecting and distinguishing between various antigens or markers. Further, certain colors are more easily distinguished from other colors, such that a range of options is desirable.
  • IHC assays It is additionally advantageous to automate IHC assays. This increases the number of assays that can be performed in a given amount of time, reduces the likelihood of human error, and allows for standardization of assays. Histological examination of biological samples, including tissue samples, is routinely conducted through manual or automated processes. In an automated assay, however, multiplexing typically benefits from all chromogens being used having the same dehydration and rehydration workflow. However, the use of blue chromogens in IHC assays is currently limited due to differences in solubility of available blue chromogens, which are alcohol soluble.
  • chromogenic reagent systems comprising a first solution comprising a blue salt, and a second solution comprising a naphthol phosphate, and methods of preparing same. Further disclosed are methods of staining a biological sample, comprising contacting a biological sample with a solution comprising a blue salt, and a solution comprising a naphthol phosphate. Further disclosed are multiplex IHC methods for detecting at least two target antigens, wherein at least one chromogen used is a blue chromogen. Kit for use in IHC methods are further provided.
  • FIG. l is a schematic illustrating an exemplary blue chromogen reaction using alkaline phosphatase (AP) enzyme, N-(2,4,-dimethylphenyl)-3-phosphonooxy)-2- naphthalenecarboxamide (Naphthol AS-MX), and RR Salt (also referred to as 4-Benzoylamino- 2,5-dimethoxybenzenediazonium chloride hemi (zinc chloride) salt, Azoic Diazo No. 24, available from Sigma- Aldrich®), and the resulting blue precipitate formed from the reaction on a tissue sample.
  • Alkaline phosphatase induces dephosphorylation of the phosphate group on the Naphthol AS-MX, which then reacts with the active diazonium ion on the RR salt to form the insoluble, blue precipitate.
  • FIG. 2 depicts a flow chart of an example of an Immunohistochemistry (IHC) Assay Protocol for use with the disclosed blue chromogen.
  • IHC Immunohistochemistry
  • FIG. 3 depicts the results of an exemplary blue chromogen reaction using alkaline phosphatase enzyme, Naphthol AS-MX, and RR Salt, in a single plex staining protocol on tissue from the colon, using anti-CD3 and anti-CDX2 primary antibodies.
  • FIG. 4 depicts the results of an exemplary multiplex staining protocol using alkaline phosphatase enzyme, Naphthol AS-MX, and RR Salt in combination with a red chromogen (e.g., Fast Red) and a green chromogen (e.g., available from 42 Life Sciences).
  • the tissues shown are tonsil, Diffuse Large B-Cell Lymphoma, and follicular lymphoma.
  • the primary antibodies used are anti-CD5 antibody, anti-CD20 antibody, anti-CD3 antibody, and anti-Ki67 antibody.
  • FIG. 5 depicts the results of an exemplary multiplex staining protocol using alkaline phosphatase enzyme, Naphthol AS-MX, and RR Salt, in combination with a red chromogen (e.g., Fast Red) and a green chromogen (e.g., available from 42 Life Sciences) on normal tonsil.
  • the primary antibodies used are anti-CD5 antibody, anti-BCL2 antibody, and anti-cyclin DI antibody.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
  • naphthol phosphate is intended to encompass naphthalene-based compounds, whereby the alcohol group is substituted with a phosphate group, commonly referred to as a “naphthol phosphate”, such as the compound known as “naphthol- AS-GR phosphate”. “Naphthol phosphate” is intended to further include salts and hydrates thereof.
  • the naphthol phosphate is selected from the group comprising naphthol AS phosphate, naphthol AS-OL phosphate, naphthol AS-E phosphate, naphthol AS-MX phosphate, naphthol AS-TR phosphate, naphthol AS-BI phosphate, naphthol AS-BS phosphate and naphthol AS-GR phosphate, the salts and hydrates thereof and mixtures thereof.
  • Suitable salts include lithium, sodium, potassium, ammonium, magnesium calcium and alkylammonium salts.
  • a “diagnostic marker” is a specific biochemical in the body which has a particular molecular feature that makes it useful for detecting a disease, measuring the progress of disease or the effects of treatment, or for measuring a process of interest.
  • epitope as used herein is defined as small chemical groups on the antigen molecule that is bound to by an antibody.
  • An antigen can have one or more epitopes.
