Blue chromogens and methods of using same

Abstract

Disclosed are 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 blue salt and 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. Kits for use in IHC methods are further provided.

Description

BLUE CHROMOGENS AND METHODS OF USING SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Appliation Serial No. 63 / 566,236, entitled “Blue Chromogens and Methods of Using Same,” filed on March 16, 2024, the disclosure of which is incorporated by reference herein.BACKGROUND

[0002] In immunohistochemistry (IHC) assays, 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.

[0003] The use of multiple chromogens of different colors is desirable, particularly with multiplex IHC assays. For example, in 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.

[0004] 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 alcoholsoluble. Consequently, samples subjected to an IHC assay using a blue chromogen must be removed from the workflow and dried separately, to avoid being solubilized in an alcohol treatment used for other IHC assays. This complicates the seamless integration of assays and reagents employing blue chromogens into existing automated systems.

[0005] Thus, there is a need for blue chromogens which can be used in IHC assays, but which are not soluble in alcohol and which can be successfully incorporated into an automated protocol. The present disclosure addresses one or more of the aforementioned needs in the art.BRIEF SUMMARY

[0006] Disclosed are 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.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0008] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0009] 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 NaphtholAS-MX, which then reacts with the active diazonium ion on the RR salt to form the insoluble, blue precipitate.

[0010] FIG. 2 depicts a flow chart of an example of an Immunohistochemistry (IHC) Assay Protocol for use with the disclosed blue chromogen.

[0011] 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.

[0012] 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.

[0013] 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.DETAILED DESCRIPTIONI. DEFINITIONS

[0014] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0015] All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

[0016] The headings provided herein are not limitations of the various aspects or embodiments of the invention. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual (Table of Contents available at www.cshlpress.com / pdf / sample / 2013 / MC4 / MC4FM.pdf).

[0018] As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0019] 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.

[0020] The term “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. In one embodiment, the naphthol phosphate is selected from the group comprising naphthol AS phosphate, naphtholAS-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.

[0021] 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.

[0022] The term “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. 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.

[0023] 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.

[0024] As used herein, the term “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”.

[0025] As used herein, the term “formalin-fixed paraffin embedded (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.

[0026] ‘Molecule of interest,” “target molecule,” “target,” and “target antigen” each refers to a molecule for which the presence, location and / or concentration is to be determined. Examples of molecules of interest include proteins and nucleic acid sequences. In one aspect, the target antigen may be a diagnostic marker.

[0027] The term “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. The detectable label can be directly conjugated to the target-specific binding agent (e.g., a primary antibody) or may be conjugated to a secondary reagent that binds specifically to an unlabeled target-specific reagent (e.g., a secondary antibody). In some cases, the target-specific reagent is itself detectable, and thus no additional attached label is needed.

[0028] 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. Examples of 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.

[0029] As used herein, the term “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). Examples of target-specific binding agents include antibodies, receptors, and ligands, or target-binding fragments thereof, polynucleotide probes, and the like.

[0030] As used herein, the term “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.

[0031] As used herein, the terms “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 basicstructural 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. These terms also include 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.

[0032] As used herein, the terms “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.

[0033] The term “specific 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).

[0034] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of + / — 15%, or alternatively 10%, or alternatively 5%, or alternatively 2%. It is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0035] It is to be inferred without explicit recitation and unless otherwise intended, that when the present technology relates to a polypeptide, protein, polynucleotide or antibody, an equivalent or a biologically equivalent of such is intended within the scope of the present technology.II. Blue Chromogen CompositionsIII. Methods for Detecting a Target in a Sample Using a Blue Chromogen Composition

[0036] Disclosed are methods, compositions, and kits utilizing a blue chromogen.

[0037] In one aspect, a chromogenic reagent system is disclosed. The chromogenic reagent system may comprise a first solution comprising a blue salt, and a second solution comprising a naphthol phosphate. Referring to FIG. 1, the blue salt may comprise a diazonium salt of Fast Blue RR. In one aspect, 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. In one aspect, the naphthol phosphate is N-(2,4,-dimethylphenyl)-3- phosphonooxy)-2-naphthalenecarboxamide, or (Naphthol AS-MX), CAS: 1596-56-1 . Each of the two 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. In one aspect, a blue chromogen composition is disclosed, the composition comprising RR Salt and Naphthol AS-MX. As shown in FIG. 1, when alkaline phosphatase (AP Enzyme) and AS-MX are brought into contact, 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.

