MICROWAVE ANTIGEN RETRIEVAL IN NON-AQUEOUS SOLVENTS

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of the earlier filing date of U.S. Provisional Application No. 61/017,414, filed on December 28, 2007, which is incorporated herein by reference.

FIELD

This application relates to methods of high temperature antigen retrieval for immunohistochemistry, in particular examples using an antigen retrieval solution that includes at least one non-aqueous solvent and heating using microwave energy.

BACKGROUND

Immunohistochemistry (IHC) is a technique involving the use of specific binding agents, such as antibodies and antibody fragments, to detect specific antigens that may be present in a tissue sample. IHC is widely used in clinical and diagnostic settings, for example to diagnose particular disease states or conditions, such as a cancer. For example, a diagnosis of a particular type of cancer can be made based on the presence of a particular marker antigen present in a sample from a patient. Additional clinical applications of IHC include identification of infectious agents (such as Hepatitis B virus or H eliobacter pylori) and in the diagnosis of other diseases (such as Alzheimer disease or Creutzfeldt- Jacob disease).

IHC requires that sample cells or tissues undergo preparatory procedures that may include fixing the sample with chemicals such as an aldehyde, formalin substitutes, or alcohol. The sample may also be embedded in inert materials such as paraffin, celloidin, agars, polymers, resins, cryogenic media, or a variety of plastic embedding media. Other sample tissue or cell preparations require physical manipulation such as freezing or aspiration through a fine needle (fine needle aspiration). Without preservation, tissue samples rapidly deteriorate such that their use in diagnostics is compromised shortly after removal from their host. In 1893, Ferdinand Blum discovered that formaldehyde could be used to preserve or fix

tissue so that it could be used in histochemical procedures. Formalin is still the most widely used fixative for preparing samples for IHC. Some antigens may be detected after formalin fixation, but for many antigens, immunodetection after formalin treatment is lost or markedly reduced. Loss of antigen immunoreactivity is most noticeable at antigen epitopes that are discontinuous, i.e. amino acid sequences where the formation of the epitope depends on the confluence of portions of the protein sequence that are not contiguous.

Antigen retrieval (AR) refers to the attempt to reverse the structural changes that treatment of tissue with a fixative induces in the antigens within that tissue. Although the exact mechanism of AR is unknown, it is clear that modification of protein structure by formalin is reversible under conditions such as high-temperature heating. It is also clear that several factors affect antigen retrieval: heating, pH, molarity and metal ions in solution (Shi et ah, J. Histochem. Cytochem. 45:327-43 (1997)). The need exists for methods of antigen retrieval that are rapid and reproducible.

SUMMARY

Methods are disclosed herein for high temperature AR for use in immunohistochemistry. In particular examples, the method includes contacting a sample that may include one or more target antigens with an AR solution that includes a non-aqueous solvent and heating the AR solution using microwave energy. In some examples, the method includes heating the antigen retrieval solution to a temperature of at least 100 ⁰ C, such as 110 ⁰ C to 130 ⁰ C.

In further examples, the non-aqueous solvent includes at least one polar solvent. In particular examples, the polar solvent includes a polar protic solvent, a polar aprotic solvent, or a combination of polar solvents. In additional examples, the non-aqueous solvent includes at least one ionic liquid. In particular embodiments, the ionic liquid includes a deep eutectic solvent.

The foregoing and other objects and features of the disclosure will become more apparent from the following detailed description.

DETAILED DESCRIPTION I. Abbreviations and Terms

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular forms "a," "an," and "the" refer to one or more than one, unless the context clearly dictates otherwise. For example, the term "including a sample" includes single or plural samples and is considered equivalent to the phrase "including at least one sample." The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein,

"comprises" means "includes." Thus, "comprising A or B," means "including A, B, or A and B," without excluding additional elements. For example, the phrase "mutations or polymorphisms" or "one or more mutations or polymorphisms" means a mutation, a polymorphism, or combinations thereof, wherein "a" can refer to more than one.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Common terms in immunohistochemistry may be found in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988).

AR: antigen retrieval

IHC: immunohistochemistry

Aldehyde: An organic compound containing at least one terminal carbonyl group, also called an aldehyde, formyl, or methanoyl group. Aldehydes are amine reactive and can be used to cross-link proteins and/or cells containing an amine, for example to preserve biological samples, such as tissue samples from a subject. In one example, an aldehyde is formaldehyde.

Antibody: A polypeptide ligand that includes at least a light chain or heavy chain immunoglobulin variable region and specifically binds an epitope of an antigen. Antibodies can include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, or fragments of antibodies, and the like. In some examples, an antibody is labeled with a detectable label such as an enzyme or fluorophore.

The term "specifically binds" refers to, with respect to an antigen, the preferential association of an antibody or other ligand, in whole or part, with a specific polypeptide. A specific binding agent binds substantially only to a defined target. It is recognized that a minor degree of non-specific interaction may occur between a molecule, such as a specific binding agent, and a non-target polypeptide. Nevertheless, specific binding can be distinguished as mediated through specific recognition of the antigen. Although selectively reactive antibodies bind antigen, they can do so with low affinity. Specific binding typically results in greater than 2- fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody or other ligand (per unit time) to a target polypeptide, such as compared to a non-target polypeptide. A variety of immunoassay formats are appropriate for selecting antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the

antigen recognized by the antibody. This includes intact immunoglobulins and the variants and portions of them well known in the art, such as Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes recombinant forms such as chimeric antibodies (for example, humanized murine antibodies) and heteroconjugate antibodies (such as bispecifϊc antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

A "monoclonal antibody" is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody- forming cells from a fusion of myeloma cells with immune spleen cells. These fused cells and their progeny are termed "hybridomas." Monoclonal antibodies include humanized monoclonal antibodies.

Antigen: A molecule that stimulates an immune response, for example in a mammal. Antigens are usually proteins or polysaccharides. An epitope is an antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response. An antibody binds a particular antigenic epitope. The binding of an antibody to a particular antigen or epitope of an antigen can be used to localize the position of the antigen, for example in or on a biological sample, or to determine if the particular antigen is present in a biological sample.

Antigen retrieval (AR): A process for recovering antigenicity in fixed processed tissue samples. AR is also sometimes referred to as epitope retrieval, target retrieval, or target unmasking. Fixation modifies the tertiary structure of proteins and makes many antigens undetectable by specific antibodies by IHC. AR reverses these structural changes, making antigens detectable by specific antibodies.

Various methods are used for AR, including enzymatic treatment, heat treatment, or a combination of heat and enzyme treatments. Enzymatic AR utilizes protease digestion, such as by trypsin, proteinase K, pronase, or pepsin, to unmask antigens. Heat-induced (or high temperature) AR unmasks antigens by heat treatment, although the mechanism by which this occurs is unclear. In one example, high temperature AR includes heating using microwave energy for about 5 to about 20 minutes, such as about 10 minutes. In a further example, high temperature AR includes heating a sample in contact with an AR solution to at least 95 ⁰ C or at least 100 ⁰ C, such as about 100 ⁰ C to about 200 ⁰ C, such as about 110 ⁰ C to about 130 ⁰ C, or about 120 ⁰ C.

An antigen retrieval solution is a solution in which the process of AR occurs. A sample for IHC which has been fixed, such as fixation by formalin treatment, may be contacted with an AR solution. The sample and AR solution may then be incubated at a temperature suitable for AR to occur. In one example the AR solution includes a non-aqueous solvent, such as a polar solvent (e.g., propylene glycol) or an ionic liquid (e.g., a deep eutectic solvent including urea and choline chloride).

Contacting: Placement in direct physical association, for example both in solid form and/or in liquid form. In one example, the placement of a sample, such as a tissue sample affixed to a slide, in physical association with a solution, such as an AR solution.