  • epitopes One skilled in the art understands that generally the overall three-dimensional structure or the specific linear sequence of the molecule can be the main criterion of antigenic specificity.
  • a “subject” of diagnosis or treatment is a plant or animal, including a human.
  • Nonhuman animals subject to diagnosis or treatment include, for example, livestock and pets.
  • the term “individual” or “patient” is used interchangeably.
  • tissue section refers to a piece of tissue that has been obtained from a subject and mounted on a planar surface, e.g., a microscope slide. The sample may be fixed and/or sectioned as desired.
  • a “tumor tissue sample” or “tumor tissue biopsy sample” includes cells derived from a tumor in a subject, e.g., a human subject having a malignancy. Such tissue samples are sometimes referred to simply as a “biopsy”.
  • FFPE tissue section refers to a piece of tissue, e.g., a biopsy that has been obtained from a subject, fixed in formaldehyde (e.g., 3%-5% formaldehyde in phosphate buffered saline) or Bouin solution, embedded in wax, cut into thin sections, and then mounted on a planar surface, e.g., a microscope slide.
  • formaldehyde e.g., 3%-5% formaldehyde in phosphate buffered saline
  • Bouin solution embedded in wax
  • Target molecule refers to a molecule for which the presence, location and/or concentration is to be determined.
  • molecules of interest include proteins and nucleic acid sequences.
  • the target antigen may be a diagnostic marker.
  • staining includes binding a target (e.g., an antigen) in a cellular sample with a target-specific binding agent (e.g., an antibody) and then detecting the presence of the targetspecific binding agent on the cells of the cellular sample using a detectable label or chromogen.
  • a “chromogen” or “chromogenic compound” and the like is a substance that can be converted into a colored compound under specific conditions, e.g., when acted upon by an enzyme or under specific chemi cal/reacti on conditions.
  • enzyme-substrate combinations include: (i) Horseradish peroxidase (HRP) with hydrogen peroxidase as a substrate, where the hydrogen peroxidase oxidizes a dye precursor; and ii) alkaline phosphatase (AP). Numerous other enzyme-substrate combinations are available to those skilled in the art. For a general review of these, see, e.g., U.S. Pat. Nos. 4,275,149 and 4,318,980.
  • target-specific binding agent or “binding agent” or “binding moiety” means any agent that specifically binds to a target or analyte of interest, e.g., a target of interest that is present in a tissue section as described herein (e.g., a polypeptide).
  • target-specific binding agents include antibodies, receptors, and ligands, or target-binding fragments thereof, polynucleotide probes, and the like.
  • multiplexing refers to using more than one label, stain, and/or chromogen for the simultaneous or sequential detection and measurement of a target in a sample, e.g., a tissue section.
  • Multiplexing includes “triplexing” (using three labels, stains, and/or chromogens for the simultaneous or sequential detection and measurement of a target in a sample, e.g., a tissue section) or more than three labels, stains, and/or chromogens for simultaneous detection.
  • Single plex refers to an assay in which only one label, stain, and/or chromogen is used for the detection of a single target antigen.
  • antibody and “immunoglobulin” are used interchangeably and are well understood by those in the field. Those terms refer to a protein consisting of one or more polypeptides that specifically binds an antigen.
  • One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of antibody chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.
  • fragments of antibodies which retain specific binding to antigen or target including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bi-specific hybrid antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.
  • primary antibody and “secondary antibody” refer to different antibodies, where a primary antibody is a polyclonal or monoclonal antibody from one species (rabbit, mouse, goat, donkey, etc.) that specifically recognizes an antigen (e.g., a biomarker) in a sample (e.g., a human tissue sample) under study, and a secondary antibody is an antibody (usually polyclonal) from a different species that specifically recognizes the primary antibody, e.g., in its Fc region.
  • a primary antibody is a polyclonal or monoclonal antibody from one species (rabbit, mouse, goat, donkey, etc.) that specifically recognizes an antigen (e.g., a biomarker) in a sample (e.g., a human tissue sample) under study
  • an antigen e.g., a biomarker
  • a secondary antibody is an antibody (usually polyclonal) from a different species that specifically recognizes the primary antibody, e.g., in its Fc
  • telomere binding refers to the ability of a binding agent to preferentially bind to a particular analyte that is present in a homogeneous mixture of different analytes. In certain aspects, a specific binding interaction will discriminate between desirable and undesirable analytes in a sample, in some aspects more than about 10 to 100-fold or more (e g., more than about 1000- or 10,000-fold).
