Synthetic controls for immunohistochemistry

Abstract

To provide reliable positive and negative IHC controls that are highly sensitive, specific, and adaptable to a wide range of targets, including targets for which no suitable biological controls exist.SOLUTION: Synthetic controls include a solid antigen / carrier protein gel comprising purified antigen (e.g., in a known amount) and a carrier protein, such as a serum albumin protein (e.g., a serum albumin protein), an egg white protein or a mixture of egg white proteins, gelatin, or polylysine. In some embodiments, the purified antigen is cross-linked to a carrier protein in a solid antigen / carrier protein gel.SELECTED DRAWING: Figure 3-1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority benefit of U.S. Provisional Application Serial No. 62 / 595,434, filed December 6, 2017, and U.S. Provisional Application Serial No. 62 / 730,422, filed September 12, 2018, each of which is incorporated herein by reference in its entirety.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission regarding an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing Computer Readable Form (CRF) (Filename: 146392040440SEQLIST.TXT, Recorded: December 5, 2018, Size: 3KB).

[0003] The present disclosure relates to synthetic controls useful, for example, in immunohistochemistry (IHC), and related methods of manufacture and uses. In some embodiments, the synthetic controls comprise serum albumin protein, egg white protein or a mixture of egg white proteins, or a solid gel containing purified antigen (e.g., in a known amount) cross-linked to a carrier protein such as gelatin. [Background technology]

[0004] IHC is an important tool for both research and clinical applications. Among the many techniques used to characterize protein expression, IHC is one of the few that provides information on expression levels and localization, e.g., at the cellular and / or tissue level. Therefore, IHC is an important tool used to characterize proteins of interest in research. IHC has also become an important diagnostic tool in the clinic, for example, for classifying patients for various personalized medicine applications. As an example, the HercepTest™ (Dako Denmark A / S) semi-quantitative HER2 IHC assay has been approved by the FDA for nearly 20 years for use in assessing HER2 protein status. This test allows clinicians to identify patients whose tumors overexpress HER2. While HER2 is overexpressed in many types of cancer, 25–30% of breast cancers have been shown to overexpress HER2, and this marker has been correlated with shorter disease-free intervals and overall survival. HER2-targeted therapy (e.g., anti-HER2 antibody therapy) in these patients significantly improves overall survival, response rate, duration of response, and time to disease progression. See, e.g., Slamon, DJ et al. (2001) N Engl J Med 344:783-792.

[0005] Establishing a reliable IHC assay for a specific target presents multiple challenges. First, it is necessary to identify antibodies that are specific for the target of interest and do not cross-react with other targets. This requires appropriate positive and negative controls that express the target at known levels, which can be difficult to identify if the target's expression has not been characterized.

[0006] Second, even if antibodies are available, it can be difficult to identify positive and negative controls that are reliable, readily available, and easy to mass-produce. Both tissue samples and cell lines (e.g., cell pellets) have been used as IHC controls, but both have significant limitations. For many targets, suitable tissues are unavailable. In other cases, expression in tissues may be variable, uncharacterized, or too weak to detect. Cell lines are more readily available than tissue samples (although it can be difficult to grow specific cell lines on an industrial scale). However, like expression in tissue samples, expression in cell lines may be variable, uncharacterized, or too weak to detect. While cell lines can be engineered to overexpress a target of interest, overexpression can be significantly higher than expression in the actual tissue, resulting in artificial subcellular localization. Overexpression can also be heterogeneous within a population of cultured cells. While cell lines can be produced in batches, these can quickly become exhausted, necessitating the production of new batches that may have different properties.

[0007] Various approaches aimed at establishing a standardized approach for generating IHC controls have been attempted. Over 45 years ago, Brandtzaeg described a method for producing "artificial tissue" samples, i.e., millimeter-sized blocks of glutaraldehyde-fixed rabbit serum dispersed with human immunoglobulin fractions or whole serum (Brandtzaeg, P. (1972) Immunology;22(1):177-183). This general technique was revisited and expanded with apparent success over the next few decades (Millar and Williams (1982) Histochem J. 14(4):609-620; Schipper and Tilders (1983) J Histochem Cytochem. 31(1):12-18; Valnes et al. (1984) J Histochem Cytochem. 33(8):755-61; Valnes and Brandtzaeg (1985) Histochemistry. 81(4):313-9), but it has also been described as prone to uneven and nonspecific staining (Shi et al. (2005) J Histochem Cytochem. 53(9):1167-1170) and is rarely used in modern practice. More widely used are clonal cell lines containing abundant, well-characterized target proteins (Mohd Omar et al. (2010) Acta Histochem. 112(6):519-28). While these are invaluable in many settings, cell line controls can exhibit significantly heterogeneous expression of a particular target across individual subclones, different passages of a single clone, or even within a single culture population, defeating the goal of creating a uniform, reproducible standard.

[0008] Sompuram, SR et al. (2002) Clin Chem 48:410-420 describes spotting peptides directly onto glass slides or coupling peptides to glass beads (see also Sompuram et al. (2015) J. Histochem Cytochem 63:681-690). However, implementing this approach presented technical challenges. For example, because it was difficult for technicians to see the spots where the peptides were applied to the glass (they were invisible before staining), many slides were stained with an insufficient amount of reagent to cover all the controls, thereby introducing staining artifacts (see Bogen, SA et al. (2009) Appl Immunohistochem Mol Morphol 17:239-246). Because these peptide spots were much thinner than the actual tissue sections, achieving strong positive control staining was difficult. Other groups have attempted to mix targets of interest into lysozyme solutions that can be prepared like formalin-fixed, paraffin-embedded tissue sections (see Fowler, CB et al. (2007) Lab Invest 87:836-846). They noted that gel formation depends on the protein concentration and isoelectric point (Fowler et al., (2007) Lab Invest. 87(8):836-46). However, this approach did not work for many targets, such as peptides, which can leak out of lysozyme gelatin. Agarose has also been tested as a potential tissue surrogate. However, the peptides dispersed in the agarose were not uniform. Additionally, these agarose-based peptide gels were typically subjected to antigen retrieval, which involves boiling the agarose to melt it and then separating it on a glass slide.

[0009] Therefore, there is a need for an approach that provides reliable positive and negative IHC controls that are sensitive, specific, and applicable to a wide range of targets, including those for which no appropriate biological controls exist. Such an approach would also provide a useful assay for determining antibody specificity, which is particularly advantageous when screening large numbers of antibodies to identify and validate new antibodies specific to a target of interest, and when optimizing IHC staining protocols.

[0010] All references cited herein, including patent applications, patent publications, non-patent literature, and UniProtKB / Swiss-Prot accession numbers, are hereby incorporated by reference in their entirety, as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0011] To meet these and other needs, methods are provided herein for producing solid antigen / carrier protein gels. These solid gels can be processed like tissue samples or other biological samples, following standard IHC or electron microscopy (EM) processing methods (including fixation, sectioning, antigen retrieval, etc.). Because the gels contain known amounts of the antigen of interest, they can be used to generate a series of gels with known concentrations of antigen, for example, to standardize staining with specific antibodies or to screen for antibodies that specifically recognize the antigen of interest and are suitable for IHC / EM analysis. These methods are believed to provide a general platform for mimicking multiple levels of expression using known concentrations of antigen, allowing for control staining of any antigen of interest.

[0012] Certain aspects of the present disclosure relate to methods for producing a solid antigen / carrier protein gel (e.g., for IHC or EM analysis), the method comprising: (a) mixing a purified antigen with a liquid solution comprising a carrier protein selected from the group consisting of albumin protein (e.g., serum albumin protein), egg white protein, or a mixture of egg white proteins, and gelatin to produce the antigen / carrier protein liquid solution; and (b) heating the antigen / carrier protein liquid solution to form the solid antigen / carrier protein gel. In some embodiments, the method comprises: (a) mixing a purified antigen with a liquid solution comprising a carrier protein selected from the group consisting of albumin protein (e.g., serum albumin protein), egg white protein, or a mixture of egg white proteins, gelatin, and polylysine to produce the antigen / carrier protein liquid solution; and (b) heating the antigen / carrier protein liquid solution to form the solid antigen / carrier protein gel. In some embodiments, the method further comprises, after (b): dehydrating the solid antigen / carrier protein gel and embedding the dehydrated solid antigen / carrier protein gel in a paraffin block. In some embodiments, the method further comprises, after embedding the dehydrated solid antigen / carrier protein gel in the paraffin block: transferring a core containing the antigen / carrier protein gel from the paraffin block to a recipient tissue microarray (TMA) block. In some embodiments, the method further comprises, after (b): incubating the solid antigen / carrier protein gel in a liquid embedding medium and freezing the solid antigen / carrier protein gel in the embedding medium. In some embodiments, the method further comprises, after (b): embedding the solid antigen / carrier protein gel in a plastic resin. In some embodiments, the method further comprises, after (b): sectioning the solid antigen / carrier protein gel into one or more solid antigen / carrier protein gel sections having a thickness of about 30 nm to about 50 μm. In some embodiments, the solid antigen / carrier protein gel is sliced ​​into one or more solid antigen / carrier protein gel sections having a thickness of about 2 μm to about 30 μm.In some embodiments, the solid antigen / carrier protein gel is sectioned into one or more solid antigen / carrier protein gel slices having a thickness of about 30 nm to about 100 nm. In some embodiments, the method further comprises, prior to (b), including a fixative in the antigen / carrier protein liquid solution. In some embodiments, the fixative comprises formaldehyde. In some embodiments, the antigen / carrier protein liquid solution comprises formaldehyde at a final concentration of at least about 1%. In some embodiments, the fixative comprises glutaraldehyde, Davidson's fixative, Bouin's fixative, half-strength Karnovski's fixative, or a zinc salt. In some embodiments, the method further comprises subjecting the solid antigen / carrier protein gel to antigen retrieval. In some embodiments, subjecting the solid antigen / carrier protein gel to antigen retrieval comprises heating the solid antigen / carrier protein gel in the liquid solution. In some embodiments, the method further comprises: prior to (b), including a fixative in the antigen / carrier protein liquid solution; after (b), dehydrating the solid antigen / carrier protein gel; embedding the dehydrated solid antigen / carrier protein gel in a paraffin block; slicing the paraffin block embedded in the antigen / carrier protein gel into one or more sections having a thickness of about 30 nm to about 50 μm; subjecting one or more sections of the embedded solid antigen / carrier protein gel to antigen retrieval; and after antigen retrieval, blocking one or more sections of the embedded solid antigen / carrier protein gel. In some embodiments, the method further comprises, after (b), incubating the solid antigen / carrier protein gel in a liquid embedding medium; freezing the solid antigen / carrier protein gel in the embedding medium; slicing the frozen antigen / carrier protein gel into one or more sections having a thickness of about 30 nm to about 50 μm; and blocking the one or more sections of the frozen antigen / carrier protein gel. In some embodiments, the antigen is a polypeptide antigen. In some embodiments, the antigen comprises an N-terminal tyrosine, a C-terminal cysteine, or both.In some embodiments, the method further comprises crosslinking the antigen to a carrier protein (e.g., albumin protein, egg white protein or a mixture of egg white proteins, or gelatin) using a cysteine-reactive reagent prior to (b). In some embodiments, the antigen comprises a non-polypeptide antigen. In some embodiments, the carrier protein is an albumin protein, such as bovine, caprine, equine, or human serum albumin. In some embodiments, the carrier protein is egg white protein or a mixture of egg white proteins. In some embodiments, the antigen / carrier protein liquid solution produced in (a) comprises a carrier protein concentration of 2% (w / v) or greater. In some embodiments, the antigen / carrier protein liquid solution produced in (a) comprises a carrier protein at a final concentration of about 25% (w / v) or less. In some embodiments, the carrier protein is gelatin, and the method further comprises, after (b), cooling the heated antigen / carrier protein liquid solution to form a solid antigen / carrier protein gel. In some embodiments, the method further comprises, after cooling the heated antigen / carrier protein liquid solution, incubating the solid antigen / carrier protein gel with a fixative to form an immobilized antigen / carrier protein gel, and dehydrating the immobilized antigen / carrier protein gel. In some embodiments, the antigen / carrier protein liquid solution produced in (a) comprises a carrier protein at a concentration of about 0.5% (w / v) or greater. In some embodiments, the antigen / carrier protein solution is heated to at least about 65°C in (b). In some embodiments, the antigen / carrier protein solution is heated to at least about 65°C in (b) for at least 6 minutes. In some embodiments, polylysine is present in the antigen / carrier protein solution produced in (a) at a concentration of about 14 mg / mL.

[0013] Further provided herein is a solid antigen / carrier protein gel produced by a method according to any one of the above embodiments.

[0014] Further provided herein is a tissue microarray (TMA) comprising at least a first solid antigen / carrier protein gel produced by a method according to any one of the above embodiments and a second solid antigen / carrier protein gel produced by a method according to any one of the above embodiments.

[0015] Certain aspects of the present disclosure relate to a method of immunohistochemistry (IHC) staining of an antigen, comprising: providing a solid antigen / carrier protein gel produced by the method of any one of the above-described embodiments, wherein the solid antigen / carrier protein gel comprises an antigen; providing a sample; contacting the solid antigen / carrier protein gel and the sample with a primary antibody that specifically binds to the antigen; after contacting the solid antigen / carrier protein gel and the sample with the primary antibody, contacting the solid antigen / carrier protein gel and the sample with a secondary antibody that specifically binds to the primary antibody, wherein a detectable moiety is conjugated to the secondary antibody; detecting a signal of the detectable moiety from the solid antigen / carrier protein gel; and detecting a signal of the detectable moiety from the sample, wherein detection of a signal from the sample compared to the signal detected from the solid antigen / carrier protein gel indicates the presence of the antigen in the sample. In some embodiments, the detectable moiety comprises an enzyme, and detecting the signal of the detectable moiety comprises exposing the detectable moiety to a chromogenic, fluorogenic, or chemiluminescent substrate of the enzyme and detecting a signal from the substrate upon reaction with the enzyme. In some embodiments, the detectable moiety comprises a fluorophore, a metal particle, a metal ion, a radioisotope, a nucleic acid, an electrochemiluminescent reporter, or a "quantum dot" (a solid, semiconductor, or carbon-based fluorescent nanoparticle). In some embodiments, the sample is a tissue sample.

[0016] A particular aspect of the present disclosure is a method of immunohistochemistry (IHC) staining of a control antigen, comprising providing first and second solid antigen / carrier protein gels, each of which is produced by the method of any one of the above-described embodiments, wherein the first solid antigen / carrier protein gel comprises an antigen at a first concentration and the second solid antigen / carrier protein gel comprises the antigen at a second concentration higher than the first concentration; contacting the first and second solid antigen / carrier protein gels with a primary antibody that specifically binds to the antigen; The method includes contacting the second solid antigen / carrier protein gel with a primary antibody, followed by contacting the first and second solid antigen / carrier protein gels with a secondary antibody that specifically binds to the primary antibody, wherein a detectable moiety is conjugated to the secondary antibody; detecting a first signal of the detectable moiety from the first solid antigen / carrier protein gel; and detecting a second signal of the detectable moiety from the second solid antigen / carrier protein gel, wherein detection of the second signal being greater than the first signal indicates IHC staining of a control antigen. In some embodiments, the detectable moiety comprises an enzyme, and detecting the signal of the detectable moiety comprises exposing the detectable moiety to a chromogenic, fluorogenic, or chemiluminescent substrate of the enzyme and detecting a signal from the substrate upon reaction with the enzyme. In some embodiments, the detectable moiety comprises a fluorophore, a metal particle, a metal ion, a radioisotope, a nucleic acid, an electrochemiluminescent reporter, or a "quantum dot" (a solid, semiconductor, or carbon-based fluorescent nanoparticle). In some embodiments, the first concentration is 0 nM and detection of the absence of a first signal indicates IHC staining of the control antigen.In some embodiments, the method further comprises providing a sample; contacting the sample with a primary antibody; after contacting the sample with the primary antibody, contacting the sample with a secondary antibody, wherein a detectable moiety is conjugated to the secondary antibody; detecting a third signal of the detectable moiety from the sample; and comparing the third signal to the first and second signals, wherein the amount of the third signal compared to the amount of the first and second signals indicates the amount of antigen present in the sample relative to the amount of antigen in the first and second solid antigen / carrier protein gels.

[0017] Certain aspects of the present disclosure include a method of immunohistochemistry (IHC) staining with a control secondary antibody, comprising providing first and second solid antigen / carrier protein gels, each of the first and second solid antigen / carrier protein gels produced by the method of any one of the above-described embodiments, wherein the first solid antigen / carrier protein gel comprises a first antibody having a first isotype and the second solid antigen / carrier protein gel comprises a second antibody having a second isotype different from the first isotype; and detecting a lack of a detectable moiety signal from the second solid antigen / carrier protein gel, wherein the detection of a signal associated with the first solid antigen / carrier protein gel and a lack of a signal associated with the second solid antigen / carrier protein gel indicates control staining by the secondary antibody. In some embodiments, the detectable moiety comprises an enzyme, and detecting the signal of the detectable moiety comprises exposing the detectable moiety to a chromogenic, fluorogenic, or chemiluminescent substrate of the enzyme and detecting a signal from the substrate upon reaction with the enzyme. In some embodiments, the detectable moiety comprises a fluorophore, a metal particle, a metal ion, a radioisotope, a nucleic acid, an electrochemiluminescent reporter, or a "quantum dot" (a solid, semiconductor, or carbon-based fluorescent nanoparticle).