[0038] 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. In other aspects, a multiplex assay may be carried out using the disclosed chromogenic reagent compositions. In this aspect, 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). Each IHC assay can be carried out sequentially on the same tissue sample, separated by washes.

[0039] FIG. 2 depicts an exemplary Immunohistochemistry Assay Protocol (100) for use with the disclosed compositions. For example, as a first step of an IHC assay, 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. An exemplary protocol is the BOND Dewax protocol, which removes paraffin wax from the tissue section before rehydration and staining.

[0040] The disclosed methods employ IHC assays as generally understood in the art. Referring to FIG. 2, 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. In aspects, 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.

[0041] Following epitope retrieval solution incubation step (110) and prior to primary antibody step (120), the tissue section may optionally be subjected to a hydrogen peroxide treatment toquench 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.

[0042] A primary antibody step (120) is then carried out. In 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. For example, 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.

[0043] Following primary antibody step (120) and prior to secondary antibody application step 130, the tissue section may then subjected to post-primary linker reagent. Post-primary linkers 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. In one aspect, 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.

[0044] Following primary antibody step (120) and the optional post-primary linker step, 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. In general, the secondary antibody is an anti-species antibody specific to the Fc region of the primary antibody and / or the post-primary linker. For example, a goat anti-mouse antibody, or a goat anti-rabbit antibody, in which the primary antibody is a mouse species or rabbit species, respectively. In general, the enzyme coupled to the secondary antibody is one configured to react with a later-applied chromogen reagent. In one aspect, the secondary antibody is conjugated to an alkaline phosphatase (AP) enzyme. AP enzyme, usuallyisolated from calf intestine, is a 140-kDa enzyme that catalyzes the hydrolysis of phosphate groups from a substrate molecule.

[0045] The chromogen application step (140) follows the application of the secondary antibody step (130). During chromogen application step (140), 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. In aspects, 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. In aspects, 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. Upon contact with the RR Salt and Naphthol AS-MX or the mixture of RR Salt and Naphthol AS-MX with the tissue sample comprising bound secondary antibody conjugated to the AP enzyme, 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.

[0046] The disclosed 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. In general, the multiplex assay uses a blue chromogen as described, and a second, non-blue chromogen. In this aspect, following the chromogen application step (140) described above, 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. In aspects, 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 about10 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. In further aspects, 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. For example, a second IHC assay may be performed on the tissue sample for detection of a second target antigen. In this aspect, 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.

[0047] Following the one or more IHC assays described above, the tissue section may then be counterstained with a suitable counterstain in a post-processing step. For example, 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).

[0048] It should be understood that 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%. Such 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.

[0049] Incubation times of any of the described steps may vary. For example, any dispense cycle in which a reagent is applied to the sample may include an incubation. Incubations aregenerally 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.IV. Example Automated Method Using a Slide-Staining Instrument

[0050] A slide-staining instrument (i.e., 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.

[0051] In one aspect, disclosed are methods for the preparation of samples for IHC detection methods which may employ, in whole or in part, the slide-staining instrument described herein. It should be understood that other instruments may be used to carry out the disclosed methods, for example that described in U.S. Patent 11493411, U.S. Patent Publication No. 2021 / 0293670, and U.S. Patent Publication No. 2023 / 0022299. For example, 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. To supply reagents, 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 becommunicated to group fluid dispenser and / or each slide staining assembly. Examples of 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. Additionally, 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.

[0052] 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. For example, the first solution comprising a blue chromogen and / or the second solution comprising a naphthol may be dispensed via this assembly. Alternatively, 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. In aspects, 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.V. Kits

[0053] Further disclosed are IHC assay 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.

[0054] In one aspect, the kit may comprise a first solution comprising a blue salt and a second solution comprising a naphthol phosphate. For example, the first solution may comprise an RR Salt and one or more buffers whereas the second solution may comprise a naphthol phosphate. In one aspect, the second solution comprises naphthol AS-MX phosphate and one or more buffers.

[0055] 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.

[0056] The kit may further comprise a secondary antibody conjugated to an enzyme. For example, 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. In aspects, 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.

[0057] 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. In one aspect, 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.

[0058] 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.

[0059] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.VI. EXAMPLESExample 1

[0060] Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections of 3 pm thickness were mounted onto charged slides. Slides were baked at 60 °C for 30 minutes. All steps were completed on the BOND-Rx automated staining instrument (software version 7) utilizing the sequential staining method. All steps unless otherwise stated are completed at ambient temperature.