  • a chromogenic reagent system may comprise a first solution comprising a blue salt, and a second solution comprising a naphthol phosphate.
  • the blue salt may comprise a diazonium salt of Fast Blue RR.
  • a suitable diazonium salt of Fast Blue RR is 4-Benzoylamino-2,5- dimethoxybenzenediazonium chloride hemi(zinc chloride) salt (CAS: 14726-29-5) also referred to as RR Salt and Azoic Diazo No. 24, available from Sigma-Adrich®.
  • the second solution may comprise a naphthol phosphate selected from naphthol AS phosphate, naphthol AS-OL phosphate, naphthol AS-E phosphate, naphthol AS-MX phosphate, naphthol AS-TR phosphate, naphthol AS- BI phosphate, naphthol AS-BS phosphate and naphthol AS-GR phosphate, the salts and hydrates thereof and mixtures thereof.
  • the naphthol phosphate is N-(2,4,-dimethylphenyl)-3- phosphonooxy)-2-naphthalenecarboxamide, or (Naphthol AS-MX), CAS: 1596-56-1 .
  • compositions may be supplied individually, and may be supplied in a buffer composition.
  • the compositions may further comprise one or more of sodium chloride, magnesium chloride, a buffer, a preservative, and antimicrobial, and combinations thereof.
  • a blue chromogen composition is disclosed, the composition comprising RR Salt and Naphthol AS-MX.
  • AS-MX alkaline phosphatase
  • AS-MX is dephosphorylated.
  • the hydrazine of RR Salt reacts with the free oxygen group on the phosphate to create a blue precipitate. Applying the IHC methods described herein, the blue precipitate deposits on the treated sample, allowing for visualization of the target.
  • the disclosed chromogenic reagent compositions may be used to carry out an immunohistochemistry assay protocol, for example a single plex or multiplex IHC assay.
  • Single plex assays may be used for the detection of a single antigen, such as a single diagnostic marker, and may use only the RR Salt-AS-MX-based reagent systems.
  • a multiplex assay may be carried out using the disclosed chromogenic reagent compositions.
  • the RR Salt-AS-MX-based reagent system may be used for detection of a first target antigen (e.g., a first diagnostic marker), and an alternative chromogenic system utilizing a non-blue chromogen (e.g., red, green, brown/DAB) may be used for the detection of a second, third, or fourth target antigen (e g., a second, third, or fourth diagnostic marker).
  • a first target antigen e.g., a first diagnostic marker
  • an alternative chromogenic system utilizing a non-blue chromogen e.g., red, green, brown/DAB
  • a second, third, or fourth target antigen e.g., a second, third, or fourth diagnostic marker.
  • Each IHC assay can be carried out sequentially on the same tissue sample, separated by washes.
  • FIG. 2 depicts an exemplary Immunohistochemistry Assay Protocol (100) for use with the disclosed compositions.
  • a tissue section may be prepared from a tissue sample obtained from an individual using methods known in the art, such as via a standard biopsy method.
  • the tissue sample may be a formalin fixed paraffin embedded (FFPE) tissue section which is placed on a slide and deparaffinized.
  • FFPE formalin fixed paraffin embedded
  • An exemplary protocol is the BOND Dewax protocol, which removes paraffin wax from the tissue section before rehydration and staining.
  • the IHC assay (100) generally begins with an epitope retrieval solution incubation step (110) in which the tissue section is incubated with an epitope retrieval solution.
  • the epitope retrieval solution assists in exposing the desired antigen.
  • Exemplary epitope retrieval solutions are known in the art, and may include, for example, a solution comprising EDTA, Tris Base, Sodium Citrate, Proteinase K, Trypsin, and Pepsin.
  • the epitope retrieval solution is BOND Epitope Retrieval Solution 1, a ready to use, citrate-based pH 6 epitope retrieval solution for the heat-induced epitope retrieval (HIER) of formalin-fixed, paraffin-embedded tissue on the BOND automated system, which restores epitopes that have been modified by formalin fixation, allowing accessibility of the primary antibody to the epitope.
  • the epitope retrieval solution incubation step (110) may be carried out at a temperature sufficient to expose the antigens of interest and may be carried out for a period of time ranging from about 30 seconds to about 5 minutes, or from about 1 minute to about 10 minutes, or about 2 minutes.