Cross-linking agent: A homo- or hetero-multifunctional reagent with at least two identical or non-identical groups that are reactive to functional groups present in proteins, such as sulfhydryls and/or amine groups. In some examples, a protein cross-linker is amine reactive, meaning it is capable of forming a covalent bond with an amine group, such as an amine group present in a protein, for example an amine group present on a lysine or arginine residue. Cross-linking agents which are used in histology to prepare tissue samples for IHC act by creating covalent bonds between cellular components in the sample, such as within or between proteins, within or between nucleic acids, or between proteins and nucleic acids.

Cross-linking agents include aldehyde cross-linking agents and non-aldehyde cross- linking agents. Aldehyde cross-linking agents include formaldehyde,

paraformaldehyde, glyoxal, glutaraldehyde, adipaldehyde, succinaldehyde, and suberaldehyde. In a particular example, the cross-linking agent comprises formaldehyde. Non-aldehyde cross-linking agents include cyanuric chloride, carbodiimides, diisocyanates, diimido esters, diethylpyrocarbonate, and maleimides. Deep eutectic solvent: An ionic liquid formed by reacting an amine salt and an organic compound capable of forming a hydrogen bond with the anion of the amine salt and which exhibits a reduced melting point compared to either component in the pure state. A deep eutectic solvent can include a mixture of a substituted quaternary ammonium salt and a suitable hydrogen bond donor (e.g., see WO 02/26701, incorporated herein by reference). A suitable substituted quaternary ammonium salt includes choline chloride (hydroxyethyl-trimethyl ammonium chloride). Suitable hydrogen bond donors include amides (such as urea, thiourea, and acetamide), carboxylic acids (such as oxalic acid, benzoic acid, and citric acid), alcohols, phenols, and sugars (such as fructose). In one example, a deep eutectic solvent is a 1 :2 molar mixture of choline chloride and urea.

Embedding medium: An inert material in which tissues and/or cells are embedded to help preserve them for future analysis. Embedding also enables tissue samples to be sliced into thin sections. Embedding media include paraffin, celloidin, OCT™ compound, agar, plastics, or acrylics. Many embedding media are hydrophobic, therefore the inert material may need to be removed prior to histological or cytological analysis, which utilizes primarily hydrophilic reagents. The term deparaffinization or dewaxing is broadly defined herein to refer to the removal of any type of embedding medium from a biological sample. For example, paraffin embedded tissue sections are dewaxed by passage through organic solvents, such as toluene, xylene, limonene, or other suitable solvents.

Fixation: A process which preserves cells and tissue constituents in as close to a life-like state as possible and allows them to undergo preparative procedures without significant change. Fixation arrests the autolysis and bacterial decomposition processes which begin upon cell death, and stabilizes the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing, such as for IHC or in situ hybridization.

Tissues can be fixed either by perfusion or by submersion in a fixative. Fixatives can be classified as cross-linking agents (such as aldehydes, e.g., formaldehyde, paraformaldehyde, and glutaraldehyde, as well as non-aldehyde cross-linking agents), oxidizing agents (e.g., metallic ions and complexes, such as osmium tetroxide and chromic acid), protein-denaturing agents (e.g., acetic acid, methanol, and ethanol), fixatives of unknown mechanism (e.g., mercuric chloride, acetone, and picric acid), combination reagents (e.g., Carnoy's fixative, methacarn, Bouin's fluid, B5 fixative, Rossman's fluid, and Gendre's fluid), microwaves, and miscellaneous fixatives (e.g., excluded volume fixation and vapor fixation). Additives may also be included in the fixative, such as buffers, detergents, tannic acid, phenol, metal salts (such as zinc chloride, zinc sulfate, and lithium salts), and lanthanum.

The most commonly used fixative in preparing samples for IHC is formaldehyde, generally in the form of a formalin solution (4% formaldehyde in a buffer solution, referred to as 10% buffered formalin). In one example, the fixative is 10% neutral buffered formalin.

Immunohistochemistry (IHC): A method of determining the presence or distribution of an antigen in a sample by detecting interaction of the antigen with a specific binding agent, such as an antibody. A sample including an antigen (such as a target antigen) is incubated with an antibody under conditions wherein antibody- antigen binding can occur. Antibody-antigen binding is determined by means of a detectable label conjugated to the antibody (direct detection) or by means of a detectable label conjugated to a secondary antibody, which is raised against the primary antibody (indirect detection). Detectable labels include, but are not limited to, radioactive isotopes, enzymes (such as peroxidase and alkaline phosphatase), fluorochromes (such as fluorescein, fluorescein isothiocyanate, and rhodamine), and biotin.

Ionic liquid: An organic salt that includes a cation and an anion that has a melting point below about 100° C. In some examples, the ionic liquid is liquid at ambient or near ambient temperatures (e.g., having a melting point at or below about 20° C). An ionic liquid can include two or more different salts, such as mixtures of salts including two or more different cations, anions, or both. Properties of ionic

liquids include low toxicity, low vapor pressure, non-flammability, and thermal stability over a wide range of temperatures (e.g., stable to over 200° C). For example, the absence of volatility of ionic liquids offers lower toxicity than low- boiling point solvents. Ionic liquids are safe for use in microwave methods, as sudden pressure surges do not occur. The dipole characteristics of ionic liquids results in rapid excitation by microwaves, and consequently, faster reactions (see, e.g., Ionic Liquids in Synthesis, Wasserscheid and Welton, Wiley- VCH Verlag, 2002; Chemistry in Alternative Reaction Media, Adams, et al., Eds., Wiley, 2003). Ionic liquids can be based on ammonium, imidazolium, phosphonium, pyridinium, pyrrolidinium, or sulfonium. In a particular example an ionic liquid includes ethyl ammonium nitrate. Ionic liquids containing eutectic mixtures of salts include mixtures of a substituted quaternary ammonium salt and a metal salt (referred to as a metal-based ionic liquid) or mixtures of a substituted quaternary ammonium salt and a hydrogen bond donor (referred to as a deep eutectic solvent). Metal-based ionic liquids include mixtures of choline chloride (2-hydroxyethyl- trimethylammonium chloride) with CoCl ₂ , CrCl ₃ , ZnCl ₂ , or Zn(NO ₃ ) ₂ . Deep eutectic solvents include mixtures of choline chloride with urea, 1 -methyl urea, 1,3- dimethyl urea, thiourea, or acetamide. Additional deep eutectic solvents include mixtures of choline chloride with phenol, glycerol, trifluoroacetic acid, ethylene glycol, oxalic acid, or citric acid. In a particular example a deep eutectic solvent includes a eutectic mixture of choline chloride and urea in a 1 :2 molar ratio.

Label: An agent capable of detection, for example by spectrophotometry, flow cytometry, or microscopy. For example, a label can be attached to a specific binding agent, such as an antibody, thereby permitting detection of the specific binding agent and hence an antigen bound by the specific binding agent. Examples of labels include, but are not limited to, radioactive isotopes, enzyme substrates, co- factors, ligands, chemiluminescent agents, fluorophores (such as small molecule fluorophores or semiconductor nanocrystals), haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning:

A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).

Microwave energy: Non-ionizing electromagnetic radiation with a frequency between about 300 megahertz (MHz) and about 300 gigahertz (GHz). Commercially available microwave ovens for laboratory or home use typically utilize microwave radiation of a frequency of about 2.45 GHz. These microwave ovens generally operate from about 20 W to about 1000 W of power.

Microwave energy may generate heat by dielectric heating. A substance possessing a dipole moment is sensitive to external electric fields and will attempt to align itself with the field by rotation. The frequency of the microwave energy is not high enough for this rotation to precisely follow the field. This results in increased molecular friction and collisions, giving rise to heating. Microwave energy may also generate heat by ionic heating. Ions will move through the solution under the influence of an electric field generated by the microwaves, resulting in an increased collision rate and giving rise to heating.

Non-aqueous solvent: A solvent other than water. A non-aqueous solvent includes both organic non-aqueous solvents (such as ethanol, formamide, and propylene glycol) and inorganic non-aqueous solvents (such as liquid ammonia and inorganic acids). In some examples, the non-aqueous solvent is an organic solvent. In additional examples, the non-aqueous solvent includes more than one nonaqueous solvent, such as two, three, four, or five non-aqueous solvents. In a particular example, the non-aqueous solvent includes propylene glycol.