[0018] Certain aspects of the present disclosure relate to solid antigen / carrier protein gels (e.g., for immunohistochemistry (IHC) staining) comprising a purified antigen crosslinked to a carrier protein selected from the group consisting of albumin protein (e.g., serum albumin protein), egg white protein or a mixture of egg white proteins, and gelatin. Some embodiments relate to solid antigen / carrier protein gels (e.g., for immunohistochemistry (IHC) staining) comprising a purified antigen crosslinked to a carrier protein selected from the group consisting of albumin protein (e.g., serum albumin protein), egg white protein or a mixture of egg white proteins, gelatin, and polylysine. In some embodiments, the solid gel has a thickness of about 30 nm to about 50 μm. In some embodiments, the solid gel has a thickness of about 2 μm to about 30 μm. In some embodiments, the solid gel has a thickness of about 30 nm to about 100 nm. In some embodiments, the solid gel is frozen in an embedding medium. In some embodiments, the solid gel is embedded in paraffin. In some embodiments, the solid gel is embedded in a plastic resin. In some embodiments, the solid gel is fixed to a solid substrate. In some embodiments, the solid gel is fixed in a fixative. In some embodiments, the fixative comprises formaldehyde. In some embodiments, the fixative comprises formaldehyde at a concentration of at least about 1%. In some embodiments, the fixative comprises glutaraldehyde, Davidson's fixative, Bouin's fixative, half-strength Karnovski's fixative, or zinc salt. In some embodiments, the solid gel has been subjected to antigen retrieval. In some embodiments, the antigen is a polypeptide antigen. In some embodiments, the antigen comprises an N-terminal tyrosine, a C-terminal cysteine, or both. In some embodiments, the N-terminal tyrosine and / or the C-terminal cysteine ​​are crosslinked to a carrier protein. In some embodiments, the antigen comprises a non-polypeptide antigen. In some embodiments, the carrier protein is an albumin protein, such as bovine, caprine, equine, or human serum albumin. In some embodiments, the carrier protein is an egg white protein or a mixture of egg white proteins.In some embodiments, the solid gel comprises a carrier protein at a concentration of 2% or greater. In some embodiments, the solid gel comprises a carrier protein at a concentration of about 25% or less. In some embodiments, the carrier protein is gelatin. In some embodiments, the solid gel comprises a carrier protein at a concentration of 0.5% or greater. In some embodiments, the solid gel comprises an antigen at a concentration of at least about 25 nM. In some embodiments, the solid gel comprises polylysine at a concentration of about 14 mg / mL.

[0019] Certain aspects of the present disclosure relate to tissue microarrays (TMA) comprising at least first and second solid antigen / carrier protein gels, both of which comprise a purified antigen cross-linked to a carrier protein selected from the group consisting of albumin protein, egg white protein or a mixture of egg white proteins, and gelatin. In some embodiments, the present disclosure relates to tissue microarrays (TMA) comprising at least first and second solid antigen / carrier protein gels, both of which comprise a purified antigen cross-linked to a carrier protein selected from the group consisting of albumin protein, egg white protein or a mixture of egg white proteins, gelatin, and polylysine. In some embodiments, the first solid antigen / carrier protein gel comprises a first purified antigen and the second solid antigen / carrier protein gel comprises a second purified antigen that is different from the first purified antigen. In some embodiments, the first solid antigen / carrier protein gel comprises a first purified antigen at a first concentration, and the second solid antigen / carrier protein gel comprises the first purified antigen at a second concentration that is different from the first concentration.

[0020] Certain aspects of the present disclosure relate to a method of immunohistochemistry (IHC) staining of an antigen, comprising: providing a solid antigen / carrier protein gel according to any one of the above-described embodiments, wherein the solid antigen / carrier protein gel comprises an antigen; providing a sample; contacting the solid antigen / carrier protein gel and the sample with a primary antibody that specifically binds to the antigen; after contacting the solid antigen / carrier protein gel and the sample with the primary antibody, contacting the solid antigen / carrier protein gel and the sample with a secondary antibody that specifically binds to the primary antibody, wherein a detectable moiety is conjugated to the secondary antibody; detecting a signal of the detectable moiety from the solid antigen / carrier protein gel; and detecting a signal of the detectable moiety from the sample, wherein detection of a signal from the sample compared to the signal detected from the solid antigen / carrier protein gel indicates the presence of the antigen in the sample. In some embodiments, the detectable moiety comprises an enzyme, and detecting the signal of the detectable moiety comprises exposing the detectable moiety to a chromogenic, fluorogenic, or chemiluminescent substrate of the enzyme and detecting a signal from the substrate upon reaction with the enzyme. In some embodiments, the detectable moiety comprises a fluorophore, a metal particle, a metal ion, a radioisotope, a nucleic acid, an electrochemiluminescent reporter, or a "quantum dot" (a solid, semiconductor, or carbon-based fluorescent nanoparticle). In some embodiments, the sample is a tissue sample.

[0021] A particular aspect of the present disclosure is a method of immunohistochemistry (IHC) staining of a control antigen, comprising providing first and second solid antigen / carrier protein gels according to the method of any one of the above-described embodiments, wherein the first solid antigen / carrier protein gel comprises the antigen at a first concentration and the second solid antigen / carrier protein gel comprises the antigen at a second concentration higher than the first concentration; contacting the first and second solid antigen / carrier protein gels with a primary antibody that specifically binds to the antigen; and detecting a second signal of the detectable moiety from the second solid antigen / carrier protein gel, wherein detection of the second signal, which is greater than the first signal, indicates IHC staining of the control antigen. In some embodiments, the detectable moiety comprises an enzyme, and detecting the signal of the detectable moiety comprises exposing the detectable moiety to a chromogenic, fluorogenic, or chemiluminescent substrate of the enzyme and detecting a signal from the substrate upon reaction with the enzyme. In some embodiments, the detectable moiety comprises a fluorophore, a metal particle, a metal ion, a radioisotope, a nucleic acid, an electrochemiluminescent reporter, or a "quantum dot" (a solid, semiconductor, or carbon-based fluorescent nanoparticle). In some embodiments, the first concentration is 0 nM, and detection of the absence of a first signal indicates IHC staining of a control antigen. In some embodiments, the method further includes providing a sample; contacting the sample with a primary antibody; contacting the sample with a secondary antibody after contacting the sample with the primary antibody; detecting a third signal of a detectable moiety from the sample; and comparing the third signal to the first and second signals, wherein the amount of the third signal compared to the amount of the first and second signals indicates the amount of antigen present in the sample relative to the amount of antigen in the first and second solid antigen / carrier protein gels.

[0022] Certain aspects of the present disclosure relate to a method of immunohistochemistry (IHC) staining with a control secondary antibody, comprising: providing first and second solid antigen / carrier protein gels according to any one of the above-described embodiments, wherein the first solid antigen / carrier protein gel comprises a first antibody having a first isotype and the second solid antigen / carrier protein gel comprises a second antibody having a second isotype different from the first isotype; contacting the first and second solid antigen / carrier protein gels with a secondary antibody that specifically binds to the first isotype, wherein a detectable moiety is conjugated to the secondary antibody; detecting a detectable moiety signal from the first solid antigen / carrier protein gel; and detecting a lack of a detectable moiety signal from the second solid antigen / carrier protein gel, wherein detection of a signal associated with the first solid antigen / carrier protein gel and a lack of a signal associated with the second solid antigen / carrier protein gel indicates staining with the control secondary antibody. In some embodiments, the detectable moiety comprises an enzyme, and detecting the signal of the detectable moiety comprises exposing the detectable moiety to a chromogenic, fluorogenic, or chemiluminescent substrate of the enzyme and detecting a signal from the substrate upon reaction with the enzyme. In some embodiments, the detectable moiety comprises a fluorophore, a metal particle, a metal ion, a radioisotope, a nucleic acid, an electrochemiluminescent reporter, or a "quantum dot" (a solid, semiconductor, or carbon-based fluorescent nanoparticle).

[0023] Certain aspects of the present disclosure relate to a kit (e.g., for immunohistochemistry (IHC) staining of a control antigen) comprising: a first solid gel comprising a purified antigen cross-linked to a carrier protein selected from the group consisting of albumin protein (e.g., serum albumin protein), egg white protein or a mixture of egg white proteins, and gelatin, wherein the purified antigen is present in the first solid gel at a first concentration; and a second solid gel comprising the purified antigen cross-linked to a carrier protein, wherein the purified antigen is present in the second solid gel at a second concentration different from the first concentration. In some embodiments, the kit includes a first solid gel comprising a purified antigen cross-linked to a carrier protein selected from the group consisting of albumin protein (e.g., serum albumin protein), egg white protein or a mixture of egg white proteins, gelatin, and polylysine, where the purified antigen is present in the first solid gel at a first concentration, and a second solid gel comprising the purified antigen cross-linked to a carrier protein, where the purified antigen is present in the second solid gel at a second concentration different from the first concentration. In some embodiments, the first and second solid gels are each sliced ​​to a thickness of about 30 nm to about 50 μm. In some embodiments, the first and second solid gels are immobilized on one or more solid substrates. In some embodiments, the kit further includes a third solid gel comprising the carrier protein, where the third solid gel does not comprise the purified antigen. In some embodiments, the third solid gel is sliced ​​to a thickness of about 30 nm to about 50 μm. In some embodiments, the third solid gel is immobilized on a solid substrate.

[0024] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows.

[0025] The patent or 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. [Brief explanation of the drawings]