  • the duration and temperature used for incubation of the reagents during the epitope retrieval solution incubation step (110) may be predetermined and programmed into a slide-staining instrument (for example, the Leica® BOND-III Fully Automated IHC and ISH Staining System) and may depend on the specific epitope retrieval solution used.
  • a slide-staining instrument for example, the Leica® BOND-III Fully Automated IHC and ISH Staining System
  • the tissue section may optionally be subjected to a hydrogen peroxide treatment to quench endogenous peroxidase activity.
  • the hydrogen peroxide treatment step may employ, for example, a solution of about 3% to 4% hydrogen peroxide solution applied to the tissue section for about 30 seconds to about 5 minutes, or from about 1 minute to about 10 minutes, or about 2 minutes.
  • a primary antibody step (120) is then carried out.
  • primary antibody step (120) primary antibody having specific binding to an antigen of interest is applied to the tissue section and incubated for a time and temperature sufficient to allow specific binding to the target.
  • the primary antibody may be incubated with the tissue section for an incubation period of about 1 to about 30 minutes or about 5 to about 25 minutes, or about 10 to about 20 minutes, or about 15 minutes.
  • post-primary linker reagent serve to amplify a signal in an immunohistochemistry assay.
  • Suitable post-primary linkers will be understood by one of ordinary skill in the art and may include, for example, reagents containing multiple binding sites that simultaneously interact with both the primary antibody and the secondary antibody, reagents that alleviate steric hindrance by altering the orientation of the primary binding antibody or by exposing additional binding sites and improving secondary antibody binding, or the like.
  • an a-mouse IgG linker is used to localize a mouse primary antibody, and is incubated with the tissue sample for about 5 minutes to about 30 minutes, or about 10 minutes to about 20 minutes, or about 15 minutes.
  • a secondary antibody step (130) is carried out in which a secondary antibody coupled to an enzyme is contacted with the tissue section.
  • the secondary antibody has specific binding to the primary antibody and/or the post-primary linker.
  • the secondary antibody is an anti-species antibody specific to the Fc region of the primary antibody and/or the post-primary linker.
  • a goat anti-mouse antibody, or a goat anti-rabbit antibody in which the primary antibody is a mouse species or rabbit species, respectively.
  • the enzyme coupled to the secondary antibody is one configured to react with a later-applied chromogen reagent.
  • the secondary antibody is conjugated to an alkaline phosphatase (AP) enzyme.
  • AP enzyme usually isolated from calf intestine, is a 140-kDa enzyme that catalyzes the hydrolysis of phosphate groups from a substrate molecule.
  • the chromogen application step (140) follows the application of the secondary antibody step (130).
  • an RR Salt (4-Benzoylamino-2,5- dimethoxybenzenediazonium chloride hemi(zinc chloride) salt, Azoic Diazo No. 24, available from Sigma-Aldrich®) and a naphthol phosphate (e.g., N-(2,4,-dimethylphenyl)-3- phosphonooxy)-2-naphthalenecarboxamide (Naphthol AS-MX)) are contacted with the tissue sample.
  • RR Salt 4-Benzoylamino-2,5- dimethoxybenzenediazonium chloride hemi(zinc chloride) salt, Azoic Diazo No. 24, available from Sigma-Aldrich®
  • a naphthol phosphate e.g., N-(2,4,-dimethylphenyl)-3- phosphonooxy-2-naphthalenecarboxamide (Naph
  • a first solution comprising an RR Salt and a second solution comprising a Naphthol AS-MX are mixed to form a third solution comprising the RR Salt and the Naphthol AS- MX.
  • the third solution may be mixed at least 1 minute, or at least five minutes, or at least 10 minutes, or at least 20 minutes, or at least 30 minutes or at least one hour, or at least 90 minutes, or at least 120 minutes, or at least 300 minutes prior to contact with the tissue sample.
  • the RR Salt and Naphthol AS-MX are each dispensed separately onto the tissue sample and mixed in situ.
  • the chromogen application step (140) may be carried out for a time and temperature sufficient to allow precipitation of the chromogen, for example, for about 5 minutes to about 20 minutes, or about 7 minutes to about 15 minutes, or about 10 minutes.
  • a detectable, insoluble blue precipitate is generated and deposited onto the tissue sample at the location of the bound antibody, according to the reaction shown in FIG. 1. Visualization of the blue precipitate that forms on the sample allows for detection and localization of the target antigen, as can be seen in FIGS. 1 and 3- 5.