A solvent is a compound which is present in the greatest quantity in a mixture of two or more compounds which form a homogenous liquid phase. A solute is a compound which is present in a lesser amount in the mixture. A nonaqueous solvent includes solutions containing less than about 50% water. A nonaqueous solvent can include water, for example about 2% water, about 5%, 10%, 20%, 30%, 40%, or about 50% water. In some examples, the non-aqueous solvent includes about 10% to about 30% water. Polar solvent: A solvent having a dielectric constant of about 6 ε or greater.

A polar protic solvent includes polar solvents capable of forming hydrogen bonds. Polar protic solvents include solvents that contain O-H and/or N-H groups, such as

water, ethanol, methanol, glycerol, ethylene glycol, propylene glycol, and formamide. A polar aprotic solvent includes polar solvents not capable of forming hydrogen bonds. Polar aprotic solvents include 2-methoxyethyl ether (diglyme), pyridine, acetone, N-methylpyrrolidone, dimethylformamide, sulfolane, dimethyl sulfoxide, and propylene carbonate.

Sample: A sample, such as a biological sample, includes biological materials (such as nucleic acid molecules and proteins) obtained from an organism or a part thereof, such as a plant, animal, bacteria, and the like. In particular embodiments, the biological sample is obtained from an animal subject, such as a human subject. A biological sample is any solid or fluid sample obtained from, excreted by, or secreted by any living organism, including without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas, among others, and multicellular organisms (such as plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as cancer). A biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease, such as a rheumatoid arthritis, osteoarthritis, gout or septic arthritis). A biological sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can include a cell (whether a primary cell or cultured cell) or medium conditioned by any cell, tissue or organ. In some examples, a sample is a tissue or tumor biopsy.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals (such as laboratory or veterinary subjects).

II. Methods of Antigen Retrieval Using Non-Aqueous Solvent and Microwave Energy

Methods are provided for AR in a sample to be used for IHC. The disclosed methods can be used to detect antigens of interest (also referred to as target antigens)

that may be present in a biological sample. In particular examples, the method includes contacting a sample that includes an antigen (for example is suspected of containing a target antigen) that has been fixed by a cross-linking agent, with an AR solution, and heating the AR solution using microwave energy, thereby retrieving antigens in the sample. In one example, the sample includes an antigen that has been fixed by an aldehyde cross-linking agent, such as formaldehyde, for example 10% neutral buffered formalin. In some examples, the sample has been processed by embedding the sample in an inert material and/or cutting the sample into thin sections. In further examples, the sample is affixed to a solid support, such as a microscope slide (e.g., glass slide).

In one example, the method includes submerging a sample containing an antigen that has been fixed by a cross-linking agent in an AR solution, which is then heated to a temperature at which AR may occur. In some examples, the method includes submerging a sample affixed to a microscope slide in a container, such as a jar, which includes a volume of AR solution, such that the sample is completely covered by the AR solution. In further examples, a small volume of AR solution is placed directly in contact with a sample affixed to a microscope slide. Furthermore, the sample and AR solution may be covered to prevent evaporation, such as by a glass or plastic coverslip, or Liquid Coverslip™ (Ventana Medical Systems, Inc., Tucson, AZ), or the entire slide, or the portion of the slide including the sample can be enclosed in a chamber that is sealed or unsealed. In some examples, the method may comprise use of an automated IHC staining system (such as NexES® IHC or Benchmark® XT systems (Ventana Medical Systems), or GenoMx™ (Biogenex Laboratories, Inc.)). In additional examples, the sample is free-floating in the AR solution (i.e. the sample is not affixed to a solid support).

The method includes contacting a sample with an AR solution that includes a non-aqueous solvent. In one example, the non-aqueous solvent includes a polar solvent. In additional examples, the polar solvent includes a solvent having a dielectric constant of at least 6 ε. In further examples, the polar solvent includes a solvent having a boiling point of about 100 ⁰ C to about 300 ⁰ C, such as about 100° C to about 300° C, about 110° C to about 200° C, or about 115° C to about 150° C. In some examples, the AR solution includes a mixture of at least two polar solvents,

such as two, three, four, or five polar solvents. In a particular example, the polar solvent comprises propylene glycol, for example about 70% propylene glycol. In additional examples, the polar solvent comprises a mixture of propylene glycol and propylene carbonate. In one example, the polar solvent includes a polar protic solvent. In particular examples, the polar protic solvent includes propylene glycol or formamide. In further examples, the polar solvent includes a polar aprotic solvent. In some examples, the polar aprotic solvent includes dimethyl formamide, sulfolane, propylene carbonate, N-methyl pyrrolidone, diglyme, or pyridine. In some examples, the AR solution includes a non-aqueous solvent which is an ionic liquid. In further examples, the AR solution includes a mixture of at least two ionic liquids, such as two, three, four, or five ionic liquids. In some examples the ionic liquid includes at least one organic salt having a melting point at or below about 100° C, such as about 100 ⁰ C to about 5°C, about 100 ⁰ C to about 10 ⁰ C, about 100 ⁰ C to about 20 ⁰ C, about 80 ⁰ C to about 20 ⁰ C, or about 60 ⁰ C to about 10 ⁰ C. In further examples, the ionic liquid includes at least one organic salt having a melting point at or below about 20° C. In a particular example, the ionic liquid includes ethyl ammonium nitrate. In a further example, the ionic liquid includes a mixture of l-alkyl-3-methylimidazolium salts, such as l-methyl-3-methylimidazolium, 1-ethyl- 3-methylimidazolium, or l-butyl-3-methylimidazolium.

In some examples, the ionic liquid comprises a deep eutectic solvent which includes a mixture of organic salts having a decreased melting point compared to the pure compounds. In one example, the deep eutectic solvent includes a 1 :2 molar mixture of choline chloride and urea. In further examples, the AR solution comprises a mixture of one or more polar solvents and one or more ionic liquids. In a particular example, the mixture of polar solvents and ionic liquids includes propylene glycol and l-methyl-3-methyl- imidazolium.

A. Antigen Retrieval Solutions

The methods provided herein use an AR solution that includes a nonaqueous solvent. In some examples, the AR solution includes more than one non-

aqueous solvent, such as two, three, four, or five non-aqueous solvents. AR is most efficient at temperatures greater than 100° C (such as 120° C); however, it is not possible to heat aqueous solutions above 100° C unless pressure is used, such as with an autoclave or pressure cooker device. AR solutions including a non-aqueous solvent can have a boiling point greater than 100° C, therefore AR can be performed without the use of pressure, such as in a microwave oven.

Characteristics of AR solutions for use in the disclosed method include: (1) boiling point greater than 100° C, (2) miscible with water, (3) low viscosity, (4) low volatility, and (5) low flammability. In one example, the AR solutions for use in the methods disclosed herein include non-aqueous solvents having a boiling point of at least 100° C, such as about 100° C to about 400° C, about 100° C to about 300° C, about 110° C to about 200 ⁰ C, or about 115° C to about 150° C. In further examples, the boiling point of the AR solution including a non-aqueous solvent is at least 100° C, such as about 100° C to about 400° C, about 100° C to about 300° C, about 110° C to about 200° C, or about 115° C to about 150° C. In some examples, the AR solution comprises a non-aqueous solvent and water, such as about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% water, such that the boiling point of the AR solution is at least 100° C. In one example, the boiling point of the AR solution including a non-aqueous solvent is about 188° C. In another particular example, the AR solution comprises about 70% propylene glycol, such that the boiling point of the AR solution is about 125° C. In further examples, the AR solution comprises a mixture of non-aqueous solvents, such as two, three, four, or five non-aqueous solvents, wherein the boiling point of the AR solution is at least 100° C. During the IHC procedure, the sample can be moved from an aqueous environment to the AR solution, and/or from the AR solution to an aqueous environment. Therefore, the AR solution can include a solvent that is miscible with water. Generally, polar solvents are miscible with water. In one example, the AR solution includes solvents having a dielectric constant greater than about 6 ε, such as about 6 ε to about 120 ε, about 20 ε to about 100 ε, or about 30 ε to about 70 ε. In some examples, the AR solution comprises a non-aqueous solvent and water, such as about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% water,

such that the dielectric constant is greater than about 6 ε, such as about 6 ε to about 120 ε. In a particular example, the dielectric constant of the AR solution including a non-aqueous solvent is about 32 ε.