[0061] 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) 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 applied to the slide for 30 minutes. The RR salt solution and AS-MX solution is mixed on board the instrument prior to being applied to the tissue for a total of 20 minutes, and produces a blue precipitate, localized to the cellular compartment of the epitope being detected. FIG. 3 depicts an exemplary result using the blue chromogen disclosed herein, using colon tissue and anti-CD3 primary antibody and anti-CDX2 antibody.

[0062] 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. For a multiplex protocol, 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 mayinclude 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.

[0063] 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. In one aspect, 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.

[0064] Slides are incubated with BOND Epitope Retrieval Solution 2 for 5 minutes. 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 fifteen minutes. A secondary reagent a-mouse IgG linker localizing mouse primary antibodies is applied to the slide for fifteen minutes and subsequently detected using a a-rabbit polymer reagent conjugated to horse radish peroxidase for 10 minutes. The green chromogen is applied to the tissue (10 minutes) and produces a green precipitate localized to the cellular compartment of the epitope in detection.

[0065] Final protocol steps include a counterstain dispense of Hematoxylin solution for 1 minute at ambient temperature, followed by the application of BOND Wash Solution to allow bluing of the reagent to take place. 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 animmunohistochemistry assay using anti-Ki67, anti-CDlO, and anti-BCL-2 on tonsil tissue. The multiple immunohistochemistry assay of 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.

[0066] All percentages and ratios are calculated by weight unless otherwise indicated.

[0067] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[0068] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0069] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”

[0070] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. All accessioned information (e.g., as identified by PUBMED, PUBCHEM, NCBI, UNIPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known tothose skilled therein as of the date of this disclosure. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0071] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

CLAIMSWhat is claimed is:

1. A chromogenic reagent system comprising a first solution comprising a blue salt and a second solution comprising a naphthol phosphate.

2. The chromogenic reagent system of claim 1, said blue salt comprisingwherein X comprises an inorganic or organic anion.

3. The chromogenic reagent system of claim 1 or 2, said blue salt comprising RR Salt having the following structure:

4. The chromogenic reagent system of any preceding claim, said naphthol phosphate being 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.

5. The chromogenic reagent system of any preceding claim, said naphthol phosphate comprising N-(2,4,-dimethylphenyl)-3-phosphonooxy)-2-naphthalenecarboxamide (Naphthol AS-MX) having the structure:

6. The chromogenic reagent system of any preceding claim, said first solution having a pH of from about 8.7 to about 8.9.

7. The chromogenic reagent system of any preceding claim, said first solution having a pH of from about 8 to about 9.

8. The chromogenic reagent system of any preceding claim, said second solution having a pH of from about 8 to about 9.

9. The chromogenic reagent system of any preceding claim, said first and / or said second solution further comprising one or both of sodium chloride and magnesium chloride.

10. The chromogenic reagent system of any preceding claim, said first and / or said second solution further comprising a buffer.

11. The chromogenic reagent system of any preceding claim, said first and / or said second solution further comprising a preservative.

12. The chromogenic reagent system of any preceding claim, said first and / or said second solution further comprising an antimicrobial.

13. A method of preparing a chromogen composition, comprising combining a first solution according to any preceding claim with the second solution of any preceding claim to form a third solution.

14. The method of claim 13, further comprising incubating said third solution for a period of time of from about 10 minutes to about five hours.

15. The method of claim 14, said incubation being carried out at ambient temperature.

16. A method of staining a biological sample, comprising contacting a biological sample with a first solution of any of claims 1 through 13.

17. The method of claim 16, further comprising contacting said biological sample with the second solution of any of claims 1 through 12.

18. The method of claim 17, said first and second solution being combined prior to contact with said biological sample.

19. The method of claim 17, said first solution being contacted with the biological sample prior to contacting said second solution with the biological sample.

20. The method of claim 17, said first solution being contacted with the biological sample after contacting said second solution with the biological sample.

21. The method of claim 17, said first solution being contacted with the biological sample simultaneously with contacting said second solution with the biological sample.

22. The method of any of claims 16 through 21, said biological sample comprising at least one primary antibody bound to a target antigen.

23. The method of any of claims 16 through 22, said biological sample comprising at least one secondary antibody conjugated to a primary antibody bound to a target antigen.

24. The method of any of claims 16 through 23, said biological sample comprising a secondary antibody conjugated to a primary antibody bound to a target antigen, said secondary antibody being conjugated to a alkaline phosphatase enzyme.