  • compositions and methods may further be employed to carry out a multiplex assay in which at least two target antigens are detected and visualized in the same tissue sample using different chromogens.
  • the multiplex assay uses a blue chromogen as described, and a second, non-blue chromogen.
  • an elution step may be carried out to remove previously bound antibody followed by a neutralization wash (e.g. EDTA based pH 9 solution) to bring the tissue to neutral pH.
  • a neutralization wash e.g. EDTA based pH 9 solution
  • the elution may be carried out at a temperature of from about 50 °C and for a period of about 5 minutes to about 20 minutes, or about 7 minutes to about 15 minutes, or about 10 minutes.
  • a second IHC assay (for example as depicted in FIG. 2) may then be carried out, in which the same tissue sample described above is used for the detection of a second target antigen via a second primary antibody specific to the second target antigen.
  • two or more, or three or more, or four or more, or more than five target IHC assays may be carried out in which two or more, or three or more, or four or more, or more than five antigens may be detected in a tissue sample, in which at least two, or at least three, or at least four, or five or more different chromogens are used to detect the different target antigens.
  • a second IHC assay may be performed on the tissue sample for detection of a second target antigen.
  • a second IHC assay is carried out on the tissue section, using a primary antibody specific to a second target antigen distinct from that used in the first IHC assay.
  • the second IHC assay further uses a secondary antibody specific to the second target antigen.
  • the second IHC assay may employ any suitable chromogen and corresponding enzyme for catalyzing the chromogen precipitate, but which typically a color that is distinguishable from the first chromogen precipitate.
  • the second IHC assay in general, will utilize a non-blue chromogen, for example a DAB chromogen, a red chromogen, or a green chromogen, to allow for the first and second IHC assay results to be distinguishable in the tissue sample.
  • the tissue section may then be counterstained with a suitable counterstain in a post-processing step.
  • the tissue sample may be contacted with a hematoxylin solution and washed to remove excess reagent.
  • the resulting tissue sample may then be visualized for the presence of blue chromogen, in addition to further chromogens (such as brown, red, and green, or, where co-localization occurs, further colors may be identified).
  • the above protocols may include one or more washing steps between any one or more of the steps or protocols described above prior to the dehydration step with increased concentration of alcohol ranging from 70% to 100%.
  • wash steps may be performed with a variety of solutions such as deionized water, phosphate buffered saline, or other buffered solutions, or various combinations thereof.
  • any dispense cycle in which a reagent is applied to the sample may include an incubation. Incubations are generally performed with the given slide being heated to a predetermined temperature. The incubation time may be varied depending on the desired outcome and the particular reagents (for example, the particular probe) that is used. For example, the incubation time may be about 20 minutes for a dispense cycle, or about 30 minutes for a dispense cycle, or about 60 minutes for a dispense cycle. Although particular incubation times are described, it should be understood that in other examples, either a different minimum incubation time or a different maximum incubation time may be used.
  • a slide-staining instrument i.e., a slide-staining device
  • a slide-staining device may be configured to perform a variety of protocols where robotic arm and/or slide staining assemblies may automatically apply a variety of reagents to slides under various predetermined parameters.
  • Such slide staining protocols may include a series of steps where various reagents may be applied to individual slides in a predetermined order, in a predetermined volume at predetermined temperatures for predetermined periods of time.
  • the side-staining instrument may be generally configured to automatically stain tissue mounted on microscope slides.
  • the slidestaining instrument may include a group fluid dispenser (also referred to as an aspirating probe) mounted to a robotic arm and a plurality of slide staining assemblies disposed beneath robotic arm.
  • the robotic arm and slide staining assemblies may be generally configured to operate cooperatively to automatically dispense reagents, for example, IHC assay reagents such as one or more IHC compositions as disclosed herein, including chromogen reagents, stain, probes, enzymes, and the like, onto one or more microscope slides disposed within each slide staining assembly.
  • IHC assay reagents such as one or more IHC compositions as disclosed herein, including chromogen reagents, stain, probes, enzymes, and the like
  • the slide-staining instrument may include a bulk container rack and a reagent platform.
  • the bulk container rack may be configured to hold a plurality of bulk containers, which may be configured to contain a relatively large quantity of reagent that may be communicated to group fluid dispenser and/or each slide staining assembly.