In a further example, the AR solution includes a solvent having low viscosity, such that the AR solution may be easily rinsed away from the sample following the AR procedure. Viscosity refers to the internal resistance to flow of a fluid. It can be measured by methods known in the art and is expressed in units of dyne-seconds per cm ² (poise). In one example, the viscosity of the AR solution is about 0.5 centipoise to about 100 centipoise at 25° C, such as about 1 centipoise to about 75 centipoise, about 10 centipoise to about 60 centipoise, or about 20 centipoise to about 50 centipoise. In some examples, the AR solution comprises a non-aqueous solvent and water, such as about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% water, such that the viscosity of the AR solution is about 0.5 centipoise to about 100 centipoise at 25° C. In further examples, the AR solution comprises a mixture of non-aqueous solvents, such as two, three, four, or five non-aqueous solvents, such that the viscosity of the AR solution is about 0.5 centipoise to about 100 centipoise at 25° C. In a particular example, the viscosity of the AR solution including a non-aqueous solvent is about 56 centipoise at 25° C.

In further examples, the AR solution includes solvents having low volatility. Volatility is expressed as vapor pressure (mm Hg). At a given temperature, substances with a lower vapor pressure are less volatile than substances with a higher vapor pressure. In some examples, the AR solution has a vapor pressure at 20° C of less than about 5 mm Hg, such as about 0.01 mm Hg to about 5 mm Hg, such as about 0.01 mm Hg to about 3 mm Hg, or about 0.1 mm Hg to about 1 mm Hg. In some examples, the AR solution comprises a non-aqueous solvent and water, such as about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% water, such that the vapor pressure of the AR solution is less than about 5 mm Hg at 20° C. In a particular example, the AR solution including a non-aqueous solvent has a vapor pressure of about 0.13 mm Hg at 20° C. In another example, the AR solution includes solvents having low flammability. Substances having a flash point greater than 37.8° C (100° F) are considered non-flammable. In some examples the AR solution has a flash point of

about 38° C to about 200° C, such as about 50° C to about 150° C, about 70° C to about 130° C, or about 95° C to about 110° C. In some examples, the AR solution comprises a non-aqueous solvent and water, such as about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% water, such that the flash point is greater than about 38° C. In a particular example, the AR solution has a flash point of about 99° C.

In one example, the AR solution includes additional agents, such as water, buffers, detergents, and/or chelating agents. In some examples, the AR solution includes water, such as about 2 % water, about 5%, 10%, 20%, 30%, 40%, or 50% water. In further examples, the AR solution includes at least one buffering agent, such as Tris, Tris buffered saline, phosphate buffered saline, citrate, borate, acetate, or combinations thereof. In some examples, the AR solution includes at least one detergent, such as polysorbate surfactants, such as Tween® 20, or nonionic surfactants, such as Triton® X-100 or Nonidet P-40. In further examples, the AR solution includes at least one chelating agent such as ethylenediaminetetraacetic acid (EDTA), N,N'-ethylenebis[2-(2-hydroxyphenyl)-glycine] (EHPG), diethylene- triamine-pentaacetic acid (DTPA), citrate, tartaric acid and tartarate salts, 5- sulfosalicylic acid, oxalic acid and oxalate salts, pyromellitic acid and salts thereof, N,N-dimethyldecylamine N-oxide, nitrilotriacetic acid, or ethylene glycol-bis(2- aminoethylether)-λ/,λ/,N',N'-tetraacetic acid (EGTA). In additional examples, the AR solution comprises metal ions, such as divalent metal ions (for example, zinc or lead). Further additives that may be included in AR solutions for use in the disclosed methods include, but are not limited to, glycine, urea, citraconic anhydride, guanidine thiocyanate, and formic acid.

1. Polar Solvents

In some examples, the non-aqueous solvent includes a polar solvent. Polarity can be expressed in terms of the dielectric constant or the dipole moment of a compound. These can be determined by methods known in the art. The dielectric constant (ε) is an indicator of the polarity of a solvent, wherein a high dielectric constant indicates a polar solvent (such as water (80 ε) or formamide (109 ε)); a low dielectric constant indicates a non-polar solvent (such as hexane (1.9 ε) or benzene

(2.3 ε)). In some examples, a polar solvent includes a solvent having a dielectric constant of at least about 6 ε, such as about 6 ε to about 120 ε, about 20 ε to about 100 ε, or about 30 ε to about 70 ε. In some examples, the AR solution comprises a non-aqueous solvent and water, such as about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% water, such that the dielectric constant is about 6 ε to about 120 ε. In a particular example, the dielectric constant of the AR solution including a non-aqueous solvent is about 32 ε.

In some examples, the disclosed methods use a non-aqueous AR solution that includes at least one polar solvent. In some examples, the AR solution includes a combination of polar solvents, such as two, three, four, or five polar solvents.

In one example, the AR solution includes a polar solvent, such as at least one polar protic solvent. In some examples, the AR solution includes more than one polar protic solvent, such as two, three, four, or five polar protic solvents. Polar protic solvents include polar solvents capable of forming hydrogen bonds, such as solvents that contain O-H and/or N-H groups. In some examples, the polar protic solvent includes a solvent containing at least one O-H group, such as phenol, ethylene glycol, propylene glycol, glycerol, or formic acid. In an additional example, the polar protic solvent includes a solvent containing at least one N-H group, such as formamide. In a particular example, the polar protic solvent includes propylene glycol.

In some examples, the AR solution includes a polar solvent including at least one polar aprotic solvent. In additional examples the AR solution includes more than one polar aprotic solvent, such as two, three, four, or five polar aprotic solvents. Polar aprotic solvents include polar solvents which are not capable of forming hydrogen bonds. In some examples, the polar aprotic solvent includes 2- methoxyethyl ether (diglyme), pyridine, hexamethylphosphoramide, N- methylpyrrolidone, nitromethane, dimethylformamide, sulfolane, dimethyl sulfoxide, or propylene carbonate.

In additional examples, the AR solution can include a mixture of at least one polar protic solvent and at least one polar aprotic solvent. In some examples, the AR solution comprises propylene glycol and dimethyl formamide or propylene glycol and propylene carbonate.

2. Ionic Liquids

In some examples, the non-aqueous solvent includes an ionic liquid. An ionic liquid is an ionic compound, such as an organic salt, which is liquid at temperatures below about 100° C. In some examples, the ionic liquid has a melting point at or below room temperature, such as at or below 20° C. In some examples, the AR solution comprises an ionic liquid and water, such as about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% water.

In some examples, the disclosed methods use a non-aqueous AR solution that includes at least one ionic liquid. In some examples, the AR solution includes a combination of ionic liquids, such as two, three, four, or five ionic liquids. In some examples the AR solution including at least one ionic liquid has a boiling point of at least about 125° C, such as about 150° C to about 400° C, such as about 200° C to about 300° C. Ionic liquids generally exhibit very low vapor pressure. In some examples, the ionic liquid has no distinguishable vapor pressure {see e.g.,

Huddleston et al, Green Chem. 3:156-164, 2001). In further examples, the AR solution including at least one ionic liquid has a vapor pressure less than about 1 mm Hg at 25° C, such as less than 0.1 mm Hg at 25° C. For many ionic liquids, the first thermal event upon heating is thermal decomposition, rather than boiling. In some AR solutions including at least one ionic liquid, the onset of thermal decomposition is about 150° C to about 500° C, such as about 200° C to about 450° C, about 250° C to about 400° C, or about 300° C to about 350° C.