25. The method of any of claims 16 through 24, said biological sample being a formalin-fixed paraffin embedded (FFPE) tissue section.

26. The method of any of claims 16 through 25, said biological sample being a formalin-fixed paraffin embedded (FFPE) tissue section treated with an epitope retrieval solution.

27. The method of claim any of claims 16 through 26, said method being performed at ambient temperature.

28. The method of any of claims 16 through 27, said method being automated.

29. The method of any of claims 16 through 28, said contact being carried out on a device.

30. A method for carrying out single plex immunohistochemistry (IHC) assay for detecting a target antigen in a biological sample, comprising(a) contacting said biological sample with a first target-specific binding agent specific to a first target antigen;(b) contacting said biological sample with a second binding agent specific to said first binding agent, said second binding agent comprising an enzyme;(c) contacting said biological sample with a blue salt and a naphthol phosphate to form a blue precipitate; and(d) detecting said blue precipitate.

31. The method of claim 30, further comprising staining said biological sample.

32. The method of claim 30 or 31, further comprising visualizing a cellular structure in said biological sample.

33. The method of any of claims 30 through 32, further comprising visualizing and / or quantifying a target antigen in said biological sample.

34. The method of any of claims 30 through 33, said first and second binding agents being antibodies.

35. The method of any of claims 30 through 34, said enzyme being alkaline phosphatase (AP).

36. The method of any of claims 30 through 35, said biological sample being a formalin-fixed paraffin embedded (FFPE) tissue section.

37. The method of any of claims 30 through 36, said biological sample being a formalin-fixed paraffin embedded (FFPE) tissue section treated with an epitope retrieval solution.

38. The method of any of claims 30 through 37, further comprising detecting said blue precipitate.

39. The method of any of claims 30 through 38, said blue precipitate being insoluble in an organic medium.

40. The method of any of claims 30 through 39, said blue precipitate being insoluble in an aqueous medium.

41. The method of any of claims 30 through 40, said blue precipitate being insoluble in ethanol.

42. The method of any of claims 30 through 41, said method being performed at ambient temperature.

43. The method of any of claims 30 through 42, said method being automated.

44. The method of any of claims 30 through 43, said method being performed on a device.

45. A multiplex immunohistochemistry (IHC) method for detecting at least two target antigens in a biological sample, comprising(a) performing a first IHC assay for detecting a first target antigen, said first IHC assay comprising contacting a tissue sample labeled with a secondary antibody conjugated to an enzyme with first chromogen comprising a Fast Blue RR Salt and naphthol phosphate, said enzyme comprising alkaline phosphatase, to form a blue precipitate; and(b) performing a second IHC assay for detecting a second target antigen, said second IHC assay comprising contacting a tissue sample labeled with a secondary antibody conjugated to an enzyme with a second chromogen, to form a non-blue precipitate.

46. The multiplex IHC method of claim 45, the tissue sample of steps (a) and (b) being the same tissue sample.

47. The multiplex IHC method of claim 45 or 46, further comprising detecting said blue precipitate and said non-blue precipitate in the same sample.

48. The multiplex IHC method of any of claims 45 through 47, said blue precipitate being insoluble in an organic medium.

49. The multiplex IHC method of any of claims 45 through 48, said blue precipitate being insoluble in an aqueous medium.

50. The multiplex IHC method of any of claims 45 through 49, said blue precipitate being insoluble in ethanol.

51. The multiplex IHC method of any of claims 45 through 50, said method being performed on a device.

52. The multiplex IHC method of any of claims 45 through 51, said method being automated.

53. A kit comprising: a first solution comprisingwherein X' is an organic or inorganic anion; and a second solution comprising a naphthol phosphate.

54. The kit of claim 53, wherein the first solution comprises:

55. The kit of claim 53 or 54, said naphthol phosphate being 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.

56. The kit of claim 53 or 55, said naphthol phosphate comprising N-(2,4,-dimethylphenyl)-3- phosphonooxy)-2-naphthalenecarboxamide (Naphthol AS-MX) having the structure:

57. The kit of any of claims 53 through 56, said first and / or second solution having a pH of from about 8.7 to about 8.9.

58. The kit of any of claims 53 through 57, said first and / or second solution having a pH of from about 8 to about 9.

59. The kit any of claims 53 through 58, further comprising at least one primary antibody.

60. The kit of any of claims 53 through 59, further comprising at least one secondary antibody.

61. The kit of any of claims 53 through 60, further comprising a positive control.

62. The kit of any of claims 53 through 61, further comprising a negative control.

Images