  • reagents that may be stored in bulk containers may include, for example, formalin solutions, wash solutions, deionized water, buffered solutions such as phosphate buffered saline (PBS), and/or alcohol, and additional reagents useful to carrying out the disclosed methods.
  • one or more of bulk containers may be configured to contain waste fluids communicated from group fluid dispenser and/or each slide staining assembly. In some examples, such waste fluids may be separated by slide-staining instrument into bulk waste and hazardous waste. Thus, separate bulk containers may be included for bulk waste and hazardous waste.
  • a plurality of syringe pumps may be included proximate bulk containers to communicate fluid to or from bulk containers relative to other portions of si ide- staining instrument.
  • the slide staining instrument may further include a reagent platform, which is generally configured to receive one or more reagent trays, which is in turn configured to hold a plurality of reagent containers.
  • the reagent containers may be configured to contain a relatively small quantity of reagent for communication to a group fluid dispenser and/or slide staining assembly.
  • the first solution comprising a blue chromogen and/or the second solution comprising a naphthol may be dispensed via this assembly.
  • the first solution comprising a blue chromogen and the second solution comprising a naphthol may be mixed to form a third solution comprising both the blue chromogen and the naphthol prior to dispensing on the slide.
  • Each reagent tray may include a particular predetermined combination of reagent containers to form a predefined reagent system.
  • the first solution comprising a blue chromogen and the second solution comprising a naphthol may be combined as a predefined reagent systems and may be prepared as a ready-to-use formulation and may be discarded upon exhaustion during use with a particular protocol.
  • kits for performing one or more steps of an IHC method.
  • the disclosed kits may comprise one or more solutions for the detection and visualization of a specific antigen within a tissue sample.
  • the kit may comprise reagents and solutions optimized for detection of a target antigen, e g., a diagnostic marker.
  • the kit may comprise a first solution comprising a blue salt and a second solution comprising a naphthol phosphate.
  • the first solution may comprise an RR Salt and one or more buffers whereas the second solution may comprise a naphthol phosphate.
  • the second solution comprises naphthol AS-MX phosphate and one or more buffers.
  • the kit may further comprise one or more antibody solutions, for example, one or more primary antibodies for detection of a target antigen, or for detection of two antigens, or for detection of at three antigens, or for detection of four antigens, or for detection of five or more antigens.
  • the kit may comprise at least two different primary antibodies, or at least three different primary antibodies, or at least four different primary antibodies, or five or more different primary antibodies for detection of at least two, or at least three, or at least four, or five or more distinct target antigens.
  • the kit may further comprise a secondary antibody conjugated to an enzyme.
  • the kit may comprise a secondary antibody conjugated to an enzyme selected from alkaline phosphatase or horseradish peroxidase.
  • the kit may further comprise at least two different secondary antibodies, or at least three different secondary antibodies, or at least four different secondary antibodies, or five or more different secondary antibodies for detection of the at least two, or at least three, or at least four, or five or more distinct primary antigens.
  • the kit may comprise secondary antibodies that are the same, for example, a goat anti-mouse secondary antibody or a goat-anti-rabbit secondary antibody.
  • the kit may further comprise additional chromogen solutions, wherein the additional chromogens are selected from a red chromogen, a green chromogen, a brown chromogen, and combinations thereof.
  • the kit comprises one or more of a solution comprising a red chromogen, a solution comprising a green chromogen, and a solution comprising a brown chromogen.
  • the kit may further comprise one or more ancillary components such as a substrate buffer for dilution of the chromogen solution, a wash buffer for removing unbound antibodies, a and blocking reagent to prevent nonspecific binding, and combinations thereof.
  • the kit may further comprise positive and/or negative control slides for validation of assay performance and accurate interpretation of results.
  • the kit may be used for either manual or automated use.
  • FFPE Formalin-Fixed Paraffin-Embedded
  • Slides are deparaffinized using the BOND Dewax protocol and heat induced epitope retrieved using BOND Epitope Retrieval Solution 2 for 20 mins at 100°C.
  • a primary antibody BOND Ready-to-Use reagent
  • a secondary reagent a-mouse IgG linker localizing mouse primary antibodies is applied to the slide for 20 minutes and subsequently detected using a a-rabbit polymer reagent conjugated to alkaline phosphatase applied to the slide for 30 minutes.