In some examples, the disclosed methods use a non-aqueous AR solution that includes at least one ionic liquid. In some examples, the AR solution includes a combination of ionic liquids, such as two, three, four, or five ionic liquids. Ionic liquids can be ammonium-based (such as ethyl ammonium nitrate, tetrabutylammonium heptadecafluorooctanesulfonate, tetrabutylammonium nonafluorobutanesulfonate, and tetrapentylammonium thiocyanate), imidazolium- based (such as l-butyl-3-methylimidazo Hum bromide, l-butyl-3-methylimidazolium tetrafluoroborate, l-butyl-3-methylimidazolium hex afluorophosphate 1 ,2-dimethyl- 3-propylimidazolium bis(trifluoromethylsulfonyl)imide, l-ethyl-3- methylimidazolium bis(trifluoromethylsulfonyl)imide, 1 -ethyl-3-methylimidazolium

bromide, l-hexyl-3-methylimidazolium chloride, and l-methyl-3-octylimidazolium chloride), phosphonium-based (such as trihexyltetradecylphosphonium bis(trifluoromethylsulfonyl)amide, trihexyltetradecylphosphonium bromide, and triisobutylmethylphosphonium tosylate), pyridinium-based (such as 1 -butyl - 3methylpyridinium bis(trifluormethylsulfonyl)imide, 1 -butyl -4-methylpyridinium chloride, l-butyl-4-methylpyridinium hexafluorophosphate, 1 -butyl -4- methylpyridinium tetrafluoroborate, and 3 -methyl- 1-propylpyridinium bis(trifluormethylsulfonyl)imide), pyrrolidinium-based (such as 1 -butyl- 1- methylpyrrolidinium chloride, 1 -butyl- 1-methylpyrrolidinium tetrafluoroborate, and 1 -butyl- 1 -methylpyrrolidinium bis(trifluoromethylsulfonyl)imide), or sulfonium- based (such as triethylsulfonium bis(trifluoromethylsulfonyl)imide). One of skill in the art can identify ionic liquids, for example lists of ionic liquids can be found in the catalog of Sigma- Aldrich (St. Louis, MO). In a particular example, the ionic liquid may be ethyl ammonium nitrate. Ionic liquids also include eutectic mixtures, wherein an ionic liquid is formed by mixing two salts and heating until a liquid is formed. The resulting composition is liquid at temperatures lower than either of the individual components. Ionic liquids which are eutectic mixtures are known in the art (e.g., WO 02/26701 and U.S. Pat. No. 6,573,405). Ionic liquids including eutectic mixtures include mixtures of a substituted quaternary ammonium salt and a metal salt (referred to as a metal- based ionic liquid) or mixtures of a substituted quaternary ammonium salt and a hydrogen bond donor (referred to as a deep eutectic solvent). Metal-based ionic liquids include mixtures of choline chloride (2-hydroxyethyl-trimethylammonium chloride) with CoCl ₂ , CrCl ₃ , ZnCl ₂ , or Zn(NO ₃ ) ₂ . Deep eutectic solvents include mixtures of choline chloride with urea, 1 -methyl urea, 1,3 -dimethyl urea, thiourea, or acetamide. Additional deep eutectic solvents include mixtures of choline chloride with phenol, glycerol, trifluoroacetic acid, ethylene glycol, oxalic acid, or citric acid. In a particular example a deep eutectic solvent includes a eutectic mixture of choline chloride and urea in a 1 :2 molar ratio.

B. Microwave Heating

The disclosed methods include contacting a sample (which may include a target antigen) with an AR solution including at least one non-aqueous solvent and heating the AR solution using microwave energy. Methods of microwave AR in water are described in U.S. Patent No. 5,244,787. In some examples, the AR solution is heated to a temperature of at least 100 ⁰ C, such as about 100° C to about 200° C, such as about 100° C to about 150° C, or about 110° C to about 130° C. In a particular example, the AR solution is heated to about 120° C.

The method can include heating the AR solution in contact with the sample for a time sufficient to unmask antigens which have been masked by fixation. In some examples, the sample is heated by microwave energy for a set period of time, such as about 1 minute to about 20 minutes, such as about 5 minutes to about 15 minutes. In a particular example, the AR is heated in contact with the sample for 10 minutes using microwave energy. In additional examples, the AR solution is heated to a desired temperature and maintained at that temperature for a predetermined period of time. In some examples, the AR solution is heated to 120° C in about 5 minutes. In one example, the AR solution is heated to a temperature of 120 ⁰ C over the course of about 5 minutes and the temperature of the AR solution is subsequently maintained at 120° C for a further 5 minutes. The AR solution may be maintained at 120° C for longer times, such as about 5 minutes to about 30 minutes. Microwave ovens commercially available for home or laboratory use can be used with the disclosed methods. Suitable home microwave ovens include GE Profile™ countertop microwave ovens (such as model number JEB1860DMWW), Kenmore countertop microwave ovens (such as model number 66462), and Amana countertop microwave ovens (such as model number AMC2206BAW). Suitable laboratory grade microwave ovens include EMS-9000 (Electron Microscopy Sciences, Hatfield, PA), H2850V Microwave Processor (EB Sciences, East Granby, CT) and Pelco Bio Wave® Pro (Ted Pella, Inc., Redding, CA). Alternatively, the disclosed methods may be used with an automated IHC processor incorporating a microwave oven (such as GenoMx™ (Biogenex Laboratories, Inc.)), or special equipment, such as microwave ovens designed specifically for the disclosed

methods, can be integrated into existing devices for automated IHC (such as NexES® IHC or Benchmark® XT systems (Ventana Medical Systems)).

The power level to achieve a particular temperature will vary depending on the equipment utilized and the composition and volume of AR solution utilized. The microwave oven can be utilized at a set power level, such as about 20 W to about 1000 W, such as about 100 W to about 800 W, about 200 W to about 700 W, about 300 W to about 600 W, or about 400 W to 500 W. In a particular example, AR solution in contact with a sample is heated using a microwave oven set at a power of 350 W. A temperature control probe can be used in conjunction with the microwave oven to determine the temperature. In some examples, the temperature control probe is used in conjunction with a programmable microwave oven, such that a particular temperature of the AR solution is maintained during the heating period. In one example, a temperature control probe can be placed in the AR solution during heating and the microwave oven may be programmed to heat the sample to a preset temperature and to maintain the set temperature for a particular period of time. In some examples, the preset temperature is about 100° C to about 200° C, such as about 120° C. In further examples, the predetermined period of time is about 1 minute to about 30 minutes, such as about 5 minutes to about 20 minutes, for example, about 10 minutes.

C. Samples

The disclosed methods can be applied to samples that are fixed by cross- linking agents, such as whole tissue or tissue biopsies including, but not limited to lymph node, gut, liver, kidney, breast, prostate, lung, tonsils, spinal cord, and cervix. Samples can also include tumor biopsies, including biopsies from a neoplasm, such as a cancer, for example breast cancer, prostate cancer, colorectal cancer, cervical cancer, lung cancer, melanoma, and ovarian cancer. Samples can be fluidic, such as bodily fluids, for example blood samples, or a cell suspension. The methods can also be applied to cells of microorganisms, such as yeast, bacteria, and fungi.

The methods can be applied to samples that are fixed by cross-linking agents which are cut into thin sections and subsequently applied to a support, such as a

microscope slide. Microscope slides suitable for use with the disclosed methods include coated or charged slides, such as poly-lysine coated slides or gel coated slides. The disclosed methods may also be carried out with free-floating samples.

D. Exemplary antigens of interest

A target antigen can be any selected antigen, such as a protein, whose presence or absence is to be determined. For example, a target antigen can be one that is associated with a disease or condition, such that detection of the antigen (or not) can be used to infer information (such as diagnostic or prognostic information for the subject from whom the sample is obtained) relating to the disease or condition. In particular examples the target antigen is from a eukaryotic organism, such as a human. In some examples, the target antigen is from a pathogen, such as a virus, bacteria, or intracellular parasite. In certain examples, the antigen of interest is a molecule associated with a neoplastic disease (or cancer). For example, the antigen of interest can include at least one at least one protein associated with cancer (e.g., HER2, c-Myc, n-Myc, AbI, Bcl2, Bcl6, RbI, p53, EGFR, TOP2A, MET, or other receptors and/or signaling molecules, etc.).