[0026] [Figure 1] 1 shows an exemplary workflow for IHC staining using slides obtained from tissue samples. [Figure 2-1] Figure 2A shows a bovine serum albumin (BSA) gel prepared in and removed from a 1.5 mL microcentrifuge tube. Figures 2B and 2C show various views of sections of a BSA gel prepared in and removed from a 1.5 mL microcentrifuge tube. Figure 2D shows a gel portion sliced ​​for dehydration and paraffinization. Figure 2E shows a sliced ​​gel portion embedded in a paraffin "donor block." Scale bar: 1 cm. Figure 2F shows tissue microarrays (TMAs) prepared from various paraffin gel donor blocks. The darkest cores are orientation references containing black and green pigments. [Figure 2-2] Figure 2A shows a bovine serum albumin (BSA) gel prepared in and removed from a 1.5 mL microcentrifuge tube. Figures 2B and 2C show various views of sections of a BSA gel prepared in and removed from a 1.5 mL microcentrifuge tube. Figure 2D shows a gel portion sliced ​​for dehydration and paraffinization. Figure 2E shows a sliced ​​gel portion embedded in a paraffin "donor block." Scale bar: 1 cm. Figure 2F shows tissue microarrays (TMAs) prepared from various paraffin gel donor blocks. The darkest cores are orientation references containing black and green pigments. [Figure 3-1]Figure 3A shows the results of IHC staining of sections from 4-micron-thick BCL2 peptide / BSA gel cylinders embedded in paraffin blocks. The inset shows 40x magnification of IHC staining for BCL2 using sections obtained from BCL2 peptide / BSA gels containing (from left to right): no peptide, 5 × 10-5 mg / mL (2.5E-8 M) BCL2 peptide, 5 × 10-4 mg / mL (2.5E-7 M) BCL2 peptide, 5 × 10-3 mg / mL (2.5E-6 M) BCL2 peptide, 5 × 10-2 mg / mL (2.5E-5 M) BCL2 peptide, or 0.5 mg / mL (2.5E-4 M) BCL2 peptide. BCL2 staining in sections corresponding to 0, 1+, 2+, or 3+ (qualitative scale of 0 to 3+) is labeled. Figure 3B shows the results of six independent experiments analyzing the sections shown in Figure 3A for optical density (OD) versus BCL2 peptide concentration. Figure 3C shows the results of six independent experiments analyzing the sections shown in Figure 3A for image intensity versus BCL2 peptide concentration. Figures 3D and 3E show quantitative immunofluorescence (IF) staining of 4-micron-thick sections of BCL2 peptide / BSA gel cylinders embedded in paraffin blocks, stained with anti-BCL2 primary antibody, and detected with a fluorescent reporter (using a Ventana Medical Systems OmniMAP anti-mouse HRP detection system followed by a Ventana Discovery Red 610 detection system). Test samples included wild-type (unmutated) BCL2 peptide, BCL2 peptides containing six different single amino acid substitutions, and a peptide from the MCL1 protein (a negative control sample not expected to bind to the anti-BCL2 antibody). Figure 3D shows the red fluorescence of the reporter. Figure 3E shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting the blue autofluorescence of the BSA core). Figures 3F and 3G show quantitative immunofluorescence (IF) staining results of 4-micron-thick sections of paraffin-embedded BCL2 peptide / BSA gel cylinders stained with a naive control primary antibody. Figure 3F shows reporter fluorescence. Figure 3G shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting autofluorescence of the BSA core).Figure 3H shows quantification of the results shown in Figure 3D (reported as mean optical emittance). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3I shows quantification of the results shown in Figure 3F (reported as mean optical emittance). The naive control antibody exhibits an almost undetectable signal, as expected. Figure 3J shows quantification of the results shown in Figure 3D (reported as mean signal intensity). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3K shows quantification of the results shown in Figure 3F (reported as mean signal intensity). The naive control antibody gives, as expected, an almost undetectable signal. [Figure 3-2]Figure 3A shows the results of IHC staining of sections from 4-micron-thick BCL2 peptide / BSA gel cylinders embedded in paraffin blocks. The inset shows 40x magnification of IHC staining for BCL2 using sections obtained from BCL2 peptide / BSA gels containing (from left to right): no peptide, 5 × 10-5 mg / mL (2.5E-8 M) BCL2 peptide, 5 × 10-4 mg / mL (2.5E-7 M) BCL2 peptide, 5 × 10-3 mg / mL (2.5E-6 M) BCL2 peptide, 5 × 10-2 mg / mL (2.5E-5 M) BCL2 peptide, or 0.5 mg / mL (2.5E-4 M) BCL2 peptide. BCL2 staining in sections corresponding to 0, 1+, 2+, or 3+ (qualitative scale of 0 to 3+) is labeled. Figure 3B shows the results of six independent experiments analyzing the sections shown in Figure 3A for optical density (OD) versus BCL2 peptide concentration. Figure 3C shows the results of six independent experiments analyzing the sections shown in Figure 3A for image intensity versus BCL2 peptide concentration. Figures 3D and 3E show quantitative immunofluorescence (IF) staining of 4-micron-thick sections of BCL2 peptide / BSA gel cylinders embedded in paraffin blocks, stained with anti-BCL2 primary antibody, and detected with a fluorescent reporter (using a Ventana Medical Systems OmniMAP anti-mouse HRP detection system followed by a Ventana Discovery Red 610 detection system). Test samples included wild-type (unmutated) BCL2 peptide, BCL2 peptides containing six different single amino acid substitutions, and a peptide from the MCL1 protein (a negative control sample not expected to bind to the anti-BCL2 antibody). Figure 3D shows the red fluorescence of the reporter. Figure 3E shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting the blue autofluorescence of the BSA core). Figures 3F and 3G show quantitative immunofluorescence (IF) staining results of 4-micron-thick sections of paraffin-embedded BCL2 peptide / BSA gel cylinders stained with a naive control primary antibody. Figure 3F shows reporter fluorescence. Figure 3G shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting autofluorescence of the BSA core).Figure 3H shows quantification of the results shown in Figure 3D (reported as mean optical emittance). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3I shows quantification of the results shown in Figure 3F (reported as mean optical emittance). The naive control antibody exhibits an almost undetectable signal, as expected. Figure 3J shows quantification of the results shown in Figure 3D (reported as mean signal intensity). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3K shows quantification of the results shown in Figure 3F (reported as mean signal intensity). The naive control antibody gives, as expected, an almost undetectable signal. [Figure 3-3]Figure 3A shows the results of IHC staining of sections from 4-micron-thick BCL2 peptide / BSA gel cylinders embedded in paraffin blocks. The inset shows 40x magnification of IHC staining for BCL2 using sections obtained from BCL2 peptide / BSA gels containing (from left to right): no peptide, 5 × 10-5 mg / mL (2.5E-8 M) BCL2 peptide, 5 × 10-4 mg / mL (2.5E-7 M) BCL2 peptide, 5 × 10-3 mg / mL (2.5E-6 M) BCL2 peptide, 5 × 10-2 mg / mL (2.5E-5 M) BCL2 peptide, or 0.5 mg / mL (2.5E-4 M) BCL2 peptide. BCL2 staining in sections corresponding to 0, 1+, 2+, or 3+ (qualitative scale of 0 to 3+) is labeled. Figure 3B shows the results of six independent experiments analyzing the sections shown in Figure 3A for optical density (OD) versus BCL2 peptide concentration. Figure 3C shows the results of six independent experiments analyzing the sections shown in Figure 3A for image intensity versus BCL2 peptide concentration. Figures 3D and 3E show quantitative immunofluorescence (IF) staining of 4-micron-thick sections of BCL2 peptide / BSA gel cylinders embedded in paraffin blocks, stained with anti-BCL2 primary antibody, and detected with a fluorescent reporter (using a Ventana Medical Systems OmniMAP anti-mouse HRP detection system followed by a Ventana Discovery Red 610 detection system). Test samples included wild-type (unmutated) BCL2 peptide, BCL2 peptides containing six different single amino acid substitutions, and a peptide from the MCL1 protein (a negative control sample not expected to bind to the anti-BCL2 antibody). Figure 3D shows the red fluorescence of the reporter. Figure 3E shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting the blue autofluorescence of the BSA core). Figures 3F and 3G show quantitative immunofluorescence (IF) staining results of 4-micron-thick sections of paraffin-embedded BCL2 peptide / BSA gel cylinders stained with a naive control primary antibody. Figure 3F shows reporter fluorescence. Figure 3G shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting autofluorescence of the BSA core).Figure 3H shows quantification of the results shown in Figure 3D (reported as mean optical emittance). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3I shows quantification of the results shown in Figure 3F (reported as mean optical emittance). The naive control antibody exhibits an almost undetectable signal, as expected. Figure 3J shows quantification of the results shown in Figure 3D (reported as mean signal intensity). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3K shows quantification of the results shown in Figure 3F (reported as mean signal intensity). The naive control antibody gives, as expected, an almost undetectable signal. [Figure 3-4]Figure 3A shows the results of IHC staining of sections from 4-micron-thick BCL2 peptide / BSA gel cylinders embedded in paraffin blocks. The inset shows 40x magnification of IHC staining for BCL2 using sections obtained from BCL2 peptide / BSA gels containing (from left to right): no peptide, 5 × 10-5 mg / mL (2.5E-8 M) BCL2 peptide, 5 × 10-4 mg / mL (2.5E-7 M) BCL2 peptide, 5 × 10-3 mg / mL (2.5E-6 M) BCL2 peptide, 5 × 10-2 mg / mL (2.5E-5 M) BCL2 peptide, or 0.5 mg / mL (2.5E-4 M) BCL2 peptide. BCL2 staining in sections corresponding to 0, 1+, 2+, or 3+ (qualitative scale of 0 to 3+) is labeled. Figure 3B shows the results of six independent experiments analyzing the sections shown in Figure 3A for optical density (OD) versus BCL2 peptide concentration. Figure 3C shows the results of six independent experiments analyzing the sections shown in Figure 3A for image intensity versus BCL2 peptide concentration. Figures 3D and 3E show quantitative immunofluorescence (IF) staining of 4-micron-thick sections of BCL2 peptide / BSA gel cylinders embedded in paraffin blocks, stained with anti-BCL2 primary antibody, and detected with a fluorescent reporter (using a Ventana Medical Systems OmniMAP anti-mouse HRP detection system followed by a Ventana Discovery Red 610 detection system). Test samples included wild-type (unmutated) BCL2 peptide, BCL2 peptides containing six different single amino acid substitutions, and a peptide from the MCL1 protein (a negative control sample not expected to bind to the anti-BCL2 antibody). Figure 3D shows the red fluorescence of the reporter. Figure 3E shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting the blue autofluorescence of the BSA core). Figures 3F and 3G show quantitative immunofluorescence (IF) staining results of 4-micron-thick sections of paraffin-embedded BCL2 peptide / BSA gel cylinders stained with a naive control primary antibody. Figure 3F shows reporter fluorescence. Figure 3G shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting autofluorescence of the BSA core).Figure 3H shows quantification of the results shown in Figure 3D (reported as mean optical emittance). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3I shows quantification of the results shown in Figure 3F (reported as mean optical emittance). The naive control antibody exhibits an almost undetectable signal, as expected. Figure 3J shows quantification of the results shown in Figure 3D (reported as mean signal intensity). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3K shows quantification of the results shown in Figure 3F (reported as mean signal intensity). The naive control antibody gives, as expected, an almost undetectable signal. [Figure 3-5]Figure 3A shows the results of IHC staining of sections from 4-micron-thick BCL2 peptide / BSA gel cylinders embedded in paraffin blocks. The inset shows 40x magnification of IHC staining for BCL2 using sections obtained from BCL2 peptide / BSA gels containing (from left to right): no peptide, 5 × 10-5 mg / mL (2.5E-8 M) BCL2 peptide, 5 × 10-4 mg / mL (2.5E-7 M) BCL2 peptide, 5 × 10-3 mg / mL (2.5E-6 M) BCL2 peptide, 5 × 10-2 mg / mL (2.5E-5 M) BCL2 peptide, or 0.5 mg / mL (2.5E-4 M) BCL2 peptide. BCL2 staining in sections corresponding to 0, 1+, 2+, or 3+ (qualitative scale of 0 to 3+) is labeled. Figure 3B shows the results of six independent experiments analyzing the sections shown in Figure 3A for optical density (OD) versus BCL2 peptide concentration. Figure 3C shows the results of six independent experiments analyzing the sections shown in Figure 3A for image intensity versus BCL2 peptide concentration. Figures 3D and 3E show quantitative immunofluorescence (IF) staining of 4-micron-thick sections of BCL2 peptide / BSA gel cylinders embedded in paraffin blocks, stained with anti-BCL2 primary antibody, and detected with a fluorescent reporter (using a Ventana Medical Systems OmniMAP anti-mouse HRP detection system followed by a Ventana Discovery Red 610 detection system). Test samples included wild-type (unmutated) BCL2 peptide, BCL2 peptides containing six different single amino acid substitutions, and a peptide from the MCL1 protein (a negative control sample not expected to bind to the anti-BCL2 antibody). Figure 3D shows the red fluorescence of the reporter. Figure 3E shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting the blue autofluorescence of the BSA core). Figures 3F and 3G show quantitative immunofluorescence (IF) staining results of 4-micron-thick sections of paraffin-embedded BCL2 peptide / BSA gel cylinders stained with a naive control primary antibody. Figure 3F shows reporter fluorescence. Figure 3G shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting autofluorescence of the BSA core).Figure 3H shows quantification of the results shown in Figure 3D (reported as mean optical emittance). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3I shows quantification of the results shown in Figure 3F (reported as mean optical emittance). The naive control antibody exhibits an almost undetectable signal, as expected. Figure 3J shows quantification of the results shown in Figure 3D (reported as mean signal intensity). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3K shows quantification of the results shown in Figure 3F (reported as mean signal intensity). The naive control antibody gives, as expected, an almost undetectable signal. [Figure 3-6]Figure 3A shows the results of IHC staining of sections from 4-micron-thick BCL2 peptide / BSA gel cylinders embedded in paraffin blocks. The inset shows 40x magnification of IHC staining for BCL2 using sections obtained from BCL2 peptide / BSA gels containing (from left to right): no peptide, 5 × 10-5 mg / mL (2.5E-8 M) BCL2 peptide, 5 × 10-4 mg / mL (2.5E-7 M) BCL2 peptide, 5 × 10-3 mg / mL (2.5E-6 M) BCL2 peptide, 5 × 10-2 mg / mL (2.5E-5 M) BCL2 peptide, or 0.5 mg / mL (2.5E-4 M) BCL2 peptide. BCL2 staining in sections corresponding to 0, 1+, 2+, or 3+ (qualitative scale of 0 to 3+) is labeled. Figure 3B shows the results of six independent experiments analyzing the sections shown in Figure 3A for optical density (OD) versus BCL2 peptide concentration. Figure 3C shows the results of six independent experiments analyzing the sections shown in Figure 3A for image intensity versus BCL2 peptide concentration. Figures 3D and 3E show quantitative immunofluorescence (IF) staining of 4-micron-thick sections of BCL2 peptide / BSA gel cylinders embedded in paraffin blocks, stained with anti-BCL2 primary antibody, and detected with a fluorescent reporter (using a Ventana Medical Systems OmniMAP anti-mouse HRP detection system followed by a Ventana Discovery Red 610 detection system). Test samples included wild-type (unmutated) BCL2 peptide, BCL2 peptides containing six different single amino acid substitutions, and a peptide from the MCL1 protein (a negative control sample not expected to bind to the anti-BCL2 antibody). Figure 3D shows the red fluorescence of the reporter. Figure 3E shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting the blue autofluorescence of the BSA core). Figures 3F and 3G show quantitative immunofluorescence (IF) staining results of 4-micron-thick sections of paraffin-embedded BCL2 peptide / BSA gel cylinders stained with a naive control primary antibody. Figure 3F shows reporter fluorescence. Figure 3G shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting autofluorescence of the BSA core).Figure 3H shows quantification of the results shown in Figure 3D (reported as mean optical emittance). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3I shows quantification of the results shown in Figure 3F (reported as mean optical emittance). The naive control antibody exhibits an almost undetectable signal, as expected. Figure 3J shows quantification of the results shown in Figure 3D (reported as mean signal intensity). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3K shows quantification of the results shown in Figure 3F (reported as mean signal intensity). The naive control antibody gives, as expected, an almost undetectable signal. [Figure 3-7]Figure 3A shows the results of IHC staining of sections from 4-micron-thick BCL2 peptide / BSA gel cylinders embedded in paraffin blocks. The inset shows 40x magnification of IHC staining for BCL2 using sections obtained from BCL2 peptide / BSA gels containing (from left to right): no peptide, 5 × 10-5 mg / mL (2.5E-8 M) BCL2 peptide, 5 × 10-4 mg / mL (2.5E-7 M) BCL2 peptide, 5 × 10-3 mg / mL (2.5E-6 M) BCL2 peptide, 5 × 10-2 mg / mL (2.5E-5 M) BCL2 peptide, or 0.5 mg / mL (2.5E-4 M) BCL2 peptide. BCL2 staining in sections corresponding to 0, 1+, 2+, or 3+ (qualitative scale of 0 to 3+) is labeled. Figure 3B shows the results of six independent experiments analyzing the sections shown in Figure 3A for optical density (OD) versus BCL2 peptide concentration. Figure 3C shows the results of six independent experiments analyzing the sections shown in Figure 3A for image intensity versus BCL2 peptide concentration. Figures 3D and 3E show quantitative immunofluorescence (IF) staining of 4-micron-thick sections of BCL2 peptide / BSA gel cylinders embedded in paraffin blocks, stained with anti-BCL2 primary antibody, and detected with a fluorescent reporter (using a Ventana Medical Systems OmniMAP anti-mouse HRP detection system followed by a Ventana Discovery Red 610 detection system). Test samples included wild-type (unmutated) BCL2 peptide, BCL2 peptides containing six different single amino acid substitutions, and a peptide from the MCL1 protein (a negative control sample not expected to bind to the anti-BCL2 antibody). Figure 3D shows the red fluorescence of the reporter. Figure 3E shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting the blue autofluorescence of the BSA core). Figures 3F and 3G show quantitative immunofluorescence (IF) staining results of 4-micron-thick sections of paraffin-embedded BCL2 peptide / BSA gel cylinders stained with a naive control primary antibody. Figure 3F shows reporter fluorescence. Figure 3G shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting autofluorescence of the BSA core).Figure 3H shows quantification of the results shown in Figure 3D (reported as mean optical emittance). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3I shows quantification of the results shown in Figure 3F (reported as mean optical emittance). The naive control antibody exhibits an almost undetectable signal, as expected. Figure 3J shows quantification of the results shown in Figure 3D (reported as mean signal intensity). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3K shows quantification of the results shown in Figure 3F (reported as mean signal intensity). The naive control antibody gives, as expected, an almost undetectable signal. [Figure 3-8]Figure 3A shows the results of IHC staining of sections from 4-micron-thick BCL2 peptide / BSA gel cylinders embedded in paraffin blocks. The inset shows 40x magnification of IHC staining for BCL2 using sections obtained from BCL2 peptide / BSA gels containing (from left to right): no peptide, 5 × 10-5 mg / mL (2.5E-8 M) BCL2 peptide, 5 × 10-4 mg / mL (2.5E-7 M) BCL2 peptide, 5 × 10-3 mg / mL (2.5E-6 M) BCL2 peptide, 5 × 10-2 mg / mL (2.5E-5 M) BCL2 peptide, or 0.5 mg / mL (2.5E-4 M) BCL2 peptide. BCL2 staining in sections corresponding to 0, 1+, 2+, or 3+ (qualitative scale of 0 to 3+) is labeled. Figure 3B shows the results of six independent experiments analyzing the sections shown in Figure 3A for optical density (OD) versus BCL2 peptide concentration. Figure 3C shows the results of six independent experiments analyzing the sections shown in Figure 3A for image intensity versus BCL2 peptide concentration. Figures 3D and 3E show quantitative immunofluorescence (IF) staining of 4-micron-thick sections of BCL2 peptide / BSA gel cylinders embedded in paraffin blocks, stained with anti-BCL2 primary antibody, and detected with a fluorescent reporter (using a Ventana Medical Systems OmniMAP anti-mouse HRP detection system followed by a Ventana Discovery Red 610 detection system). Test samples included wild-type (unmutated) BCL2 peptide, BCL2 peptides containing six different single amino acid substitutions, and a peptide from the MCL1 protein (a negative control sample not expected to bind to the anti-BCL2 antibody). Figure 3D shows the red fluorescence of the reporter. Figure 3E shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting the blue autofluorescence of the BSA core). Figures 3F and 3G show quantitative immunofluorescence (IF) staining results of 4-micron-thick sections of paraffin-embedded BCL2 peptide / BSA gel cylinders stained with a naive control primary antibody. Figure 3F shows reporter fluorescence. Figure 3G shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting autofluorescence of the BSA core).Figure 3H shows quantification of the results shown in Figure 3D (reported as mean optical emittance). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3I shows quantification of the results shown in Figure 3F (reported as mean optical emittance). The naive control antibody exhibits an almost undetectable signal, as expected. Figure 3J shows quantification of the results shown in Figure 3D (reported as mean signal intensity). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3K shows quantification of the results shown in Figure 3F (reported as mean signal intensity). The naive control antibody gives, as expected, an almost undetectable signal. [Figure 3-9]Figure 3A shows the results of IHC staining of sections from 4-micron-thick BCL2 peptide / BSA gel cylinders embedded in paraffin blocks. The inset shows 40x magnification of IHC staining for BCL2 using sections obtained from BCL2 peptide / BSA gels containing (from left to right): no peptide, 5 × 10-5 mg / mL (2.5E-8 M) BCL2 peptide, 5 × 10-4 mg / mL (2.5E-7 M) BCL2 peptide, 5 × 10-3 mg / mL (2.5E-6 M) BCL2 peptide, 5 × 10-2 mg / mL (2.5E-5 M) BCL2 peptide, or 0.5 mg / mL (2.5E-4 M) BCL2 peptide. BCL2 staining in sections corresponding to 0, 1+, 2+, or 3+ (qualitative scale of 0 to 3+) is labeled. Figure 3B shows the results of six independent experiments analyzing the sections shown in Figure 3A for optical density (OD) versus BCL2 peptide concentration. Figure 3C shows the results of six independent experiments analyzing the sections shown in Figure 3A for image intensity versus BCL2 peptide concentration. Figures 3D and 3E show quantitative immunofluorescence (IF) staining of 4-micron-thick sections of BCL2 peptide / BSA gel cylinders embedded in paraffin blocks, stained with anti-BCL2 primary antibody, and detected with a fluorescent reporter (using a Ventana Medical Systems OmniMAP anti-mouse HRP detection system followed by a Ventana Discovery Red 610 detection system). Test samples included wild-type (unmutated) BCL2 peptide, BCL2 peptides containing six different single amino acid substitutions, and a peptide from the MCL1 protein (a negative control sample not expected to bind to the anti-BCL2 antibody). Figure 3D shows the red fluorescence of the reporter. Figure 3E shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting the blue autofluorescence of the BSA core). Figures 3F and 3G show quantitative immunofluorescence (IF) staining results of 4-micron-thick sections of paraffin-embedded BCL2 peptide / BSA gel cylinders stained with a naive control primary antibody. Figure 3F shows reporter fluorescence. Figure 3G shows the fluorescence of the same TMA imaged in the DAPI filter channel (detecting autofluorescence of the BSA core).Figure 3H shows quantification of the results shown in Figure 3D (reported as mean optical emittance). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3I shows quantification of the results shown in Figure 3F (reported as mean optical emittance). The naive control antibody exhibits an almost undetectable signal, as expected. Figure 3J shows quantification of the results shown in Figure 3D (reported as mean signal intensity). The wild-type BCL2 peptide exhibits the strongest signal at each concentration; the negative control MCL1 peptide exhibits the smallest signal, as expected. The six BCL2 peptides containing single amino acid substitutions exhibit variable signals generally intermediate between those of wild-type BCL2 and the negative control MCL1 peptide. Figure 3K shows quantification of the results shown in Figure 3F (reported as mean signal intensity). The naive control antibody gives, as expected, an almost undetectable signal. [Figure 4-1] Figure 4A shows control IHC staining using a donkey anti-mouse secondary antibody on sections of BSA gel containing mouse IgG (left) or rat IgG (right). Figure 4B shows control IHC staining using a donkey anti-rat secondary antibody on sections of BSA gel containing mouse IgG (left) or rat IgG (right). Figure 4C shows the effect on IHC staining of amino acid substitutions (indicated) introduced into the anti-BCL2 clone 124 antibody epitope of human BCL2 (amino acids 41-54 of the human BCL2 sequence described in UniProt accession number P10415). This image is a 4-micron-thick section cut from a paraffin-embedded tissue microarray constructed using BCL2 peptide / BSA gel cylinders (each 1 mm in diameter), stained with anti-BCL2 antibody clone 124, and detected with the chromogenic reagent and method. Figure 4D shows quantification of the results shown in Figure 4C (reported as optical density (OD)). [Figure 4-2]Figure 4A shows control IHC staining using a donkey anti-mouse secondary antibody on sections of BSA gel containing mouse IgG (left) or rat IgG (right). Figure 4B shows control IHC staining using a donkey anti-rat secondary antibody on sections of BSA gel containing mouse IgG (left) or rat IgG (right). Figure 4C shows the effect on IHC staining of amino acid substitutions (indicated) introduced into the anti-BCL2 clone 124 antibody epitope of human BCL2 (amino acids 41-54 of the human BCL2 sequence described in UniProt accession number P10415). This image is a 4-micron-thick section cut from a paraffin-embedded tissue microarray constructed using BCL2 peptide / BSA gel cylinders (each 1 mm in diameter), stained with anti-BCL2 antibody clone 124, and detected with the chromogenic reagent and method. Figure 4D shows quantification of the results shown in Figure 4C (reported as optical density (OD)). [Figure 4-3] Figure 4A shows control IHC staining using a donkey anti-mouse secondary antibody on sections of BSA gel containing mouse IgG (left) or rat IgG (right). Figure 4B shows control IHC staining using a donkey anti-rat secondary antibody on sections of BSA gel containing mouse IgG (left) or rat IgG (right). Figure 4C shows the effect on IHC staining of amino acid substitutions (indicated) introduced into the anti-BCL2 clone 124 antibody epitope of human BCL2 (amino acids 41-54 of the human BCL2 sequence described in UniProt accession number P10415). This image is a 4-micron-thick section cut from a paraffin-embedded tissue microarray constructed using BCL2 peptide / BSA gel cylinders (each 1 mm in diameter), stained with anti-BCL2 antibody clone 124, and detected with the chromogenic reagent and method. Figure 4D shows quantification of the results shown in Figure 4C (reported as optical density (OD)). [Figure 5-1]Figures 5A-5D show the results of screening 27 different antibodies obtained from mice immunized with human KSR2 by IHC using protein / BSA gels (compared to staining with naive mouse IgG in Figure 5D). The top row of each shows the results of IHC staining using each antibody on a BSA gel without protein. The bottom row shows the results of IHC staining using each antibody on a BSA gel embedded with human KSR2 protein, which was used to immunize the mice. Mouse antibody clones are indicated in each column. Checkmarks indicate the best-performing clones, as evidenced by a strong signal in the target protein sample (bottom row) and minimal signal in the negative control sample (top row). [Figure 5-2] Figures 5A-5D show the results of screening 27 different antibodies obtained from mice immunized with human KSR2 by IHC using protein / BSA gels (compared to staining with naive mouse IgG in Figure 5D). The top row of each shows the results of IHC staining using each antibody on a BSA gel without protein. The bottom row shows the results of IHC staining using each antibody on a BSA gel embedded with human KSR2 protein, which was used to immunize the mice. Mouse antibody clones are indicated in each column. Checkmarks indicate the best-performing clones, as evidenced by a strong signal in the target protein sample (bottom row) and minimal signal in the negative control sample (top row). [Figure 5-3]Figures 5A-5D show the results of screening 27 different antibodies obtained from mice immunized with human KSR2 by IHC using protein / BSA gels (compared to staining with naive mouse IgG in Figure 5D). The top row of each shows the results of IHC staining using each antibody on a BSA gel without protein. The bottom row shows the results of IHC staining using each antibody on a BSA gel embedded with human KSR2 protein, which was used to immunize the mice. Mouse antibody clones are indicated in each column. Checkmarks indicate the best-performing clones, as evidenced by a strong signal in the target protein sample (bottom row) and minimal signal in the negative control sample (top row). [Figure 5-4] Figures 5A-5D show the results of screening 27 different antibodies obtained from mice immunized with human KSR2 by IHC using protein / BSA gels (compared to staining with naive mouse IgG in Figure 5D). The top row of each shows the results of IHC staining using each antibody on a BSA gel without protein. The bottom row shows the results of IHC staining using each antibody on a BSA gel embedded with human KSR2 protein, which was used to immunize the mice. Mouse antibody clones are indicated in each column. Checkmarks indicate the best-performing clones, as evidenced by a strong signal in the target protein sample (bottom row) and minimal signal in the negative control sample (top row). [Figure 6] The results show the effect of temperature and heating time on BSA gel formation. Temperature indicates the temperature to which a liquid 25% BSA solution was heated. Time indicates the time the BSA solution was heated and tested for solid-liquid phase before cooling to room temperature. S = solid; L = liquid; semi-S = semi-solid. [Figure 7] Results evaluated the effects of time, temperature, and BSA concentration on BSA gel formation. Temperature indicates the temperature to which the liquid BSA solution was heated. Time indicates the time the BSA solution was heated and tested for solid-liquid phase before cooling to room temperature. Percentages indicate the concentration of BSA in the BSA / PBS liquid solution. S = solid; L = liquid; o / n = overnight. [Figure 8] Shown are IHC staining of gels made from BSA, egg white protein, or gelatin with (bottom row) or without (top row) rabbit IgG, detected with an anti-rabbit IgG secondary antibody. [Figure 9] Heterogeneous gels made from lactalbumin, liver protein powder, or soy flour with (bottom row) or without (top row) rabbit IgG are shown. [Figure 10] Gels made from casein or non-fat dry milk with (bottom row) or without (top row) rabbit IgG did not adhere to glass slides for IHC staining. [Figure 11] Figures 11A-11D show the results of two separate experiments using a chromogenic assay to examine antigen distribution within the gel. Four-micron-thick "donor block" sections of BSA gels containing 5 × 10 M BCL2 peptide (top row in Figures 11A and 11C) or no peptide (bottom row in Figures 11A and 11C) were stained with anti-BCL2 clone 124 in two separate experiments (Experiment 1: left column in Figures 11A and 11C; Experiment 2: right column in Figures 11A and 11C). Digital images of the stained sections were quantified along the horizontal axis (Figure 11A) and vertical axis (Figure 11C) using the plot profile function in ImageJ. The results are shown in Figures 11B and 11D, respectively. Experiments 1 and 2 are shown in black and gray, respectively. Data from control sections containing BCL2 peptide and BSA only are shown as solid and dashed lines, respectively. [Figure 12]Figures 12A-12E show the results of IHC analysis of BCL2 using a BCL2 peptide TMA (Figure 12A). Tissue microarray sections stained with anti-BCL2 clone 124 contain duplicate TMA cores without additional peptide (BSA), a dilution series of a peptide encoding amino acids 41-54 of the BCL2 protein, or a negative control peptide derived from the human MCL1 protein at the indicated concentrations. Images of the BCL2 and MCL1 rows are different fields from the same TMA section. The diameter of the TMA cores is 1 mm. Serial sections from the same TMA described in (Figure 12A) are stained with anti-BCL2 clone 124 using immunofluorescence detection (Figure 12B) or anti-BCL2 clone EPR17509 using chromogenic detection (Figure 12C). (Figure 12D) Quantification of signal in individual cores containing the BCL2 peptide (circles) or MCL1 peptide (squares) as shown in Figures 12A-12C. (Figure 12E) Relevant parameters of the curves shown in (Figure 12D). The HillSlope parameter conveys the steepness of the curve. ACHM is the antigen concentration at half the maximum signal. [Figure 13] Figure 13A shows consecutive sections of a single tissue microarray containing a BSA gel core with no added peptide (BSA), a peptide encoding amino acids 41-54 of the BCL2 protein, or a negative control peptide derived from the human MCL1 protein. Two slides were chromogenically stained for the BCL2 antigen using anti-BCL2 clone SP66 or clone E17. Figure 13B shows quantification of the images shown in (Figure 13A). [Figure 14]Figures 14A-14C show the reproducibility of replicate sections. (Figure 14A) Images from six serial sections of a single tissue microarray containing duplicate TMA cores with either no added peptide (BSA) or a peptide encoding amino acids 41-54 of the BCL2 protein. Each slide was stained for BCL2 using clone 124 on six separate days by two operators. Operator 1 stained run 1; operator 2 stained runs 2-6 (Figure 14B). Quantification of the images shown in Figure 14A. The average signal from duplicate cores at each peptide concentration on each of the six TMAs is displayed. (Figure 14C) A table summarizes the relevant parameters of the curves shown in Figure 14B. [Figure 15] Figures 15A-15C show IHC staining of BCL2 peptides with and without antigen retrieval. (Figure 15A) TMAs contained BSA gel cores with no added peptide (BSA) or with peptides encoding amino acids 41-54 of the BCL2 protein at the indicated concentrations. Sections were stained with (AR) or without (no AR) antigen retrieval prior to antigen retrieval. (Figure 15B) Quantification of the images shown in Figure 15A. Black squares: with antigen retrieval; squares: without antigen retrieval. The average signal from duplicate cores at each peptide concentration is shown. (Figure 15C) The table summarizes the relevant parameters of the curves shown in Figure 15B. [Figure 16] Figures 16A-16C show that the N- and C-termini of alternative BCL2 peptides affect signal intensity. (Figure 16A) Images from a single TMA section containing a double BSA gel core with either no added peptide (BSA) or 22 amino acids encoding amino acids 41-54 of the BCL2 protein (rows I-V) flanked by the three amino acid sequence GSG with the alternative N- and C-terminal amino acids (acetyl-N,C-amide) shown. (Figure 16B) Quantification of the images shown in Figure 16A. The average signal from the double cores at each peptide concentration is shown. (Figure 16C) The table summarizes the relevant parameters of the curves shown in Figure 16B. [Figure 17]Figures 17A-17E show IHC staining of BCL2 and MYC peptides. (Figure 17A) Sections of donor blocks containing 2.5 x 10 M peptides derived from human MYC, as indicated. Scale bars are 1 mm. (Figures 17B and 17C) Single TMA sections stained with anti-BCL2 clone 124 (Figure 17B) or anti-MYC clone Y69 (Figure 17C) contain duplicate cores prepared with either no peptide (BSA) or peptides encoding BCL2 amino acids 41-54 and MYC amino acids 9-24. (Figure 17D) Quantification of the images shown in Figures 17B and 17C. The average signal from duplicate cores at each peptide concentration is shown. (Figure 17E) Relevant parameters for the curves shown in Figure 17D. [Figure 18] Figures 18A-18C show dual MYC / BCL2 immunofluorescence (IF) image analysis. (Figure 18A) Single sections from a TMA containing cores containing either no peptide (BSA) or peptides encoding BCL2 amino acids 41-54 and MYC amino acids 9-24 at the indicated concentrations. Sections were sequentially stained with both anti-BCL2 clone 124 and anti-MYC clone Y69, detected with the Ventana Discovery FAM (BCL2; green) or Discovery Cy5 (MYC; red) kits, and then imaged at the appropriate wavelength for each fluorochrome. (Figure 18B) Quantification of the images shown in Figure 18A. The average signal from duplicate cores at each peptide concentration is shown. Error bars indicate ±1 standard deviation. (Figure 18C) The table summarizes the relevant parameters of the curves shown in Figure 18B. [Figure 19] Figures 19A and 19B display aggregate data for anti-BCL2 clone 124. (Figure 19A) Data from Figures 12A-12E and Figures 14A-17E (n=10 independent IHC assays, each with duplicate cores for each peptide concentration) were grouped and plotted. Error bars represent 1 standard deviation (n=10). (Figure 19B) Related parameters for the data in Figure 19A. [Figure 20-1]Figures 20A and 20B test the antibodies against human KSR2 screened in Figures 5A-5D for staining with and without antigen retrieval. Images in Figure 20A show sections of BSA gels containing no protein (BSA) or 6.3 x 10 M of the protein of interest stained with select mouse antibodies, with (AR) or without (no AR) prior antigen retrieval. Figure 20B provides quantification of the results shown in Figure 20A. [Figure 20-2] Figures 20A and 20B test the antibodies against human KSR2 screened in Figures 5A-5D for staining with and without antigen retrieval. Images in Figure 20A show sections of BSA gels containing no protein (BSA) or 6.3 x 10 M of the protein of interest stained with select mouse antibodies, with (AR) or without (no AR) prior antigen retrieval. Figure 20B provides quantification of the results shown in Figure 20A. [Figure 21] Figures 21A and 21B show BSA gels containing mouse, rat, and rabbit IgG. (Figure 21A) Serial sections of TMAs consisting of duplicate BSA gel cores containing either naive rabbit, rat, or mouse IgG (0.1 milligrams / ml; 6.7 x 10-7 M) were stained with donkey secondary antibodies specific for IgG from the indicated species. The diameter of the TMA cores is 1 mm. (Figure 21B) Quantification of the images shown in Figure 21A: rabbit IgG antigen (black bars); rat IgG (hatched bars); mouse IgG (white bars). For each specific antibody, the signal from the core containing target IgG was significantly higher than the signal from the core containing non-target IgG (p<0.0001). Error bars represent 1 SD. Note that these data are also shown in Figure 3C. [Figure 22]Figures 22A-22C show the results of generating peptide gels using alternative fixatives. (Figure 22A) Donor cores were prepared containing BCL2 or MYC peptides by heating at 85°C for 10 minutes in the presence of 18.5% formaldehyde or 50% zinc fixative. Donor blocks were prepared without peptide (BSA) or with peptides encoding BCL2 amino acids 41-54 and MYC amino acids 9-24. Sections were stained with anti-BCL2 clone 124 or anti-MYC clone Y69, as appropriate. (Figure 22B) Quantification of the images shown in Figure 22A. (Figure 22C) Relevant parameters of the curves shown in Figure 22B. [Figure 23] Figures 23A and 23B show the results of testing alternative fixatives. (Figure 23A) BSA gels containing BCL2 peptide were prepared by heating to 85°C in the presence of a zinc-containing fixative (Zn and heat) or concentrated formaldehyde (37% formaldehyde and heat), or heated to 85°C in the absence of a fixative and then fixed at room temperature with a zinc-containing fixative (heat, Zn), 4% PFA (heat, 4% PFA), or neutral buffered formalin (heat, NBF). (Figure 23B) Quantification of the images in Figure 23A. [Figure 24] Figures 24A-24C show images of mass cytometry analysis of BCL2 peptide TMAs. (Figure 24A) Representative fields (approximately 150 microns square) of TMA sections stained with anti-BCL2 clone EPR17509 conjugated to a 146Nd mass spectrometry tag and imaged on a Fluidigm Hyperion scanning mass spectrometer. Duplicate TMA cores contained no additional peptide (BSA), a peptide encoding amino acids 41-54 of BCL2 protein at the indicated concentrations, or a negative control peptide from human MCL1 protein. (Figure 24B) Quantification of the images shown in Figure 24A. Error bars indicate standard deviation. (Figure 24C) A table summarizes relevant parameters of the curves shown in Figure 24B. The HillSlope parameter conveys the steepness of the curve. ACHM is the antigen concentration at half-maximum signal. DETAILED DESCRIPTION OF THE INVENTION