  • FIG. 3 depicts an exemplary result using the blue chromogen disclosed herein, using colon tissue and anti-CD3 primary antibody and anti-CDX2 antibody.
  • FIGS. 4 and 5 depict the results of an exemplary multiplex protocol using the blue chromogen disclosed herein in combination with a red chromogen and a green chromogen.
  • an antibody elution step may be carried out prior to initiating a second immunohistochemistry assay for detection of a second target antigen.
  • the elution method may include incubation of the elution buffer with the tissue section at a heated temperature of 50 °C for about 10 minutes, followed by a neutralization wash sufficient to return the tissue to a neutral pH.
  • the second immunohistochemistry assay may comprise contacting the tissue sample with a primary antibody having specific binding to a second target antigen, followed by optional contact with a post-primary linker reagent, and contact with a secondary antibody having specific binding to the primary antibody and which is conjugated to an enzyme.
  • the enzyme selected is determined based on the chromogen to be used.
  • the second IHC assay is carried out using a red, green, or brown chromogen.
  • the specimen is incubated with a primary antibody (BOND Ready-to-Use reagent) of monoclonal mouse or rabbit origin is applied following an incubation of 15 minutes.
  • a secondary reagent a-mouse IgG linker localizing mouse primary antibodies is applied to the slide for 20 minutes and subsequently detected using a a-rabbit polymer reagent conjugated to alkaline phosphatase for 30 minutes.
  • the Refine red chromogen is applied to the tissue (10+5 minutes) and produces a red precipitate localized to the cellular compartment of the epitope in detection.
  • An antibody elution step is applied to the tissue before moving onto the next sequential staining round. This elution method includes a heated temperature of 50°C for 10 minutes, followed by a neutralization wash to ensure the tissue is brought back to a neutral pH.
  • FIG. 4 depicts exemplary results of an immunohistochemistry assay using anti-CD5, anti-CD20, and anti-CD3 antibody on tonsil tissue (top left panel), on tissue positive for Diffuse Large B-cell Lymphoma (DLBCL) (bottom left panel), and tissue positive for follicular lymphoma (bottom right).
  • FIG. 4 top right panel depicts exemplary results of an immunohistochemistry assay using anti-Ki67, anti-CDlO, and anti-BCL-2 on tonsil tissue. The multiple immunohistochemistry assay of FIG.
  • FIG. 4 utilizes the blue chromogen of the current disclosure, in combination with a red chromogen and a green chromogen.
  • FIG. 5 depicts exemplary results of an immunohistochemistry assay using anti-CD5, anti-BCL2, and anti-Cyclin DI antibody on normal tonsil tissue (left panel), anti-CD5, anti-CD3, and anti-CD20 on normal tonsil tissue (middle panel), and anti-CD3, anti-CD20, and anti-Ki67 on normal tonsil tissue (middle panel).
  • the multiple immunohistochemistry assays of FIG. 5 utilizes the blue chromogen of the current disclosure, in combination with a red chromogen, and a green chromogen.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne des systèmes de réactifs chromogènes comprenant une première solution contenant un sel bleu, et une seconde solution contenant un phosphate de naphtol, et leurs procédés de préparation. L'invention concerne en outre des procédés de coloration d'un échantillon biologique, comprenant la mise en contact d'un échantillon biologique avec un sel bleu et un phosphate de naphtol. L'invention concerne en outre des procédés IHC multiplex pour détecter au moins deux antigènes cibles, au moins un chromogène utilisé étant un chromogène bleu. L'invention concerne en outre des kits destinés à être utilisés dans des procédés IHC.