Other exemplary antigens correlated with neoplastic transformation and which are useful in the disclosed methods also include EGFR (7pl2; e.g., GENBANK™ Accession No. AAH94761 and encoded by NC 000007, nucleotides 55054219-55242525), HER2 (17q21.1; e.g., GENBANK™ Accession Nos. NP 004439 and NP OO 1005862 and encoded by NC 000017, nucleotides 35097919-35138441), AbI (9q34.1; e.g., GENBANK™ Accession Nos. NP 009297 and NP 005148 and encoded by NC 000009, nucleotides 132579089-132752883), MET (7q31; e.g., GENBANK™ Accession No.

NP 000236 and encoded by NC 000007, nucleotides 116099695-116225676), the C-MYC gene (8q24.21; e.g., GENBANK™ Accession No. NP_002458 and encoded by NC 000008, nucleotides 128817498-128822856), D5S271 (5pl5.2), lipoprotein lipase (LPL) gene (8p22; e.g., GENBANK™ Accession No. AAHl 1353 and encoded by NC 000008, nucleotides 19841058-19869049), RBl (13ql4; e.g., GENBANK™ Accession No. NP_000312 and encoded by NC_000013, nucleotides 47775912-47954023), p53 (17pl3.1; e.g., GENBANK™

Accession No. NP 000537 and encoded by NC 000017, complement, nucleotides 7512464-7531642), N-MYC (2p24; e.g., GENBANK™ Accession No. AAA36371 and encoded by NC 000002, complement, nucleotides 151835231-151854620), CHOP (12ql3; e.g., GENBANK™ Accession No. NP 004074 and encoded by NC OOOO 12, complement, nucleotides

56196638-56200567), FUS (16pl l.2; e.g., GENBANK™ Accession No. NP 004951 and encoded by NC OOOO 16, nucleotides 31098954-31110601 ), FKHR (13pl4; e.g., GENBANK™ Accession No. AAC39591 and encoded by NC_000013, complement, nucleotides 40027817-40138734), as well as, for example: ALK (2p23; e.g., GENBANK™ Accession No. NP_004295 and encoded by NC 000002, complement, nucleotides 29269144-29997936), Ig heavy chain, CCNDl (I lql3; e.g., GENBANK™ Accession No. CAG38775 and encoded by NC OOOOI l, nucleotides 69165054..69178423), BCL2 (18q21.3; e.g., GENBANK™ Accession No. NP_000624, NP_000648, and encoded by NC 000018, complement, nucleotides 58941559-59137593), BCL6 (3q27; e.g., GENBANK™ Accession No. NP_001697, NP_620309, and encoded by NC 000003, complement, nucleotides 188921859-188946169), MALFl, API (Iρ32-ρ31; e.g., GENBANK™ Accession No. NP_002219 and encoded by NC OOOOOl, complement, nucleotides 59019051-59022373), TOP2A (17q21-q22; e.g., GENBANK™ Accession No. NP_001058 and encoded by NC_000017, complement, nucleotides 35798321-35827695), TMPRSS (21q22.3; e.g., GENBANK™ Accession No. NC_000021, complement, nucleotides 41758351-41801948), ERG (21q22.3; e.g., GENBANK™ Accession No. NP 891548 and encoded by NC 000021, complement, nucleotides 38675671-38955488); ETVl (7ρ21.3; e.g., GENBANK™ Accession No. NP 004947 and encoded by NC 000007, complement, nucleotides 13897379-13995289), EWS (22ql2.2; e.g., GENBANK™ Accession No. NP_053733, NP_005234, and encoded by NC_000022, nucleotides 27994271-28026505); FLIl (I lq24.1-q24.3; e.g., GENBANK™ Accession No. NP 002008 and encoded by NC OOOOI l, nucleotides 128069199-128187521),

PAX3 (2q35-q37; e.g., GENBANK™ Accession No. NP_852122 and encoded by NC_000002, complement, nucleotides 222772851-222871944), PAX7 (Ip36.2-

p36.12; e.g., GENBANK™ Accession No. NP_039236, NP_002575, and encoded by NC OOOOOl, nucleotides 18830087-18935219, PTEN (10q23.3; e.g., GENBANK™ Accession No. NP 000305 and encoded by NC OOOO 10, nucleotides 89613175-89716382), AKT2 (19ql3.1-ql3.2; e.g., GENBANK™ Accession No. NP OO 1617 and encoded by NC OOOO 19, complement, nucleotides 45431556-45483036), MYCLl (Ip34.2; e.g., GENBANK™ Accession No. NP 001028253, NP 001028254 and encoded by NP 005367, NC OOOOOl, complement, nucleotides 40133685-40140274), REL (2pl3-pl2; e.g., GENBANK™ Accession No. NP 002899 and encoded by NC 000002, nucleotides 60962256-61003682) and CSFlR (5q33-q35; e.g., GENBANK™

Accession No. NP 005202 and encoded by NC 000005, complement, nucleotides 149413051-149473128).

In other examples, a target antigen is from a virus or other microorganism associated with a disease or condition. Detection of the virus or microorganism antigen in a cell or tissue sample is indicative of the presence of the organism. For example, the antigen can be one found in an oncogenic or pathogenic virus, a bacterium or an intracellular parasite (such as Plasmodium falciparum and other Plasmodium species, Leishmania (sp.), Cryptosporidium parvum, Entamoeba histolytica, and Giardia lamblia, as well as Toxoplasma, Eimeria, Theileria, and Babesia species).

Other exemplary viruses and corresponding protein sequences (GENBANK™ RefSeq Accession No. in parentheses) include human adenovirus A (NC OO 1460), human adenovirus B (NC 004001), human adenovirus C (NC OO 1405), human adenovirus D (NC 002067), human adenovirus E (NC 003266), human adenovirus F (NC OO 1454), human astrovirus

(NC 001943), human BK polyomavirus (VOl 109; GL60851) human bocavirus (NC 007455), human coronavirus 229E (NC 002645), human coronavirus HKUl (NC 006577), human coronavirus NL63 (NC 005831), human coronavirus OC43 ( NC 005147), human enterovirus A (NC OO 1612), human enterovirus B (NC_001472), human enterovirus C (NC_001428), human enterovirus D (NC 001430), human erythrovirus V9 (NC 004295), human foamy virus (NC 001736), human herpesvirus 1 (Herpes simplex virus type 1) (NC 001806),

human herpesvirus 2 (Herpes simplex virus type 2) (NC_001798), human herpesvirus 3 (Varicella zoster virus) (NC_001348), human herpesvirus 4 type 1 (Epstein-Barr virus type 1) (NC_007605), human herpesvirus 4 type 2 (Epstein- Barr virus type 2) (NC 009334), human herpesvirus 5 strain AD 169 (NC_001347), human herpesvirus 5 strain Merlin Strain (NC_006273), human herpesvirus 6A (NC OO 1664), human herpesvirus 6B (NC 000898), human herpesvirus 7 (NC_001716), human herpesvirus 8 type M (NC_003409), human herpesvirus 8 type P (NC 009333), human immunodeficiency virus 1 (NC_001802), human immunodeficiency virus 2 (NC_001722), human metapneumovirus (NC 004148), human papillomavirus- 1 (NC 001356), human papillomavirus- 18 (NC OO 1357), human papillomavirus-2 (NC OO 1352), human papillomavirus-54 (NC OO 1676), human papillomavirus-61 (NC OO 1694), human papillomavirus-cand90 (NC 004104), human papillomavirus RTRX7 (NC 004761), human papillomavirus type 10 (NC 001576), human papillomavirus type 101 (NC 008189), human papillomavirus type 103 (NC 008188), human papillomavirus type 107 (NC 009239), human papillomavirus type 16 (NC OO 1526), human papillomavirus type 24 (NC_001683), human papillomavirus type 26 (NC_001583), human papillomavirus type 32 (NC_001586), human papillomavirus type 34 (NC 001587), human papillomavirus type 4 (NC 001457), human papillomavirus type 41 (NC OO 1354), human papillomavirus type 48 (NC OO 1690), human papillomavirus type 49 (NC 001591), human papillomavirus type 5 (NC 001531), human papillomavirus type 50 (NC 001691), human papillomavirus type 53 (NC OO 1593), human papillomavirus type 60 (NC OO 1693), human papillomavirus type 63 (NC OO 1458), human papillomavirus type 6b (NC OO 1355), human papillomavirus type 7 (NC_001595), human papillomavirus type 71 (NC_002644), human papillomavirus type 9 (NC OO 1596), human papillomavirus type 92 (NC 004500), human papillomavirus type 96 (NC 005134), human parainfluenza virus 1 (NC 003461), human parainfluenza virus 2 (NC 003443), human parainfluenza virus 3 (NC OO 1796), human parechovirus (NC OO 1897), human parvovirus 4 (NC 007018), human parvovirus Bl 9 (NC 000883), human