[0027] I. Definition All patents, applications, published applications, and other publications are incorporated by reference in their entirety. 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.

[0028] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. Polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The terms also encompass amino acid polymers that are altered, either naturally or by intervention, including, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling moiety or a toxin. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. As used herein, the terms "polypeptide" and "protein" specifically include antibodies.

[0029] The term "antigen" as used herein is used in the broadest sense and includes various forms of antigens, both polypeptide and non-polypeptide, including, but not limited to, small peptide antigens, full-length protein antigens, carbohydrate antigens, lipid antigens, and nucleic acid antigens.

[0030] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein.

[0031] A "purified" antigen refers to an antigen that has increased purity when it is present in a purer form than it is in its natural environment and / or when it is first produced and / or synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily imply absolute purity. In some embodiments, the antigen is purified to at least 90%, at least 95%, or at least 99% purity.

[0032] II. SOLID ANTIGEN / CARRIER PROTEIN GELS AND METHODS OF PRODUCTION Certain aspects of the present disclosure relate to solid antigen / carrier protein gels and methods of producing solid antigen / carrier protein gels.

[0033] In some embodiments, the present disclosure provides solid antigen / carrier protein gels comprising a purified antigen and a carrier protein. In some embodiments, the carrier protein is selected from albumin protein (e.g., serum albumin protein), egg white protein or a mixture of egg white proteins, gelatin, and polylysine. In some embodiments, the purified antigen is crosslinked to the carrier protein. As described below, these gels may find use, for example, as controls for IHC staining or EM image analysis.

[0034] The present disclosure demonstrates that multiple types of carrier proteins can be used in solid antigen / carrier protein gels. In some embodiments, the carrier protein is an albumin protein. In some embodiments, the carrier protein is a serum albumin protein. In some embodiments, the serum albumin protein is a mammalian serum albumin protein. Examples of serum albumin proteins include, but are not limited to, serum albumin from mice, rats, guinea pigs, rabbits, pigs, cows, goats, sheep, horses, and humans. In some embodiments, the carrier protein is an egg white protein. In some embodiments, the carrier protein is a mixture of two or more egg white proteins. In some embodiments, the carrier protein is gelatin. In some embodiments, the carrier protein is polylysine.

[0035] In some embodiments, the solid antigen / carrier protein gel comprises carrier protein at a concentration of 0.5%, 1%, 2%, 5%, 7%, 10%, 15%, or 20% (w / v) or more. In some embodiments, the solid antigen / carrier protein gel comprises carrier protein at a concentration of 25%, 20%, 15%, 10%, 7%, 5%, 2%, or 1% (w / v) or less. That is, the solid antigen / carrier protein gel may comprise carrier protein at any concentration within a range of concentrations having an upper limit of 25%, 20%, 15%, 10%, 7%, 5%, 2%, or 1% (w / v) and an independently selected lower limit of 0.5%, 1%, 2%, 5%, 7%, 10%, 15%, or 20% (w / v), where the lower limit is less than the upper limit. In some embodiments, the carrier protein comprises an albumin protein (e.g., serum albumin protein), and the solid antigen / carrier protein gel comprises more than 2% (w / v), e.g., 2% to 25% (w / v), of the carrier protein. In some embodiments, the carrier protein comprises an egg white protein or a mixture of egg white proteins, and the solid antigen / carrier protein gel comprises more than 2% (w / v), e.g., 2% to 25% (w / v), of the carrier protein. In some embodiments, the carrier protein comprises gelatin, and the solid antigen / carrier protein gel comprises more than 0.5% (w / v), e.g., 0.5% to 25% (w / v) or about 10% (w / v) of the carrier protein.

[0036] In some embodiments, the solid antigen / carrier protein gel comprises antigen at a concentration of at least about 25 nM. In other embodiments, the solid antigen / carrier protein gel comprises antigen at a concentration of about 25 nM. In yet other embodiments, the solid antigen / carrier protein gel comprises antigen at a concentration of about 15, 20, 25, 30, 35, 40, 50, 60, 75, 80, 90, or 100 nM, or more. In other embodiments, the solid antigen / carrier protein gel comprises antigen at a concentration of at least about 10 nM, 15 nM, 20 nM, or 25 nM to about 100 nM. As described herein, the solid antigen / carrier protein gels of the present disclosure can comprise antigen at a wide range of concentrations, depending, for example, on the sensitivity of the detection method, the desired use of the gel, etc.

[0037] In one embodiment, the method includes one or more antigen / carrier protein gels. In one embodiment, two gels are used, each with an antigen of a different concentration, as needed. In another embodiment, three gels are used, each with an antigen of a different concentration, as needed. In fact, multiple gels containing antigens of different concentrations may be useful, for example, as controls for IHC staining to represent a range of antigen concentrations.