PCT/IB2025/000104 2024-03-16 2025-03-14 Chromogènes bleus et leurs procédés d'utilisation Pending WO2025196504A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463566236P 2024-03-16 2024-03-16
US63/566,236 2024-03-16

Publications (1)

Publication Number Publication Date
WO2025196504A1 true WO2025196504A1 (fr) 2025-09-25

Family

ID=95446665

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2025/000104 Pending WO2025196504A1 (fr) 2024-03-16 2025-03-14 Chromogènes bleus et leurs procédés d'utilisation

Country Status (1)

Country Link
WO (1) WO2025196504A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4275149A (en) 1978-11-24 1981-06-23 Syva Company Macromolecular environment control in specific receptor assays
US4318980A (en) 1978-04-10 1982-03-09 Miles Laboratories, Inc. Heterogenous specific binding assay employing a cycling reactant as label
US8603765B2 (en) * 2004-02-24 2013-12-10 Biocare Medical, Llc. Immunoassay reagents and methods of use thereof
US9068989B2 (en) * 2009-07-29 2015-06-30 Leica Biosystems Melbourne Pty Ltd Reagent system and method
US20210293670A1 (en) 2011-11-16 2021-09-23 Leica Biosystems Melbourne Pty Ltd Biological sample treatment apparatus
US11493411B2 (en) 2017-12-21 2022-11-08 Leica Biosystems Melbourne Pty Ltd Fluid transport system

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4318980A (en) 1978-04-10 1982-03-09 Miles Laboratories, Inc. Heterogenous specific binding assay employing a cycling reactant as label
US4275149A (en) 1978-11-24 1981-06-23 Syva Company Macromolecular environment control in specific receptor assays
US8603765B2 (en) * 2004-02-24 2013-12-10 Biocare Medical, Llc. Immunoassay reagents and methods of use thereof
US9068989B2 (en) * 2009-07-29 2015-06-30 Leica Biosystems Melbourne Pty Ltd Reagent system and method
US20210293670A1 (en) 2011-11-16 2021-09-23 Leica Biosystems Melbourne Pty Ltd Biological sample treatment apparatus
US11493411B2 (en) 2017-12-21 2022-11-08 Leica Biosystems Melbourne Pty Ltd Fluid transport system
US20230022299A1 (en) 2017-12-21 2023-01-26 Leica Biosystems Melbourne Pty Ltd Fluid transport system

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIANCHI VANESSA J. ET AL: "Formation of Hyaline Cartilage Tissue by Passaged Human Osteoarthritic Chondrocytes", TISSUE ENGINEERING PART A, vol. 23, no. 3-4, 1 February 2017 (2017-02-01), US, pages 156 - 165, XP093291527, ISSN: 1937-3341, DOI: 10.1089/ten.tea.2016.0262 *
DOBSON PHILIP F. ET AL: "Mitochondrial dysfunction impairs osteogenesis, increases osteoclast activity, and accelerates age related bone loss", SCIENTIFIC REPORTS, vol. 10, no. 1, 15 July 2020 (2020-07-15), US, XP093244081, ISSN: 2045-2322, Retrieved from the Internet <URL:https://www.nature.com/articles/s41598-020-68566-2> DOI: 10.1038/s41598-020-68566-2 *
no. 14726-29-5
SIGMA-ALDRICH: "Fast Blue RR Salt -crystalline", SIGMA-ALDRICH PRODUCT SPECIFICATION, 17 July 2020 (2020-07-17), pages 1 - 1, XP093291349, Retrieved from the Internet <URL:https://www.sigmaaldrich.com/specification-sheets/330/309/F0500-BULK________SIGMA____.pdf> *

Similar Documents

Publication Publication Date Title
Hussaini et al. Immunohistochemistry and immunofluorescence
US20240328910A1 (en) Compositions and methods for simultaneous inactivation of alkaline phosphatase and peroxidase enzymes during automated multiplex tissue staining assays
US11988673B2 (en) Multiplexed immunohistochemistry using recombinant antibodies with epitope tags
JP2021517242A (ja) Ｍａｇｅａ４を検出する方法
WO2025196504A1 (fr) Chromogènes bleus et leurs procédés d&#39;utilisation
US20120003664A1 (en) Method for evaluating pre-treatment
US20250321232A1 (en) Chromogenic multiplexing methods and systems for identifying a cancer of unknown primary origin
JP2011013051A (ja) 生体試料標本の作製方法
US20060160151A1 (en) Method of quantitative immunohistochemistry and in situ hybridization
WO2025178607A1 (fr) Procédés et systèmes de multiplexage chromogène
US20250389718A1 (en) Histochemical and cytochemical methods for detecting ntrk fusion proteins
US20230358649A1 (en) Compositions and methods for removal of bound detection agents
JP7757382B2 (ja) Ｎｔｒｋ融合タンパク質を検出するための組織化学的および細胞化学的方法
JP2004132838A (ja) 被検生物学的試料の新規染色方法およびそれに使用されるアッセイ用キット
US10753935B2 (en) Prognostic method and kits useful in said method

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25719452

Country of ref document: EP

Kind code of ref document: A1