respiratory syncytial virus (NC_001781) , human rhinovirus A (NC_001617), human rhinovirus B (NC OO 1490), human spumaretrovirus (NC OO 1795), human T-lymphotropic virus 1 (NC OO 1436), human T-lymphotropic virus 2 (NC_001488). In certain examples, the target antigen is from an oncogenic virus, such as

Epstein-Barr Virus (EBV) or a Human Papilloma Virus (HPV, e.g., HPVl 6, HPVl 8). In other examples, the target antigen is from a pathogenic virus, such as a Respiratory Syncytial Virus, a Hepatitis Virus (e.g., Hepatitis C Virus), a Coronavirus (e.g., SARS virus), an Adenovirus, a Polyomavirus, a Cytomegalovirus (CMV), or a Herpes Simplex Virus (HSV).

E. Fixation

In order to preserve cell and tissue components and morphology, preparation of biological samples for IHC can include a fixation step. AR appears to be particularly necessary for IHC using samples fixed by cross-linking fixatives. For example, it is estimated that up to 85% of antigens fixed in formalin require AR to optimize IHC results (Ramos- Vara, Vet. Pathol. 42:405-426, 2005). However, high temperature AR may also improve IHC results in samples which have been fixed with alcohol, which does not produce cross-links (Ramos-Vara, Vet. Pathol. 42:405- 426, 2005). In some examples, the disclosed methods may be used for AR in samples fixed using cross-linking fixatives (such as aldehydes) or denaturing fixatives (such as alcohols).

Cross-linking fixatives include aldehydes, such as formaldehyde, paraformaldehyde, glutaraldehyde, acrolein (acrylic aldehyde), glyoxal, malonaldehyde, adipaldehyde, succinaldehyde, and suberaldehyde, 2,3-butanedione, chloral hydrate, and other polyaldehydes (such as oxalic, malonic, succinic, and adipic dialdehydes). Aldehyde fixatives form cross linking methylene bridges and Schiff bases between basic amino acid (e.g., lysine) residues of proteins. In a particular example, the cross-linking fixative includes formaldehyde, for example 10% buffered formalin. Cross-linking fixatives also include non-aldehyde fixatives. Non-aldehyde cross-linking agents include cyanuric chloride, carbodiimides, diisocyanates, diimido esters, diethylpyrocarbonate, and maleimides.

Denaturing fixatives do not produce cross-links, rather they act by denaturing proteins in the sample. Denaturing fixatives include alcohols, such as ethanol or methanol. Additional examples of denaturing fixatives include mixtures which include alcohol, such as acetic acid combined with ethanol or methanol and methacarn (which includes methanol, chloroform, and acetic acid).

F. Embedding

Fixed samples can be further prepared for IHC by a step of embedding the sample in an inert embedding medium. Embedding stabilizes the sample, which may then be stored at room temperature for extended periods. In addition, the embedding material helps to provide structural rigidity, such that thin slices of the tissue (such as about sections about 3 μm to about 20 μm thick) may be cut and affixed to a surface, such as a microscope slide. In some examples, tissues are dehydrated through a series of graded alcohols or acetone prior to embedding. Methods of embedding and sectioning are known to those of skill in the art (see e.g., Woods and Ellis, "Laboratory Histopathology: A Complete Reference" Churchill Livingstone, 1994). In one example, after fixation, sections may be dehydrated in a graded series of ethanol, cleared in xylene, and embedded in molten paraffin at 56°C. Samples can be embedded by submersion in an embedding media, either manually or using an automated specimen processing apparatus. Methods of embedding are known in the art. Embedding media include paraffin, paraffin blends (such as mixtures of paraffin with plastic polymers and/or copolymer alloys), polyester wax, polyethylene glycol, mixtures of glycols and resins (such as Tissue Freezing Medium TFM™ or TissueTek® O. C. T. compound), acrylics (such as glycol methacrylate and methyl methacrylate), celloidin, and agar. In one example, the fixed sample is embedded in paraffin.

The embedding medium may be removed from the sample prior to AR and subsequent immunohistochemical processing. Removal of the embedding media may include incubations in organic solvents (such as xylene, toluene, or limonene), incubation in graded alcohols, and/or washes with water or buffer. Methods of deparaffinization are known to those of skill in the art (see e.g., Woods and Ellis,

"Laboratory Histopathology: A Complete Reference" Churchill Livingstone, 1994). In one example, tissue samples may be dewaxed in xylene and then rehydrated through steps of 100% ethanol, 50% ethanol, and water.

G. Immunological Staining

Following AR utilizing the disclosed methods, the samples are allowed to cool passively or are actively cooled, for example for about 20 minutes. A slow cooling process can be useful, as additional AR can continue during this time, as proteins that have been thermally unfolded return to their native confirmations during this cooling period. In other cases, over-retrieval or destruction of antigens can occur if the process is not stopped by cooling, therefore a faster cooling process is desirable. In the case of a manual procedure, after sufficient cooling to allow handling, the biological samples are usually rinsed with distilled water to remove any excess AR solution from their surface. A final rinsing in a buffer prior to immunological staining is preferred in order to provide physiological conditions suitable for antigen-antibody binding to the antigens present in the biological sample. Such buffers typically have a pH of about 6.5 to about 8.5, such as about pH 6.8 to about pH 8.0, or about pH 7.0 to 7.6. Numerous physiological buffers are commercially available through biological supply houses. Specific buffers may be selected according to the antibody being used.

The immunological staining process is no different from that previously known. No modification of techniques is required for using an immunological staining process with the steps of the present disclosure, but routine optimization of the immunological staining process may be desirable. Typically, the biological sample that has been contacted with an AR solution is contacted with a specific binding agent that specifically binds the antigen of interest for a sufficient length of time to allow the specific binding agent to bind the antigen of interest if the antigen is present in the biological sample. In specific embodiments, the specific binding agent includes an antibody that is capable of specifically binding the antigen of interest if it is present in the sample.

In some examples, the primary antibody is labeled with a detectable label, such as a fluorophore or enzyme (such as horseradish peroxidase or alkaline

phosphatase). Optionally a secondary antibody that binds the primary antibody and contains a label, such as a fluorophore, horseradish peroxidase, or alkaline phosphatase, is added to the tissue section and allowed to incubate for a sufficient length of time to allow the primary antibody-secondary antibody reaction to occur, if the primary antibody is bound to the antigen present in the tissue. Once the label has been linked to the primary antibody (directly, or indirectly via a secondary antibody) it can be detected, thereby detecting the antigen bound by the primary antibody. In examples in which the label is an enzyme, a substrate is applied, which is typically a liquid substrate that is converted by the enzyme into an insoluble colored dye. The colored dye precipitates onto the tissue at the site of the primary antibody. Thus, the presence of a colored end product is indicative of a positive antigen-antibody reaction, and the absence of a colored end product is indicative of a negative antigen-antibody reaction. In examples where the specific binding agent is linked to a fluorophore, the light emitted from the fluorophore is detected, for example using a fluorescent microscope. In some specific examples, the fluorophore is a nanoparticle, such as a semiconductor nanocrystal. The tissue is typically examined microscopically to detect the presence of the label and therefore the presence of the antigen of interest (wherein generally the absence of a detectable label is indicative of a negative antigen-antibody reaction).