[0038] In some embodiments, the antigen comprises a polypeptide antigen (e.g., a peptide antigen or a full-length protein antigen). In some embodiments, the polypeptide antigen comprises an N-terminal tyrosine, a C-terminal cysteine, or both (e.g., for chemically crosslinking the antigen to a carrier protein). In some embodiments, the N-terminal tyrosine and / or the C-terminal cysteine ​​is crosslinked to the carrier protein. For example, the C-terminal cysteine ​​can be used to crosslink the antigen to the carrier protein. A variety of cysteine-reactive reagents are known in the art, including, but not limited to, sulfhydryl-reactive crosslinker reactive groups such as haloacetyl, maleimide (e.g., sulfo-SMCC and its analogs), aziridine, acryloyl, arylating agents, vinyl sulfone, pyridyl disulfide, TNB-thiol, and disulfide reducing agents.

[0039] In some embodiments, the solid antigen / carrier protein gel is fixed with a fixative, e.g., as described below. In some embodiments, the fixative is a cross-linking fixative (e.g., an aldehyde-based fixative). In some embodiments, the fixative is not a cross-linking fixative (e.g., a precipitating fixative such as Carnoy's). As described herein, solid gels comprising carrier protein and antigen can be prepared by denaturing and precipitating the carrier protein and / or antigen by heating, or by adding a precipitating fixative to the mixture and immobilizing the antigen in the protein gel. For some antigens (especially those with small molecular size), cross-linking the antigen to the carrier protein (during or after the process of denaturing the carrier protein to form the gel) is believed to retain the antigen and the resulting sections in the gel, e.g., during the embedding, sectioning, and / or staining process.

[0040] In some embodiments, the antigen comprises a non-polypeptide antigen. Examples of non-polypeptide antigens include, but are not limited to, sugars, lipids, and nucleic acids.

[0041] In some embodiments, the solid antigen / carrier protein gel has a thickness of about 30 nm to about 50 μm. The appropriate thickness of the solid antigen / carrier protein gel can depend, for example, on the application. In some embodiments, the solid antigen / carrier protein gel has a thickness of about 0.03, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 μm or more. In some embodiments, the solid antigen / carrier protein gel has a thickness of about 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, or 1 μm or less. That is, the solid antigen / carrier protein gel can have a thickness with an upper limit of 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, or 1 μm and an independently selected lower limit of 0.03, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 μm, where the lower limit is less than the upper limit. For example, a solid antigen / carrier protein gel used in IHC staining may have a thickness of, for example, about 2 μm to about 30 μm, while a solid antigen / carrier protein gel used in EM may have a thickness of, for example, about 30 nm to about 50 μm, or about 30 nm to about 100 nm. A method for slicing a solid antigen / carrier protein gel of the present disclosure is described below.

[0042] In some embodiments, the solid antigen / carrier protein gel was subjected to antigen retrieval. Exemplary methods of antigen retrieval are described in more detail below.

[0043] In some embodiments, the solid antigen / carrier protein gel is embedded in paraffin (e.g., embedded in a paraffin block); epoxy, acrylic, or plastic resin (e.g., EPON™ resin, methacrylate, LR White resin, Araldite®, Spar Plastic, Lowicryl®, etc.); synthetic wax; a blend or copolymer alloy of paraffin wax and a plastic polymer; polyethylene glycol; or a medium of the present disclosure, such as GACH embedding medium (glutaraldehyde-carbohydrazide). In some embodiments, the solid antigen / carrier protein gel is fixed to a solid substrate, including, but not limited to, a glass slide (e.g., for IHC), a support, a grid, or a stub (e.g., for EM).

[0044] In some embodiments, a method for producing a solid antigen / carrier protein gel includes mixing a purified antigen with a liquid solution containing a carrier protein to produce an antigen / carrier protein liquid solution, and heating the antigen / carrier protein liquid solution to form a solid antigen / carrier protein gel. In some embodiments, the carrier protein is selected from albumin protein (e.g., serum albumin protein), egg white protein or a mixture of egg white proteins, and gelatin.

[0045] In some embodiments, the antigen / carrier protein liquid solution comprises a carrier protein at a concentration of 0.5%, 1%, 2%, 5%, 7%, 10%, 15%, or 20% (w / v) or more. In some embodiments, the antigen / carrier protein liquid solution comprises a carrier protein at a concentration of 25%, 20%, 15%, 10%, 7%, 5%, 2%, or 1% (w / v) or less. That is, the antigen / carrier protein liquid solution may comprise a carrier protein at any concentration within a range of concentrations having an upper limit of 25%, 20%, 15%, 10%, 7%, 5%, 2%, or 1% (w / v) and an independently selected lower limit of 0.5%, 1%, 2%, 5%, 7%, 10%, 15%, or 20% (w / v), where the lower limit is less than the upper limit. In some embodiments, the carrier protein comprises an albumin protein (e.g., serum albumin protein), and the antigen / carrier protein liquid solution comprises more than 2% (w / v), e.g., 2% to 25% (w / v), of the carrier protein. In some embodiments, the carrier protein comprises an egg white protein or a mixture of egg white proteins, and the antigen / carrier protein liquid solution comprises more than 2% (w / v), e.g., 2% to 25% (w / v), of the carrier protein. In some embodiments, the carrier protein comprises gelatin, and the antigen / carrier protein liquid solution comprises more than 0.5% (w / v), e.g., 0.5% to 25% (w / v) or about 10% (w / v) of the carrier protein.

[0046] In some embodiments, the methods of the present disclosure include crosslinking the antigen to the carrier protein using a cysteine-reactive reagent (e.g., before heating the antigen / carrier protein liquid solution). A variety of cysteine-reactive reagents are known in the art and include, but are not limited to, sulfhydryl-reactive crosslinker reactive groups such as haloacetyl, maleimide (e.g., sulfo-SMCC and its analogs), aziridine, acryloyl, arylating agents, vinyl sulfone, pyridyl disulfide, TNB-thiol, and disulfide reducing agents.

[0047] In some embodiments, the methods of the present disclosure include immobilizing an antigen with a carrier protein. In some embodiments, a fixative is included in the antigen / carrier protein solution. In some embodiments, the fixative is a cross-linking fixative. In some embodiments, the fixative is a non-cross-linking fixative. Examples of suitable fixatives include, but are not limited to, formaldehyde, formalin, paraformaldehyde (PFA), glutaraldehyde, Davidson's fixative, Bouin's fixative, Karnovski's fixative (e.g., half-strength Karnovski's fixative), Zenker's solution, Helly's solution, Carnoy's solution, zinc formalin, neutral-buffered formalin (NBF), periodate-lysine-paraformaldehyde (PLP), Zamboni's fixative, dimethyl suberimidate (DMS), acetone, alcohol (e.g., methanol or ethanol), zinc salts (e.g., zinc acetate, zinc chloride, zinc trifluoroacetate, etc. In certain embodiments, the fixative comprises formaldehyde (e.g., at a concentration of at least about 1% or at least about 2%).

[0048] In some embodiments, antigens are crosslinked to carrier proteins using selective chemical reactions, including but not limited to azide-alkyne addition. In some embodiments, selective chemical reactions are used to crosslink non-protein antigens, such as sugar antigens or lipid antigens, to carrier proteins.

[0049] In some embodiments, the disclosed methods include dehydrating the solid antigen / carrier protein gel. Compounds suitable for dehydration are known in the art and may include, for example, alcohols such as ethanol or methanol. In some embodiments, the disclosed methods include embedding the solid antigen / carrier protein gel. In some embodiments, the solid antigen / carrier protein gel is embedded after dehydration. For example, in some embodiments, the solid antigen / carrier protein gel is dehydrated by exposure to an increasing or graded alcohol series (e.g., a series of increasing ethanol concentrations), followed by exchange of the alcohol with xylene, and exchange of the xylene with paraffin. A variety of media are known in the art and can be used for embedding, including, but not limited to, paraffin (e.g., embedded in a paraffin block); epoxy, acrylic, or plastic resin (e.g., EPON™ resin, methacrylate, LR White resin, Araldite®, Spar Plastic, Lowicryl®, etc.); synthetic wax; blends or copolymer alloys of paraffin wax and plastic polymers; polyethylene glycol; and GACH embedding medium (glutaraldehyde-carbohydrazide).

[0050] In some embodiments, the carrier protein comprises gelatin, and the disclosed methods include cooling the heated antigen / carrier protein liquid solution to form a solid antigen / carrier protein gel. In some embodiments, the method further includes incubating the solid antigen / carrier protein gel with a fixative and dehydrating the solid antigen / carrier protein gel. In some embodiments, the antigen / carrier protein liquid solution comprises a carrier protein concentration of about 0.5% (w / v) or greater. In some embodiments, the antigen / carrier protein solution is heated to at least about 65°C (e.g., for at least 6 minutes).

[0051] In some embodiments, the carrier protein comprises polylysine, for example, at a concentration of about 14 mg / mL.

[0052] In some embodiments, the disclosed methods include incubating the solid antigen / carrier protein gel in an embedding medium (e.g., a liquid embedding medium). Various embedding media are known in the art and may include, but are not limited to, Optimum Cutting Temperature (OCT) compound (e.g., Tissue-Tek® OCT compound or Tissue-plus® OCT compound), PELCO® cryo-embedding compound, PolarStat™ or PolarStat Plus™ embedding medium, and Tissue Freezing Medium or TFM™. These embedding media may include, for example, water-soluble glycols and resins; an exemplary embedding medium OCT compound contains 5-15% polyvinyl alcohol and 1-10% polyethylene glycol. In some embodiments, the disclosed methods include freezing the solid antigen / carrier protein gel (e.g., after incubating the solid antigen / carrier protein gel in the embedding medium). In some embodiments, the disclosed methods involve freezing of an unfixed (e.g., fixative-free) solid antigen / carrier protein gel, similar to the preparation of a frozen, unfixed, freshly prepared tissue sample.

[0053] In some embodiments, the methods of the present disclosure include slicing a solid antigen / carrier protein gel into one or more solid antigen / carrier protein gel sections. In some embodiments, the one or more solid antigen / carrier protein gel sections have a thickness of about 30 nm to about 50 μm. In some embodiments, the one or more solid antigen / carrier protein gel sections have a thickness of about 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 μm or more. In some embodiments, the one or more solid antigen / carrier protein gel sections have a thickness of about 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 μm or less. That is, sections of one or more solid antigen / carrier protein gels may be separated by an upper limit of 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 μm and an upper limit of 0.03 μm. The thickness may be less than the upper limit, with an independently selected lower limit of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 μm. For example, sections of solid antigen / carrier protein gels used in IHC staining may have a thickness of, for example, about 2 μm to about 30 μm, while sections of solid antigen / carrier protein gels used in EM may have a thickness of, for example, about 30 nm to about 50 μm, or about 30 nm to about 100 nm. Instruments for slicing solid antigen / carrier protein gels of the present disclosure are known in the art and may include, but are not limited to, microtomes, cryostats (for frozen gels), knives (e.g., diamond, glass, or sapphire), and the like.In some embodiments, the solid antigen / carrier protein gel is dehydrated, embedded, fixed, frozen, or a combination thereof prior to sectioning.

[0054] In some embodiments, the methods of the present disclosure include subjecting the solid antigen / carrier protein gel to antigen retrieval. Various antigen retrieval methods are known in the art. In some embodiments, subjecting the solid antigen / carrier protein gel to antigen retrieval includes heating the solid antigen / carrier protein gel in a liquid solution, such as by boiling (e.g., heat-induced epitope retrieval). In some embodiments, the liquid solution includes a buffer such as Tris / EDTA or sodium citrate. In some embodiments, subjecting the solid antigen / carrier protein gel to antigen retrieval includes treatment with one or more proteolytic enzymes (e.g., trypsin, proteinase K, pepsin, ficin, or pronase) or antigen retrieval reagents (e.g., hydrochloric acid, formic acid, sodium dodecyl sulfate (SDS), citrate buffer, EDTA, citrate-EDTA, Tris, Tris-EDTA, Tris-HCl, Tris-buffered saline, or citraconic anhydride). For details on antigen retrieval, see, e.g., Shi, SR et al. (2011) J. Histochem. Cytochem. 59:13-32.

[0055] In some embodiments, the methods of the present disclosure include blocking the solid antigen / carrier protein gel. As is known in the art, blocking (e.g., prior to IHC staining) reduces nonspecific interactions with the antigen by preventing binding to sites not involved in specific antigen:antibody interactions. A variety of suitable blocking reagents are known in the art, including, but not limited to, serum or protein solutions (e.g., serum albumin, gelatin, nonfat dry milk, etc.).

[0056] In some embodiments, the disclosed methods include fixing the solid antigen / carrier protein gel (e.g., by including a fixative in the antigen / carrier protein solution), dehydrating the solid antigen / carrier protein gel, embedding the dehydrated solid antigen / carrier protein gel, cutting the paraffin block containing the embedded solid antigen / carrier protein gel into one or more sections, subjecting the one or more sections to antigen retrieval, and blocking the one or more sections after antigen retrieval. In other embodiments, the disclosed methods include incubating the solid antigen / carrier protein gel in a liquid embedding medium, freezing the solid antigen / carrier protein gel in the embedding medium, sectioning the frozen antigen / carrier protein gel into one or more sections, and blocking the one or more sections.

[0057] Other aspects of the present disclosure relate to tissue microarrays (TMAs) comprising one, two, or more solid antigen / carrier protein gel(s) of the present disclosure. For example, a TMA of the present disclosure may be prepared by preparing a solid antigen / carrier protein gel of the present disclosure (e.g., embedded in a paraffin block as described above), punching out a core containing the solid antigen / carrier protein gel, and transferring the core to a recipient TMA. In some embodiments, the core has a diameter of about 1 mm. An exemplary method for preparing a TMA is described below, for example, by transferring a core(s) from a donor paraffin block to a recipient TMA block, followed by heating (e.g., at 37°C overnight, then at 70°C for 10 minutes), cooling, and slicing the recipient TMA block. In some embodiments, a TMA comprises two or more solid antigen / carrier protein gels of the present disclosure comprising different antigens. In some embodiments, a TMA comprises two or more solid antigen / carrier protein gels of the present disclosure comprising different concentrations of the same antigen. In some embodiments, the TMA further comprises one or more directional reference(s), e.g., a colored pigment for identification. In some embodiments, the TMA is a TMA section of a histology slide. Without wishing to be bound by theory, it is believed that the solid antigen / carrier protein gels of the present disclosure are particularly advantageous for use in TMAs, e.g., to provide a range of epitope concentrations and / or types. This range can also be provided adjacent to the tissue section, which can provide convenient referencing for quantitative analysis.

[0058] III. Uses for Solid Antigen / Carrier Protein Gels Solid antigen / carrier protein gels (e.g., as described in Section II above and / or illustrated in the Examples below) can be used in a variety of applications, including, but not limited to, controls for IHC or EM analysis.

[0059] In some embodiments, a method for immunohistochemistry (IHC) staining of a control antigen comprises providing a plurality of solid antigen / carrier protein gels of the present disclosure (e.g., two or more, three or more, four or more, five or more, etc.) representing different concentrations of the antigen of interest. For example, the method may comprise providing two solid antigen / carrier protein gels made with different concentrations of purified antigen. In some embodiments, the method comprises contacting the solid antigen / carrier protein gel with a primary antibody that specifically binds to the antigen and is coupled to a detectable moiety. In other embodiments, the method comprises contacting the solid antigen / carrier protein gel with a primary antibody that specifically binds to the antigen, and then contacting the solid antigen / carrier protein gel with a secondary antibody that specifically binds to the primary antigen and is coupled to a detectable moiety. In some embodiments, the method comprises detecting a detectable moiety signal(s) from one or more of the plurality of solid antigen / carrier protein gels. The amount of signal detected from the multiple solid antigen / carrier protein gels can then be determined and, if desired, compared to the amount or concentration of antigen present in each of the multiple solid antigen / carrier protein gels to provide various levels of control IHC staining of the antigen.

[0060] In some embodiments, one of the antigen / carrier protein gels contains no antigen (e.g., 0 nM), and the lack of detectable signal from IHC staining of this gel indicates a negative control or background IHC staining of the sample. In some embodiments, the method includes contacting the sample with a primary antibody that specifically binds to the antigen and is bound to a detectable moiety, or with a primary antibody that specifically binds to the antigen and a secondary antibody that specifically binds to the primary antibody, and detecting the signal of the detectable moiety from the sample. The amount of signal detected from the sample can then be compared to the amount of signal(s) detected from the solid antigen / carrier protein gel(s), e.g., the amount of antigen present in the sample can be compared to the concentration or amount of a known antigen present in the solid antigen / carrier protein gel(s).

[0061] In some embodiments, a method for control secondary antibody immunohistochemistry (IHC) staining includes providing a plurality of solid antigen / carrier protein gels (e.g., two or more, three or more, four or more, five or more, etc.) of the present disclosure representing different antibody isotypes. For example, the method may include providing two solid antigen / carrier protein gels made with antibodies representing different antibody isotypes. In some embodiments, the method includes contacting the solid antigen / carrier protein gels with a secondary antibody that specifically binds to one of the antibody isotypes and is conjugated to a detectable moiety. Control secondary antibody staining allows for detection of a signal from the plurality of solid antigen / carrier protein gels. In this example, the secondary antibody specifically binds to the cognate antibody isotype, and the signal detected from the gel lacking the cognate antibody isotype represents background staining. Thus, detection of a signal associated with the solid antigen / carrier protein gel containing the cognate antibody isotype and a lack of signal associated with the solid antigen / carrier protein gel lacking the cognate antibody isotype indicates staining with the control secondary antibody.

[0062] In some embodiments, a method for immunohistochemistry (IHC) staining of an antigen includes providing one or more solid antigen / carrier protein gels containing an antigen and a sample; contacting the solid antigen / carrier protein gel and sample with a primary antibody that specifically binds to the antigen and is bound to a detectable moiety, or contacting the solid antigen / carrier protein gel and sample with a primary antibody that specifically binds to the antigen and contacting the solid antigen / carrier protein gel and sample with a secondary antibody that specifically binds to the primary antibody and is bound to a detectable moiety; detecting a signal of the detectable moiety from the solid antigen / carrier protein gel; and detecting a signal of the detectable moiety from the sample. Thus, the one or more solid antigen / carrier protein gels can be used as a positive control for IHC staining of a sample.

[0063] An exemplary flowchart of a process 100 for IHC staining of antigens, such as antigens in a sample and a solid antigen / carrier protein gel of the present disclosure (used as a control for staining of antigens in the sample), is shown in FIG. 1. In block 102, tissue is collected to provide a sample for analysis, and / or a solid antigen / carrier protein gel of the present disclosure is generated (e.g., as described in Section II above). In some embodiments, the solid antigen / carrier protein gel contains an antigen of interest (e.g., a purified antigen) that may or may not be present in the sample. Optionally, in block 104, the sample and / or solid antigen / carrier protein gel are fixed (e.g., as described in Section II above). In some embodiments, the solid antigen / carrier protein gel is incubated with a fixative. In other embodiments, the antigen / carrier protein liquid solution used to create the solid gel includes a fixative. Optionally, in block 106, the sample and / or solid antigen / carrier protein gel are embedded (e.g., as described in Section II above). As an optional alternative to block 104, the sample and / or solid antigen / carrier protein gel can be frozen in an embedding medium in block 106. Optionally, the sample and / or solid antigen / carrier protein gel is sectioned (e.g., as described above in Section II) in block 108. Optionally, the sample and / or solid antigen / carrier protein gel is subjected to antigen retrieval and blocking (e.g., as described above in Section II) in block 110.

[0064] In some embodiments, the sample and solid antigen / carrier protein gel are contacted with a primary antibody that specifically binds to the antigen, as shown in block 112. In some embodiments, the primary antibody comprises a detectable moiety. In other embodiments, the sample and solid antigen / carrier protein gel are contacted with a secondary antibody that specifically binds to the primary antibody, as shown in block 114. In some embodiments, the secondary antibody comprises a detectable moiety (biotin in this example) conjugated to the secondary antibody.

[0065] In some embodiments, the detectable moiety signal is detected from the solid antigen / carrier protein gel and / or the sample. A variety of detectable moieties are contemplated, as described below. In this example, a chromogenic detection method is used. In block 116, the sample and solid antigen / carrier protein gel are contacted with an enzyme conjugate that binds to a secondary antibody (e.g., a streptavidin-conjugated enzyme conjugate that binds to a biotinylated secondary antibody). In block 118, the sample and solid antigen / carrier protein gel are contacted with a chromogenic substrate or chromogen solution that, upon incubation with the enzyme conjugate, forms a colored precipitate in the solid antigen / carrier protein gel and, if present, in the sample, indicating the presence of the antigen.

[0066] In some embodiments, the presence of an antigen in a solid antigen / carrier protein gel of the present disclosure is detected by direct detection, e.g., by incubating the gel with an antibody or other antigen-binding moiety that specifically binds to the antigen and is bound to a detectable moiety. In other embodiments, the presence of an antigen in a solid antigen / carrier protein gel of the present disclosure is detected by indirect detection, e.g., by incubating the gel with a first antibody or other antigen-binding moiety that specifically binds to the antigen and a second antibody or other antigen-binding moiety that specifically binds to the first antibody or other antigen-binding moiety, where the second antibody or other antigen-binding moiety is bound to a detectable moiety.

[0067] A variety of detectable moieties are contemplated for use with the solid antigen / carrier protein gels of the present disclosure. In some embodiments, the detectable moiety comprises an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The signal is then detected by exposing the detectable moiety to a chromogenic substrate for the enzyme and detecting a signal from the chromogenic substrate upon reaction with the enzyme (e.g., as described above for blocks 116 and 118). Examples of chromogenic substrates include, but are not limited to, 3,3'-diaminobenzidine (DAB), which is converted to a brown product by HRP, and 3-amino-9-ethylcarbazole (AEC), which is converted to a red product by AP. Alternatively, peroxidase can chemically activate a tyramide moiety conjugated to one of several reporters (e.g., a fluorescent molecule); the activated tyramide conjugate covalently binds to a nearby molecule in the sample, generating a detectable signal at the location of the peroxidase. Indirect detection can also be used with chromogenic staining. For example, a biotinylated secondary antibody can be incubated with an avidin- or streptavidin-labeled enzyme conjugate, a polymer such as dextran can be conjugated to one or more secondary antibodies and one or more enzyme conjugates, or an enzyme conjugate can be directly polymerized onto a secondary antibody. In some embodiments, the detectable moiety comprises a fluorophore, such as fluorescein (FITC), rhodamine or its derivatives, TRITC, cyanine (Cy3), phycoerythrin (R-PE), CF™, etc. For example, the fluorophore can be conjugated to a primary or secondary antibody, or to a tyramide moiety that can be covalently bound to a sample after activation by peroxidase. In some embodiments, the detectable moiety comprises a metal particle, such as gold. For example, gold particles can be conjugated to a primary or secondary antibody and imaged, for example, by immuno-EM. In some embodiments, the detectable moiety comprises a metal particle, such as gold. 35 S, 125 I, or 131The detectable moiety may include a radioisotope such as I, and may be imaged, for example, by radioimmunodetection. For example, the radioisotope may be conjugated to a primary or secondary antibody. In some embodiments, the detectable moiety includes a nucleic acid, such as DNA or RNA. For example, the nucleic acid may be conjugated to a primary or secondary antibody. The primary or secondary antibody may be bound to a metal ion that can be detected by a mass spectrometer or mass cytometer (CYTOF and MIBI; see Giesen et al. Nat Methods. 2014 Apr;11(4):417-22 and Angelo et al., Nat Med. 2014 Apr;20(4):436-42). The primary or secondary antibody may be bound to a solid-state, semiconductor, or carbon-based nanoparticle (e.g., a "quantum dot"), and may be detected by fluorescent imaging (see Wu et al., Nat Biotechnol. 2003 Jan;21(1):41-6). The primary or secondary antibody can be conjugated to a peroxidase, such as horseradish peroxidase, which is detected by incubation with a chemiluminescent substrate. The primary or secondary antibody can be conjugated to an electrochemiluminescent reporter (e.g., ruthenium; see Valenti et al., J Am Chem Soc. 2017 Nov 8).

[0068] IV. KITS AND PRODUCTS Certain aspects of the present disclosure relate to kits or articles of manufacture comprising a solid antigen / carrier protein gel.

[0069] In some embodiments, a kit of the present disclosure comprises a first solid antigen / carrier protein gel of the present disclosure comprising a purified antigen and a second solid antigen / carrier protein gel of the present disclosure comprising a purified antigen at a different concentration than the first solid antigen / carrier protein gel.

[0070] In some embodiments, the first and / or second solid antigen / carrier protein gel(s) are sectioned, e.g., as described above in Section II. In some embodiments, the first and / or second solid antigen / carrier protein gel(s) are immobilized on a solid substrate, e.g., as described above in Section II.

[0071] In some embodiments, the kits of the present disclosure further include a third solid antigen / carrier protein gel of the present disclosure that does not include purified antigen (e.g., for use as a negative control). In some embodiments, the third solid antigen / carrier protein gel is sliced, e.g., as described above in Section II. In some embodiments, the third solid antigen / carrier protein gel is immobilized on a solid substrate, e.g., as described above in Section II.

[0072] In some embodiments, the kit may further include instructions for using the kit, for example, according to any of the methods described in Section III above. [Example]

[0073] The present disclosure will be more fully understood by reference to the following examples. However, the examples should not be construed as limiting the scope of the disclosure. It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art, and are within the spirit and scope of this application and the appended claims.

[0074] Example 1: Validating BSA gel as a platform approach to IHC control 1 shows an exemplary workflow 100 for IHC staining in tissue samples. Importantly, IHC staining reveals not only the expression level of a target, but also its cellular and tissue distribution.

[0075] In the following example, we describe a novel platform approach for generating IHC controls that can be adapted to a wide range of targets. This approach was validated using human BCL2, a target for which 3+ positive control tissues can be identified (e.g., human chronic lymphocytic leukemia tumor samples are often scored as 3+ in this assay), but which are difficult to obtain or limited by ethical considerations for routine use as staining controls. The generality of this approach was demonstrated using mouse and rat IgG and the human protein of interest.

[0076] method Approximately 0~10 -4 Approximately 0-0.5 mg of antigen containing the target epitope of M was mixed with 0.5 mL of 25% BSA in PBS in a 1.5 mL microcentrifuge tube. 0.5 mL of 37% formaldehyde was added to the tube. The tube was heated at 85 °C for 10 min and, if a fixative (e.g., formalin) was included, left at room temperature overnight to solidify. The resulting gel was removed from the tube and placed in 10% neutral-buffered formalin (NBF). Subsequently, the gel was dehydrated through graded alcohols to xylene, infiltrated with warm paraffin wax, embedded in a paraffin block, sectioned on a microtome, and stained according to standard IHC methods using the primary mouse monoclonal anti-BCL2 antibody 124 and a horseradish peroxidase (HRP) / 3,3'-diaminobenzidine (DAB) chromogenic enzyme / substrate system.

[0077] The IF detection assay was developed using a Ventana Discovery Ultra automated stainer. Four-micron sections containing the target BSA / peptide samples were cut, deparaffinized, and pretreated with CC1 cell conditioning solution. The primary mouse monoclonal anti-BCL2 antibody 124 was incubated for 16 minutes at 37°C and detected with the OmniMAP anti-mouse HRP detection system for 8 minutes, followed by detection with the Ventana Discovery Red 610 detection system for 16 minutes.

[0078] Whole-slide brightfield and immunofluorescence images were acquired at a scanning resolution of 0.46 microns / pixel using a Hamamatsu Nanozoomer-XR digital slide scanner equipped with a 20x 0.75 NA objective and a fluorescence module. Brightfield imaging was performed and used for focusing. When applied, immunofluorescence signals were acquired using a TRITC filter at 1x exposure (3.4 ms photon collection) and 1x gain. Autofluorescence was captured using a DAPI (2x exposure, 6.8 ms photon collection, 2x gain) and CFP filter (4x exposure, 13.6 ms photon collection, 2x gain). Light power was set to 50% for all immunofluorescence acquisitions. Image analysis was performed using Matlab version 9.3. Regions of interest (ROIs) were either manually marked up (brightfield scans only) or generated using thresholding and morphological filtering in the autofluorescence channel and transferred to the TRITC channel for intensity measurements. Mean grayscale intensity was calculated at 8-bit depth. The mean optical absorbance (bright-field DAB labels) or emittance (fluorescent labels) was calculated by summing all pixel absorbance or emittance values ​​within the ROI (calculated using the respective natural logarithm formulas below) and normalized to the total ROI pixel count. All zero pixel grayscale intensity values ​​were approximated using a value of 1. Bright-field pixel absorbance = log(255 / pixel grayscale intensity). Fluorescent pixel emittance = log(pixel grayscale intensity).

[0079] In validation experiments, BSA gel samples were generated in microcentrifuge tubes as described above, embedded in paraffin blocks, and tissue microarrays were created of 600-micron diameter cylinders cut from the paraffin blocks and re-embedded into arrays using standard tissue microarray techniques.

[0080] For BCL2 experiments, the sequence Ac- A peptide containing TIFF2026012670000002.tif12170-amide (SEQ ID NO: 1) was used. The underlined amino acids correspond to wild-type (unmutated) amino acids 41-54 of the human BCL2 sequence, as described in UniProt accession number P10415. This peptide includes an amino-terminal linker sequence (Ac-YGSG) (SEQ ID NO: 6) containing acetyl-Y (tyrosine), which allows conjugation with aldehydes (e.g., formaldehyde), and a carboxy-terminal linker sequence (GSGC-amide) (SEQ ID NO: 7) containing C (cysteine)-amide, which is reactive with various cross-linking reagents, such as maleimide, haloacetyl, or pyridyl disulfide-containing compounds.

[0081] result BSA / peptide gels were prepared as described above. Prior to gel generation, the antigen (e.g., peptide) was mixed with BSA in solution to ensure uniform peptide mixing. The resulting gel (Figure 2A) can be constructed in any desired shape (in this example, the gel was fabricated in the shape of a 1.5 mL microcentrifuge tube). The gel can also be sliced ​​(Figures 2B and 2C) and subjected to typical IHC processing steps, such as antigen retrieval, blocking, primary / secondary antibody incubation, and detection (e.g., using an enzyme / substrate approach). For example, Figure 2D shows a sliced ​​gel portion prepared for dehydration and paraffin processing, and Figure 2E shows a sliced ​​gel portion embedded in a paraffin donor block. Figure 2F shows TMAs created from various paraffin gel donor blocks, with cores containing black and green pigments for orientation reference. Peptide antigens can be crosslinked to BSA more efficiently than lysozyme, thereby retaining the antigen in the BSA gel during IHC processing and detection.

[0082] To examine the feasibility of peptide / BSA gels as IHC controls, various concentrations of BCL2 peptides were mixed into BSA gels, which were then sectioned and prepared according to standard IHC procedures. BCL2 was detected using a BCL2 primary antibody against human BCL2 protein (Ventana Medical Systems' "CONFIRM anti-bcl-2(124) mouse monoclonal primary antibody" BCL2), a biotinylated secondary antibody, avidin-conjugated HRP as a chromogenic reporter, and DAB as a chromogenic substrate. As shown in Figure 3A, concentrations ranging from 0 to 0.5 mg / mL (2.5 × 10 -4 Successively increasing BCL2 levels up to 5 × 10 -5 mg / mL (corresponding to a 25 nM peptide concentration) to 0.5 mg / mL (2.5 × 10 -4 The BCL2 expression gradient was resolvable across the entire test range (up to 1000 M peptide concentrations), whereas a negative control lacking the peptide yielded no staining. Importantly, BCL2 staining levels of 1+, 2+, and 3+ were achieved at different peptide concentrations. Because consistent and uniform 3+ BCL2 staining is typically difficult to find in readily available tissue samples, this alternative approach provides a simpler and more standardized method for obtaining a gradient of BCL2 expression control than assaying various tissue types.

[0083] To examine the consistency of staining, six independent experiments were performed by staining BCL2 peptide / BSA gels as described above and analyzing the resulting images using MATLAB. Analysis of both optical density (OD; Figure 3B) and image intensity (Figure 3C) as a function of peptide concentration showed that staining was consistent across experiments.

[0084] The BCL2 peptide / BSA gel was also subjected to quantitative immunofluorescence staining. Similar to the results obtained using chromogenic staining, IF analysis showed a graded increase in staining intensity as a function of peptide concentration (Figure 3D). Analysis of optical emittance (Figure 3H) and signal intensity (Figure 3J) again showed a consistent graded increase in staining. Negative control staining with a naive control primary antibody (Figures 3F, 3I, and 3K) or gel autofluorescence (Figures 3E and 3G) did not show an increase in intensity, as expected.

[0085] Secondary antibody controls were also examined. Using a donkey anti-mouse secondary antibody, mouse IgG embedded in the BSA gel was readily detected, whereas rat IgG did not yield a detectable signal, as shown in Figure 4A. Similarly, using a donkey anti-rat secondary antibody, rat IgG embedded in the BSA gel was readily detected, whereas mouse IgG did not yield a detectable signal (Figure 4B). This confirms the specificity of the BSA gel approach and demonstrates that the secondary antibody does not nonspecifically bind to the peptide / BSA gel, which lacks the antibody's target.

[0086] To examine staining specificity, we synthesized six peptides with clinically relevant single amino acid substitutions in the anti-BCL2 clone 124 antibody epitope, embedded them in BSA, and stained them as described above. As shown in Figure 4C (and Figures 3D, 3H, and 3J), these mutations had distinct effects on IHC staining (i.e., varying degrees of reduction in signal intensity, depending on the specific amino acid substitution), demonstrating the practicality of quantifying the effect of specific amino acid substitutions on staining intensity.

[0087] Next, the generality of the peptide / BSA gel platform was examined by screening several uncharacterized antibodies for specific detection of the target of interest. Twenty-seven antibody clones isolated from mice immunized with human KSR2 were screened by IHC for detection of positive and negative controls (Figures 5A-5D). For the negative control, a BSA gel without added protein or peptide was used. For the positive control, a BSA gel was generated with the human KSR2 protein used to immunize the mice. As expected, because the candidate antibody clones had been selected through a separate process to bind unfixed human target protein, most antibody clones were able to detect human protein on the protein / BSA gel (evidenced by a DAB signal above background intensity). However, the most suitable antibodies for use in IHC assays will show little or no detectable signal in the negative control while simultaneously showing strong staining in the positive control. Among the antibodies tested, clones 5, 6, 7, 11, 12, 15, and 23 were the most promising, as they exhibited the highest ratios of specific staining of the target antigen to nonspecific staining in the negative control samples. In contrast, clones 4, 13, 26, and 27 showed ineffective staining of the target antigen, while antibodies 10 and 20 showed weak but significant nonspecific staining of the negative control samples. These results help illustrate the problem that not all antibodies that can bind to their target of interest in the unfixed state are suitable for use in IHC staining. The peptide control reagents and process described here can help identify antibodies that are most suitable for use in IHC applications.

[0088] Taken together, these results demonstrate that the antigen / BSA gel approach provides a robust platform for generating IHC controls applicable to many target antigens. This approach yields controls that display a gradient of IHC staining that correlates with a range of target concentrations, demonstrating the potential for quantification of target / BSA gels. Unlike IHC controls from tissues or cell lines, peptide / BSA gels can be easily generated in the laboratory as needed, and are consistent, reproducible, inexpensive, versatile, and antigen-specific. Secondary antibody controls demonstrated the specificity of the approach. Furthermore, this approach is ideal for the process of screening hundreds to thousands of candidate antibodies, typically to identify a small number (e.g., 2–5) of leads for further characterization to identify antibodies suitable for IHC analysis of a target of interest.