The present disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Example 1 Preparation of Samples for Immunohistochemistry

This example describes the preparation of samples for immunohistochemistry, including fixation, paraffin embedding, and dewaxing of samples.

Tissue samples of breast carcinoma and tonsil were obtained and fixed in 10% neutral buffered formalin for 18 hours. The tissue was dehydrated and then embedded in paraffin. The paraffin blocks were cut into 4 μm thick sections using a microtome and applied to sialinized microscope slides. Sections were

deparaffϊnized in xylene and rehydrated in graded ethanol and water and stored until use in immunohistochemistry as described below.

Example 2 Microwave Antigen Retrieval Using Non-Aqueous Antigen Retrieval Solution

This example describes a method of antigen retrieval for immunohistochemistry utilizing a non-aqueous antigen retrieval solution and a microwave oven as the heat source and compares the use of aqueous and nonaqueous AR solutions. Methods

Samples were prepared as described in Example 1. Slides were placed in a Coplin jar containing 50 ml of AR solution, such that the tissue sections were completely submerged. The jar containing the slides was placed in a microwave oven and heated for 10 minutes at 350 W. The samples were removed from the microwave and allowed to cool for about 20 minutes at room temperature.

The slides were removed from the AR solution, washed in phosphate buffered saline (PBS), and endogenous peroxidase was blocked with 3% hydrogen peroxide. Primary antibody was applied to the tissue sections according to the manufacturer's instructions for 10 minutes. The slides were washed with PBS. Secondary antibody was applied to the slides according to the manufacturer's instructions for 10 minutes (EnVision™+ HRP kit, Dako Corp., Carpinteria, CA). The slides were washed with PBS and antibody was detected with diaminobenzidine and hydrogen peroxide.

Staining was observed using light microscopy. Staining was graded on a semi-quantitative scale of 0-4 as follows: 0 = no staining, 1 = light staining, 2 = moderate staining, 3 = intense staining, 4 = very intense staining. Results

Antigen retrieval using aqueous AR solution was carried out in 0.1 M citric acid, pH 6.0 by heating for 10 minutes using a microwave oven. The temperature of the AR solution reached 97° C. Antigen retrieval using non-aqueous AR solution was carried out in 70% propylene glycol containing 0.1 M citric acid, pH 6.0. The non-aqueous AR solution was heated using a microwave oven for 10 minutes and

the temperature of the AR solution reached 120° C. Some samples were not subjected to AR prior to immunohistochemical staining.

Breast carcinoma tissue samples were stained with antibodies against estrogen receptor (1D5, Dako Corp.) and progesterone receptor (PgR 636, Dako Corp.). Tonsil tissue samples were stained with an antibody against Ki67 (MIB 1, Dako Corp.).

Following staining, the staining intensity of each sample was assessed semi- quantitatively (Table 1). Samples which were not subjected to AR did not show any detectable staining. When aqueous AR solution was used, breast carcinoma samples exhibited moderate staining and tonsil samples exhibited light staining. Intense staining was observed for all samples when the non-aqueous AR solution was used.

Table 1. Staining Intensity for AR Solutions

Example 3

Use of Polar Aprotic Solvents in Microwave Antigen Retrieval

This example describes the use of polar aprotic solvents in a method of antigen retrieval utilizing a microwave oven.

Samples for immunohistochemistry are fixed, sectioned, and dewaxed as described in Example 1. The slides are placed in a staining jar containing an antigen retrieval solution in an amount sufficient to cover the tissue sections. The antigen retrieval solutions used are the aprotic solvents dimethyl formamide, sulfolane, propylene carbonate, N-methyl pyrrolidone, 2 methoxyethyl ether (diglyme), pyridine, or mixtures thereof. The AR solutions contain polar aprotic solvents at concentrations from 50%- 100% and some contain 0.1 M citrate and/or 0.1 M EDTA. The jar containing the slides and antigen retrieval solution is placed in a microwave oven and heated at maximum power for times from 5-30 minutes, such that the temperature of the antigen retrieval solution reaches 120° C within about 5 minutes

and the AR solution is then maintained at 120° C. The jar is removed from the microwave and allowed to cool for about 20 minutes.

The slides are washed, blocked, and exposed to a primary antibody as described in Example 2. Following further washing, the slides are exposed to a secondary antibody that is conjugated to a detectable marker. If the marker is an enzyme, the slides are exposed to the appropriate substrate prior to microscopy to observe antibody binding. If the marker is a fluorescent molecule, immunofluorescent microscopy is utilized to measure antibody binding.

Example 4

Use of Formamide in Microwave Antigen Retrieval

This example describes the use of formamide in a method of antigen retrieval utilizing a microwave oven.

Samples for immunohistochemistry are fixed, sectioned, and dewaxed as described in Example 1. The slides are placed in a staining jar containing formamide in an amount sufficient to cover the tissue sections. The AR solution contains formamide at concentrations from 50%- 100% and can contain 0.1 M citrate and/or 0.1 M EDTA. The jar containing the slides and formamide solution is placed in a microwave oven and heated at maximum power for times from 5-30 minutes, such that the temperature of the formamide solution reaches 120° C within about 5 minutes and the AR solution is then maintained at 120° C. The jar is removed from the microwave and allowed to cool for about 20 minutes.

Immunohistochemistry is carried out as described in Example 2. Antibody binding is detected using light microscopy or immunofluorescent microscopy.

Example 5

Use of Ethyl Ammonium Nitrate in Microwave Antigen Retrieval The example describes the use of ethyl ammonium nitrate in a method of antigen retrieval utilizing a microwave oven as the heat source. Samples for immunohistochemistry are fixed, sectioned, and dewaxed as described in Example 1. The slides are placed in a staining jar containing ethyl ammonium nitrate in an amount sufficient to cover the tissue sections. The AR

contains ethyl ammonium nitrate at concentrations from 50%- 100% and can contain 0.1 M citrate and/or 0.1 M EDTA. The jar containing the slides and ethyl ammonium nitrite solution is placed in a microwave oven and heated at maximum power for times from 5-30 minutes, such that the temperature of the solution reaches 120° C within about 5 minutes and the AR solution is then maintained at 120° C. The jar is removed from the microwave and allowed to cool for about 20 minutes. Immunohistochemistry is carried out as described in Example 2. Antibody binding is detected using light microscopy or immunofluorescent microscopy.

Example 6

Use of a Deep Eutectic Solvent in Microwave Antigen Retrieval

This example describes the use of a deep eutectic solvent in a method of antigen retrieval utilizing a microwave oven as the heat source.

A deep eutectic solvent of choline chloride and urea is prepared by mixing a 1 :2 molar ratio of choline chloride and urea and heating the mixture to 70° C for about 20 minutes, until a clear, colorless liquid is formed.

Samples for immunohistochemistry are fixed, sectioned, and dewaxed as described in Example 1. The slides are placed in a staining jar containing the choline chloride :urea deep eutectic solvent in an amount sufficient to cover the tissue sections. The AR solution contains the choline chloride :urea mixture at concentrations from 50%- 100% and can contain 0.1 M citrate and/or 0.1 M EDTA. The jar containing the slides and the choline chloride :urea deep eutectic solvent is placed in a microwave oven and heated at maximum power for times from 5-30 minutes, such that the temperature of the solution reaches 120° C within about 5 minutes and the AR solution is then maintained at 120° C. The jar is removed from the microwave and allowed to cool for about 20 minutes.

Immunohistochemistry is carried out as described in Example 2. Antibody binding is detected using light microscopy or immunofluorescent microscopy.

In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples of the disclosure and should not be taken as limiting the scope of

the invention. Rather, the scope of the invention is defined by the following claims. I therefore claim as my invention all that comes within the scope and spirit of these claims.