[0089] Example 2: Investigating conditions for gel formation The following examples describe testing various conditions for their effect on gel formation.

[0090] method To evaluate BSA gel formation, 1 mL of BSA solution in PBS was heated at temperatures ranging from 25 to 85°C. The BSA / PBS solution did not contain formaldehyde. The solution was continuously heated and observed at various times for liquid / solid phases under heating conditions. Solutions were tested at BSA concentrations of 25%, 20%, 15%, 10%, 5%, and 2%.

[0091] To evaluate other protein sources for making gels, 25% solutions of the following were made in PBS: casein, lactalbumin, soybean flour, and nonfat dry milk. 500 μL of 37% formaldehyde was mixed with 500 μL of each 25% protein / PBS solution and incubated at 85°C for 10 minutes.

[0092] For fixative evaluation, 500 μL of fixative was mixed with 500 μL of a 25% BSA solution in PBS and incubated for 10 minutes at 85° C. Fixatives tested included 10% neutral buffered formalin (NBF), 1 / 2 strength Karnovski (glutaraldehyde / paraformaldehyde), Methacarn, Carnoy's solution, and Bouin's.

[0093] To assess formaldehyde concentration, 500 μL of formaldehyde solution was mixed with 500 μL of 25% BSA solution in PBS and incubated at 85°C for 10 minutes. Formaldehyde solutions were tested at the following concentrations (concentrations refer to the original concentrations before dilution with BSA solution): 1%, 2%, 4.5%, 9%, and 18%. Specifically, 37% formaldehyde stock solution was mixed with distilled water to create a final volume of 500 μL containing 1%, 2%, 4.5%, 9%, or 18% formaldehyde. These solutions (each in a separate microtube) were then mixed with an equal volume (500 μL) of 25% BSA solution and heated as described above.

[0094] result Figure 6 shows the effect of heating temperature and time on 25% BSA gel formation. No gel was formed when heated at 25°C or 45°C, even after overnight heating. At 55°C, gel formation was only observed after overnight heating. However, when heated to 65°C or 85°C, solid gel formation was observed within 6 minutes and 2 minutes, respectively. These results demonstrate the effect of heating temperature and time on BSA gel formation.

[0095] Next, we examined the effect of BSA concentration on gel formation. BSA was dissolved in PBS at the following concentrations: 25%, 20%, 15%, 10%, 5%, and 2%. As shown in Figure 7 (the results for 25% BSA are shown in Figure 6), gel formation was observed at all concentrations when heated to 85°C. At BSA concentrations of 5% or higher, gel formation was observed within 10 minutes, while the 2% BSA solution formed a gel after 20 minutes. After heating to 45°C or 55°C, gel formation was not observed at any time. These results indicate that a wide range of BSA concentrations can produce BSA gels under appropriate heating conditions.

[0096] Casein, lactalbumin, soy flour, and skim milk powder were then tested for their ability to form gels. As described above, each was tested at a final concentration of 12.5% ​​in PBS after mixing with formaldehyde (final concentration: 18.5%). Under these conditions, only soy flour was observed to form a gel, although the resulting solid was not uniform. These data demonstrate the unique properties of serum albumin in promoting gel formation.

[0097] Various fixatives, including 10% NBF, 1 / 2 strength Karnovski (glutaraldehyde / paraformaldehyde), Methacarn, Carnoy's solution, and Bouin's, were then tested for their ability to form BSA gels. Of these, 10% NBF and Methacarn failed to form a solid. Carnoy's formed a solid before being mixed. 1 / 2 Karnovski and Bouin's fixatives formed distinct solids.

[0098] The effect of formaldehyde concentration on gel formation was also examined. All concentrations of formaldehyde, except for 1%, were able to promote solid gel formation. These results demonstrate the effect of fixatives on gel formation.

[0099] Example 3: Evaluation of other substrates for gel formation The following example describes the evaluation of additional proteins for their ability to form gels suitable for IHC staining.

[0100] method BSA (used at a final concentration of 25%), egg white protein or egg white protein mixture (used at a final concentration of 25%), gelatin (used at a final concentration of 10%), lactalbumin (used at a final concentration of 25%), liver protein powder, soy flour (used at a final concentration of 25%), casein (used at a final concentration of 10%), and nonfat dry milk (used at a final concentration of 25%) were evaluated for their ability to produce solid gels suitable for IHC staining. Protein solutions were mixed with 37% formaldehyde and heated to 85°C for 10 minutes.

[0101] Each type of gel was prepared with or without 0.1 mg / mL normal rabbit IgG (DA1E). Sections were stained with 5 μg / mL donkey anti-rabbit biotinylated secondary antibody and then detected with ABC-HRP.

[0102] For gelatin gels, 10% molten gelatin was mixed with 0.1 mg / mL normal rabbit IgG (DA1E), then cooled at 4°C for 1 hour, transferred to 10% NBF overnight, transferred to 70% ethanol for 2 days, and processed for IHC staining as for tissue. For lactalbumin and liver protein powder gels, Histogel™ was added to solidify the gel. For casein gels, 100 mM NaOH was added dropwise to bring the pH to 8.0, stirring as needed to form a solution.

[0103] For gel imaging, slides were scanned with a Hamamatsu Nanozoomer and digital images were captured using image viewing software.

[0104] result Additional substrate proteins were tested for their ability to form gels suitable for IHC staining, for example, by forming uniform gels that allow IHC staining and adhere to glass IHC slides. As shown in Figure 8, in addition to BSA, egg white protein or a mixture of egg white proteins and gelatin formed gels that exhibited specific IHC staining (in this case, after staining a gel containing a rabbit IgG antibody sample with an anti-rabbit secondary antibody) with low nonspecific staining (after staining a gel without rabbit IgG with an anti-rabbit secondary antibody). In contrast, gels generated from lactalbumin, liver protein powder, or soy flour failed to produce uniform gels (Figure 9). Gels generated from casein or nonfat dry milk failed to adhere to IHC slides (Figure 10). A description of each gel substrate and the resulting material from the attempted gel formation are provided in Table A.

[0105] Table A. Protein gels made from various substrates. TIFF2026012670000003.tif135170

[0106] In summary, BSA, egg white protein(s), and gelatin provided gels suitable for specific IHC staining. Other protein substrates either failed to form uniform gels or failed to adhere to the slide during IHC processing.

[0107] Example 4: Detection of antigens in gels The synthetic IHC control concept described above was further analyzed using proof-of-concept applications relevant for research and clinical use, including calibration and quality control of IHC assays.

[0108] method Peptides and Proteins The peptide was synthesized by New England Peptide (Gardner, MA) with a purity of >95%. Amino acids 41–54 of the human BCL2 protein (UniProt P10415) were extended at the N-terminus by four amino acids containing an acetylated tyrosine and the spacer sequence GSG to facilitate cross-linking with formaldehyde. The C-terminus contains a GSG spacer sequence followed by a cysteine ​​amide to facilitate cross-linking with formaldehyde (Metz, B. et al. (2004) J. Biol. Chem. 279:6235–6243; Toews, J. et al., (2008) Anal. Chim. Acta 618:168–183) or sulfhydryl-reactive reagents. The overall 22-amino acid peptide sequence is Ac-YGSGGAAPAPGIFSSQPGGSGC-amide (SEQ ID NO: 1). Additional peptides containing sequences derived from the human MYC protein (UniProt P01106) were synthesized: Ac-YGSGNRNYDLDYDSVQPYFYGSGC-amide (amino acids 9-24; SEQ ID NO: 2); Ac-YGSGDSVQPYFYCDEEENFYGSGC-amide (amino acids 17-32; SEQ ID NO: 3); Ac-YGSGQQQSELQPPAPSEDIWGSGC-amide (amino acids 35-50; SEQ ID NO: 4); and Ac-YGSGFELLPTPPLSPSRRSGGSGC-amide (amino acids 53-68; SEQ ID NO: 5). A negative control peptide containing 13 amino acids from the first exon of human MCL1 (UniProt Q07820) was synthesized with the same N- and C-terminal extensions as described above. In some experiments, arginine, serine, or tyrosine replaced the N- and C-terminal amino acids of the above peptides. Lyophilized peptides were dissolved in a minimal amount of distilled water, DMSO, or dimethylformamide. The C-terminal 301 amino acids (amino acids 650–950) of kinase inhibitor of RAS2 (KSR2; Uniprot Q6VAB6) were expressed as a 701-amino acid N-terminally [His]6-tagged maltose-binding protein (amino acids 9–399) fusion construct.Fusion proteins were expressed as baculovirus constructs in Trichoplusia niTni Pro insect cells (Expression Systems; Davis, CA) and subsequently purified sequentially by nickel nitrilotriacetic acid affinity, amylose affinity, and Sepharose S200 size-exclusion chromatography. Purified mouse IgG1 clone MOPC-31C (BD Pharmingen, San Jose, CA), rat IgG1 clone R3-34 (BD Pharmingen, San Jose, CA), and rabbit IgG clone DA1E (Cell Signaling Technology, Danvers, MA) were obtained commercially. Bovine serum albumin (BSA; Ultra Pure) was purchased from Cell Signaling Technology (Danvers, MA). Food-grade gelatin and dried egg white were from Knox (Oakbrook, IL) and Judees Gluten Free (Columbus, OH), respectively.

[0109] Protein Matrix Gel Unless otherwise stated, 0.5 mL of a solution containing the desired antigen in 25% (w / v) BSA / phosphate-buffered saline (PBS) was mixed with an equal volume of 37% formaldehyde in a 1.5 mL microtube (Electron Microscopy Sciences; Hatfield, PA), heated to 85°C for 10 minutes to solidify the solution, and fixed overnight at room temperature. The final concentrations of the reagents were 12.5% ​​(1.8 mM) BSA and 18.5% (6.2 M) formaldehyde. Peptide antigens were fixed at 2.5 × 10 nuclei in the gel. -8 M~2.5×10 -4 The gels containing naive mouse, rat, and rabbit IgG had a final concentration of 0.1 mg / ml (6.7 × 10 -7 The human [His]6-MBP-KSR2 fusion protein had a final concentration of 0.5 mg / ml (6.3 × 10 -6 The final gel concentration was 100%.

[0110] In testing of alternative fixation protocols, formalin-free zinc fixative (catalog no. 552658, BD Pharmingen; San Jose, CA) was used in place of 37% formaldehyde and NBF in the protocol described above. In other experiments, antigen in 25% BSA in PBS was solidified by heating to 85°C for 10 minutes in the absence of formaldehyde. The solidified gel was then transferred to 10% neutral buffered formalin (NBF; VWR International, LLC, Radnor, PA), 4% paraformaldehyde (PFA; VWR International, LLC, Radnor, PA), or zinc fixative and fixed overnight at room temperature.

[0111] Tissue microarray (TMA) construction Tissue microarrays (TMAs) were constructed using a TMA Grand Master tissue microarrayer (3DHISTECH, Budapest, Hungary). Double 1 mm diameter cores were punched from donor paraffin blocks containing the desired protein gels and then transferred to recipient paraffin blocks. The completed recipient TMA blocks were heated at 37°C overnight, then at 70°C for 10 minutes, cooled, and sectioned.

[0112] IHC staining The primary antibodies used were mouse anti-human BCL2 clone 124 (Ventana Medical Systems; Tucson, AZ), rabbit anti-human BCL2 clone EPR17509 (Abcam; Cambridge, MA), rabbit anti-human BCL2 clone SP66 (Ventana Medical Systems, Tucson, AZ), rabbit anti-human BCL2 clone E17 (Abcam, Cambridge, MA), rabbit anti-human MYC clone Y69 (Ventana Medical Systems, Tucson, AZ), and rabbit anti-human MCL1 clone SP143 (Ventana Medical Systems, Tucson, AZ).146 Nd antibody was purchased from Fluidigm (South San Francisco, CA). A panel of 27 mouse hybridoma antibodies against [His]6-MBP-human fusion protein was generated at Chempartner (Shanghai, China). Biotinylated donkey anti-rabbit, rat, and mouse secondary antibodies were purchased from Jackson Laboratories. Details of the staining protocol are summarized in Table 1.

[0113] Table B. IHC Protocol TIFF2026012670000004.tif252170TIFF2026012670000005.tif212170

[0114] Four-micron paraffin sections were deparaffinized and rehydrated in xylene and graded alcohols. Staining was performed using a Ventana Benchmark XT, Ventana Discovery XT, or Ventana Benchmark Ultra XT instrument (Ventana Medical Systems, Tucson, AZ) or a Dako Universal Autostainer (Agilent, Santa Clara, CA). Depending on the optimized antibody protocol, sections were pretreated with Cell Conditioning Solution 1 (CC1) (Ventana Medical Systems, Tucson, AZ) or Target Retrieval Solution, pH 6 (ready-to-use) (Dako - Agilent Technologies, Santa Clara, CA). Slides stained on Ventana instruments were counterstained with Ventana hematoxylin and bluing reagent (Ventana Medical Systems, Tucson, AZ) for 4 minutes each. Slides stained with a Dako Universal autostainer were counterstained with Mayer's hematoxylin (Rowley Biochemical, Danvers, MA) and Richard-Allen Scientific Bluing Reagent (Thermo Fisher Scientific, Waltham, MA) for 1 minute each. Stained sections were dehydrated through graded alcohols to xylene before mounting. Immunofluorescence slides were mounted with Prolong Gold mounting medium (Life Technologies, Carlsbad, CA).

[0115] Fluidigm Staining Procedure Sections were baked at 70°C for 30 min, deparaffinized, rehydrated in descending EtOH serial steps, pretreated with Target Retrieval Solution, pH 6 (Dako - Agilent Technologies, Santa Clara, CA), blocked with 10% donkey serum, 3% BSA in PBS for 30 min, and then in blocking buffer. 146 Incubated with Nd-EPR17509. Slides were incubated overnight at 4°C in a humidified sealed container without mounting, rinsed three times with PBS, post-fixed with 2% glutaraldehyde / PBS for 5 min at 20°C, rinsed with ddH2O, dehydrated in successive EtOH steps, air-dried, and stored at 20°C until imaging.

[0116] Imaging Mass Spectrometry 146 TMA cores stained with Nd-labeled EPR17509 were analyzed on a Fluidigm Hyperion imaging mass spectrometer (South San Francisco, CA) by defining a 150-micron square ablation region of interest (ROI) on each TMA core. The integrated ion counts for each ROI were converted to antibody mass using the antibody standard data below.

[0117] 1, 5, and 10 micrograms / ml 146 A control aliquot of Nd-labeled EPR17509 was prepared in 10% donkey serum in 3% BSA in PBS. One microliter of each antibody sample was spotted onto a glass slide and air-dried before ablation in a Hyperion machine (UV laser intensity = 3). The integrated ion counts for each antibody spot were used to calibrate the ion counts measured in ROIs from the stained TMA core.

[0118] Digital image acquisition and analysis Whole-slide brightfield images were acquired at a scan resolution of 0.46 microns / pixel using a Hamamatsu (Bridgewater, NJ) Nanozoomer-XR digital slide scanner equipped with a 20x 0.75 NA objective. Brightfield imaging was performed in semi-automated batch mode. Scan areas and focus points were manually created for each slide before automated high-resolution whole-slide imaging. Immunofluorescence whole-slide images were acquired using either the Nanozoomer-XR or a 3D Histech Pannoramic 250 scanner (using a 20x 0.8 NA objective with a resolution of 0.33 microns / pixel). With the Nanozoomer-XR system, the fluorescence module's light power was set to 50% and immunofluorescence signals were captured using TRITC (antibody signal at 1x exposure, 3.4 ms photon collection, 1x gain), DAPI (autofluorescence at 2x exposure, 6.8 ms photon collection, 2x gain), and CFP filters (autofluorescence at 4x exposure, 13.6 ms photon collection, 2x gain). Acquisition on the Pannoramic 250 system was performed using CY5 (antibody signal at 10 ms exposure), FITC (antibody signal at 2 ms exposure), DAPI (autofluorescence at 40 ms exposure), and CFP (autofluorescence at 200 ms exposure) filters. Image analysis was performed using Matlab version 9.3. Regions of interest (ROIs) in brightfield images were manually created and edited to exclude areas of gel with artifacts or tears. Small holes or tears within the ROIs were excluded using manual color thresholding. ROIs for immunofluorescence images were either manually created or automatically generated when sufficient signal above the gel background was obtained from autofluorescence images by thresholding (in either the DAPI or CFP channel) and morphological filtering. ROIs were transferred to images acquired in the antibody-fluorochrome channel for intensity measurements. For both brightfield and fluorescent images, the mean grayscale intensity was calculated at 8-bit depth. The plotted Y-axis value of the mean brightfield pixel intensity is 255 minus the mean pixel grayscale intensity.Digital slide scan images are displayed without altering their original intensity or contrast. Quantification of the staining intensity profile of a line drawn across the donor block section was assessed using the Analyze / Plot Profile function in ImageJ (version 1.52a; Wayne Rasband, imagej.nih.gov / ij).

[0119] statistical analysis Graphing was performed in Prism GraphPad (version 7). Signal intensity data, corrected for the background of the glass slide, were plotted as the logarithm of the formulated antigen concentration. 10 The plots were made against the mean intensity of the non-antigen core. Curve fitting used a variable slope four-parameter model constrained so that the bottom of the fitted curve was equal to the mean intensity of the non-antigen core for each assay, and is reported as "bottom" in the table associated with each graph. Other parameters reported in the table [signal maximum, span, antigen concentration at half maximum (ACHM), and HillSlope] were calculated by the software.

[0120] result As demonstrated in Examples 1-3, when incorporated into protein gels, synthetic peptides encoding antibody target epitopes can be detected using standard immunohistochemical and immunofluorescence procedures. Donor blocks containing the target peptides had relatively uniform antigen distribution as assessed by chromogenic assays (Figures 11A-11D). Tissue microarrays (TMAs) can be constructed to contain desired antigens at various antigen concentrations. Figures 12A-12E show TMAs consisting of duplicate cores containing either no peptide, serial dilutions of a negative control peptide derived from the human MCL1 protein, or dilutions of a peptide encoding amino acids 41-54 of the BCL2 protein. Parallel sections of this TMA were stained with four anti-BCL2 antibodies: clone 124, directed against the same peptide sequence used in the target peptide; SP66 and E17, both directed against peptide antigens C-terminal to the sequence in the reagent (Adam, P. et al. (2013) Human Pathology 44:1817-1826; www.abcam.com / bcl2-alpha-antibody-sp66-n-terminal-ab93884.html); and EPR17509, directed against an undisclosed BCL2 peptide antigen (www.abcam.com / BCL2-antibody-epr17509-hrp-ab209039.html). Separate slides were stained using chromogenic (all antibodies) and immunofluorescence (clone 124 only) methods.

[0121] As expected, SP66 and E17 did not detectably react with the core of either section of this TMA (Figures 13A and 13B). For both clone 124 and EPR17509, the signal in the TMA core increased with increasing concentrations of the BCL2 peptide (Figures 12A-12C), consistent with a specific interaction between the antibody and antigen within the core.

[0122] Digital image quantification allowed for a more accurate evaluation of the data (Figure 12D). The fitted curves and associated parameters reported in Figure 12E quantify the minimum and maximum signal intensities, dynamic range, antigen concentration at which the signal is half-maximal (ACHM), and the steepness (HillSlope) of the antigen concentration vs. signal intensity curve in this range. Under the conditions tested here, the nonspecific signal of core without added peptide was 2.7% of the maximum detectable signal in the chromogenic Clone 124 assay, 1000-fold lower in the fluorescent Clone 124 assay, and 23% in the EPR17509 assay. Meanwhile, for EPR17509, the ACHM value, which reflects the relative sensitivity of the assay, was approximately 8-fold lower (i.e., more sensitive) than the ACHM of the chromogenic Clone 124 assay and over 50-fold lower than the fluorescent Clone 124 assay. The immunofluorescent clone 124 assay has a slope 50-75% steeper than either chromogenic assay, reflecting a narrower range of antigen concentrations between the threshold of detection and maximum signal.

[0123] To assess the reproducibility of the peptide control across replicate assays, a second BCL2 peptide TMA was constructed using a new donor paraffin block prepared with the same target BCL2 peptide concentration as the one used to construct the TMA in Figures 12A-12E. Replicate sections from this second TMA were stained by two operators on six separate days, six months apart, using the same anti-BCL2 clone 124 chromogenic IHC protocol used in Figures 12A-12E. Quantitative digital image analysis of the stained sections showed that the signal intensity of each core was highly reproducible (Figures 14A-14C; see also Figures 3B and 3C).

[0124] In a parallel experiment, clone 124 was used to stain TMAs containing BCL2 peptide with or without prior antigen retrieval. Results showed that antigen retrieval improved signal intensity by approximately six-fold but was not required for staining (Figures 15A-15C).

[0125] Example 5: Stability of peptide crosslinks in gel matrices Without wishing to be bound by theory, it is believed that the concentration of peptide available for antibody conjugation is reduced from the formulated value by three parameters: the efficiency of cross-linking the peptide to the protein matrix, the biochemical integrity of the peptide, and the accessibility of the cross-linked peptide to the antibody. Formaldehyde was used to cross-link BSA side chains to the target peptide by reacting with the N-terminal tyrosine and C-terminal cysteine ​​residues contained in the peptide sequence. Of the internal amino acids of the BCL2 peptide (A, F, G, I, P, Q, S), only glutamine has been reported to react with formaldehyde (Metz, B. et al. (2004) J. Biol. Chem. 279:6235-6243).

[0126] To assess the effect of alternative N- and C-terminal amino acids on signal intensity, four variants of the BCL2 peptide were tested. The N-terminal tyrosine was replaced with serine, which is expected to have minimal reactivity with formalin, or with arginine, which has been reported to be 50% more reactive than tyrosine (Metz, B. et al. (2004) J. Biol. Chem. 279:6235-6243). Other variants contained tyrosine or arginine at both the N- and C-termini. TMA cores containing serial dilutions of each peptide were prepared and stained as described above.

[0127] The results showed a significantly higher signal for the peptide with an N-terminal arginine ("RC", Figures 16A-16C). The ACHM of this variant was 4.43 x 10 -7 The M peptide was 6-fold lower than the corresponding value for the original YC variant. The other variants showed a range of intensities, either similar (SC) or weaker (RR, YY) than the original YC variant. In particular, variants containing an N-terminal arginine or tyrosine and a C-terminal cysteine ​​reacted more strongly than peptides containing arginines or tyrosines at both ends.

[0128] Example 6: Generalizability of Synthetic IHC Controls Next, we performed experiments to determine whether peptides containing epitopes from proteins other than BCL2 would react similarly. The anti-MYC antibody Y69 has been reported to bind to an epitope in the N-terminal 100 amino acids of the human MYC protein (www.abcam.com / c-MYC-antibody-y69-ab32072.html). We incorporated candidate epitopes from this region into BSA gels as described above and tested for Y69 binding.

[0129] A peptide containing MYC amino acids 9–24 reacted strongly with the antibody, whereas other peptides reacted only weakly (aa 17–32) or not at all (aa 35–50 and aa 53–68) (Figure 17A). A TMA containing both the MYC aa 9–24 peptide and a BCL2 peptide double core was constructed at the aforementioned concentration range. Chromogenic detection using anti-BCL2 clone 124 and anti-MYC clone Y69 demonstrated a consistent range of signal intensities without cross-reactivity to non-target peptides (Figures 17B and 17C). Quantification of the resulting data (Figure 17D) showed that the MYC protocol had a 4-fold lower ACHM than the BCL2 protocol, with similar dynamic ranges and HillSlope parameters (Figure 17E).

[0130] Dual immunofluorescence detection on the same TMA used in Figures 17A-17E was performed using sequential incubation of both anti-BCL2 and anti-MYC primary antibodies with appropriate detection reagents (Figures 18A-18C). Qualitative results (Figures 18A and 18B) demonstrated the expected specificity, with no cross-reactivity between either antibody and non-target peptides. Isotype controls used in place of antigen-specific primary antibodies yielded no signal. Quantification of the resulting fluorescence data demonstrates increased replicate variability in the HillSlope parameter under the conditions tested compared to the chromogenic protocol.

[0131] These results confirm the broad applicability of peptide antigens as IHC controls using a variety of epitopes and detection protocols.

[0132] Example 7: Limit of Detection (LOD) and Reproducibility The quantitative data obtained allowed us to determine the limit of detection (LOD) and reproducibility of the clone 124 BCL2 IHC assay.

[0133] The experiments shown in Figures 12A, 14A-14C, 15A, and 17B represent 10 independent analyses, each with duplicate TMA cores containing no target peptide (blank) and six concentrations of BCL2 peptide. Figures 19A and 19B and Table C summarize the data. For each experiment, the average of the duplicate TMA cores at each peptide concentration was used for calculations. Values ​​are pixel intensities of the TMA cores corrected for the background intensity of the glass slide (19.2 + / - 0.5 units).

[0134] Table C. BCL2 Clone 124 IHC Assay Summary Data TIFF2026012670000006.tif104170

[0135] According to accepted clinical laboratory regulations (Armbruster and Pry, 2008), the blank limits (LOBs; = average) of these data were ブランク +1.645×SD ブランク ) is 8.0 units, and the limit of detection (LOD; = LOB + 1.645 × SD 低陽性試料 ) is 16 units, which is 2.3 × 10 -7 This concentration corresponds to a loaded antigen density of approximately 140 molecules per cubic micron of gel.

[0136] Consistent with these calculations, two-tailed t-test comparisons of the core data with increasing BCL2 peptide concentrations (Table C) showed a 2.5 × 10 -8 Cores with M peptides (below the calculated LOD) showed no significant difference from cores lacking the peptide, whereas 2.5 × 10 -7Cores with more than M peptides (above the calculated LOD) are statistically distinct neighboring cores. In particular, the signals of the cores containing the two highest peptide concentrations are statistically distinct, despite the fact that the subjective intensities of these cores are similar.

[0137] Example 8: Use of synthetic IHC controls for antibody validation As described in Example 1 above (see Figures 5A-5D), another protein was incorporated into a BSA gel and used to evaluate antibodies generated from hybridoma clones. In this case, a peptide containing the C-terminal 301 amino acids of human KSR2 (i.e., amino acids 650-950) was used. The results above suggest that the synthetic IHC control approach provides a useful method for identifying antibodies suitable for IHC analysis of a target of interest, typically in the process of screening hundreds to thousands of candidate antibodies, to identify a small number (e.g., 2-5) of leads for further characterization.

[0138] Candidate antibodies were further tested for reactivity against the antigen without prior antigen retrieval. For each antibody, antigen retrieval was required for detectable reactivity (Figures 20A and 20B). This contrasts with the results with the BCL2 peptide described in Example 4, where antigen retrieval improved signal intensity by approximately six-fold but was not required for detectable reactivity (Figures 15A-15C).

[0139] As a test of full-length proteins, rabbit, rat, and mouse full-length immunoglobulins (IgG; 0.1 mg / ml; 6.7 × 10 -7M)) was prepared and used as a technical control for an IHC assay using biotinylated donkey anti-rabbit, anti-rat, and anti-mouse secondary antibodies in the detection step. The results demonstrate the expected signal and specificity of the anti-rabbit, anti-mouse, and anti-rat secondary antibodies (Figures 21A and 21B). As shown in Figures 21A and 21B, incorporated rabbit IgG detected with the donkey anti-rabbit IgG secondary antibody exhibits a pixel intensity of 136 units. Assuming quantitative retention of the added rabbit IgG, one cubic micron (10 μm) of this sample -15 L) is 6.7 x 10 -22 moles, or approximately 400 detectable molecules of rabbit IgG.

[0140] Example 9: Testing alternative fixatives to make gels Because some epitopes are rendered unreactive by formalin-containing fixatives, a commercially available formalin-free zinc fixative was tested as an alternative to the 37% formaldehyde used in previous experiments.

[0141] Donor blocks containing BCL2 and MYC peptides in BSA gel were fixed with formalin or zinc-based fixatives while heating to 85°C (Figures 22A-22C). For both BCL2 and MYC peptides, the signal was approximately 10-fold stronger with formaldehyde fixation than with zinc fixation during heating. The loss of signal intensity correlated with heating in the presence of zinc.

[0142] An alternative procedure in which the BSA-antigen mixture was heated to 85°C in the absence of fixative and then fixed at room temperature with various fixatives (4% paraformaldehyde, neutral buffered formalin, and zinc-containing formalin-free fixative) yielded results comparable to the standard protocol described above (Figures 23A and 23B), demonstrating the adaptability of this method to various fixatives and potentially broadening the range of epitopes and antibodies that can be used.

[0143] Example 10: Imaging mass spectrometry In the experiments described above, the number of chromogen or fluorophore molecules deposited per molecule of bound antibody is unknown, and therefore the absolute concentration of the detected epitope cannot be calculated. To circumvent this limitation, a more quantitative direct detection procedure using a Hyperion mass spectrometry-based imager was tested.

[0144] Sections of the BCL2 peptide TMA 146 The TMA samples were stained with Nd-labeled anti-BCL2 antibody EPR17509 and analyzed by ultraviolet laser ablation and quantitative mass spectrometry. Quantitated TMA samples typically comprised 9 x 10 swaths of tissue samples over a 150 micron square x 4 micron thick area. 4 The results showed a graded signal that increased with increasing BCL2 peptide concentration in the target (Figures 24A-24C). The EPR17509 antibody signal in cores without peptide or with the negative control MCL1 peptide was less than 3% of the maximum signal.

[0145] The correlation between the measured ion counts and antibody concentration was determined by measuring the concentration of known amounts of antibody spotted directly on a glass slide. 146 The calibration data obtained (Table D) were determined by analyzing Nd-labeled antibodies. 146 A ratio of approximately 340 antibody molecules per Nd ion was observed.

[0146] Table D. Antibody Ion Quantification TIFF2026012670000007.tif62170

[0147] The amount of BCL2 peptide incorporated into each TMA core sample was measured. 146 The percentage of BCL2 peptide detectable in the TMA core was determined by comparing it with the amount of Nd-labeled anti-BCL2 antibody. The results (Table E) showed that, as expected, the amount of detectable BCL2 peptide increased with increasing peptide concentration.

[0148] Table E. Antibodies binding BCL2 peptide-containing cores TIFF2026012670000008.tif66170

[0149] The results also showed that the percentage of detectable added BCL2 peptide decreased as the antigen concentration increased; 2.5 × 10 -7 At M, 1.4% of the added peptides were detectable, while at 2.5 × 10 -4 In M, approximately 0.14% of the added peptide was detected.

[0150] conclusion Taken together, the results of Examples 1-10 demonstrate that antigen-containing gels can be created using materials and methods available in histology laboratories and can be embedded and sectioned to produce uniformly stained samples. These examples demonstrate that the method is compatible with a variety of fixatives and detection using chromogenic, immunofluorescent, and mass spectrometry-based methods. The choice of possible antigens is limited only by the availability of the target protein or knowledge of the linear epitope sequence. The ability to create synthetic controls of known composition offers the opportunity to more accurately characterize and control routinely used immunohistochemistry protocols, independent of the complicating factors inherent in heterogeneous tissue samples and subjective human interpretation.

[0151] These results provide proof-of-concept detection of linear peptide epitopes from BCL2, MYC, and MCL1 using antibodies specific for each protein, and further demonstrate detection of full-length IgG and the 301 amino acid human protein. The concentration of target epitopes formulated and tested here was 2.5 x 10 -8 M~2.5×10 -4 This density spans four orders of magnitude, extending to the upper end of the range of protein concentrations found in tissues. At the upper end of this range, the average intermolecular distance is less than 20 nM, approaching the distance between the two arms of a full-length IgG molecule (approximately 14 nM). Only highly abundant proteins reach this density. The protein concentrations used in this procedure are in the range found in many tissues (7-25%) (Cole, J. (2017) Scientific Reports 7:44707), and therefore recapitulate some mechanical and biochemical tissue properties.

[0152] These results further demonstrate that quantitative parameters related to in-tissue IHC assay performance—nonspecific background, detection limit, dynamic range, antigen concentration at half-maximum signal, and Hill Slope—can be assessed with objectively definable precision in any laboratory with access to a digital slide scanner and basic image analysis capabilities. The results demonstrate that these parameters can vary under different experimental conditions, such as when using a single antigen-antibody pair, when different antibodies detect the same antigen, and when using different antibody / antigen pairs. For example, the measured ACHM values ​​differed by more than 50-fold in the three assays shown in Figures 12A–12E. In a series of 10 replicate experiments using the clone 124 BCL2 assay, the calculated data parameters of maximum signal, dynamic range, log(ACHM), and Hill Slope had coefficients of variation less than 10%, but could allow for greater precision. In these experiments, the physiological range (2.5 × 10) was used. -8 ~2.5×10 -4 Although we used serial 10-fold dilutions of antigen across the M range, the accuracy of ACHM and gradient quantification could potentially be improved by including more samples across 10%–90% of the assay's dynamic range. This allows for more objective and accurate assessment of assay performance than subjective human assessment of tissue or cell pellets, allowing assay performance to be more precisely tailored to clinical needs and more tightly controlled.

[0153] Synthetic controls with varying antigen concentrations allow for the optimization, quantification, and control of IHC staining protocols using reproducible standards. The concept described here allows for quality control of IHC assays at a level intermediate between the two extremes of evaluating purified antibody-antigen interactions under controlled in vitro conditions and evaluating antibody reactivity in tissue samples through experimental optimization.

[0154] The application of this method to ongoing quantitative immunohistochemical analysis is contemplated. The synthetic antigen gel sample is homogeneous, has a uniform thickness, and contains a known number of epitope molecules, allowing correlation of signal intensity with antigen concentration. TMA sections on slides containing a useful range of epitope concentrations can easily fit adjacent to diagnostic tissue sections, allowing the technical validity of the assay or quantitative image analysis to be assessed on any slide. Because the components and procedures used in this method are fully defined, reagents produced in different laboratories should, in principle, be functionally similar. This approach allows investigators to compare and align protocols used in different laboratories, communicate more clearly when describing qualitative staining endpoints, and ultimately enable more precise management of IHC assays used in both research and patient care decision-making.

[0155] Example 11: Synthetic antigen gel made with polylysine The examples above (see, e.g., Example 3) describe matrix proteins that were tested for their ability to form gels suitable for IHC staining, e.g., by forming a uniform gel that allows for IHC staining and adheres to glass IHC slides. This example describes testing polylysine for antigen / gel matrix formation.

[0156] Polylysine offers multiple formaldehyde-reactive side chains and is commercially available in a variety of molecular weights ranging from 1-4 kDa up to 300 kDa. Because the formula weight of a polylysine monomer is 128 Da (assuming no salt counterions), commercially available polylysine molecules contain polymers ranging from 8-30 lysine monomers to over 2,300 lysine monomers.

[0157] As mentioned above, 12.5% ​​(final concentration) BSA, upon heat denaturation and reaction with formaldehyde, produces a useful matrix capable of embedding and crosslinking target antigens. Assuming a formula weight of 69,293 Da, 12.5% ​​BSA is a 1.8 mM concentration. Each BSA molecule contains 60 lysine side chains. In addition to lysines, each BSA molecule also contains 26 arginine and 21 tyrosine side chains, each of which can react with formaldehyde with some efficiency. Thus, a 12.5% ​​BSA gel contains 60 x 1.8 mM, or 108 mM, lysine side chains.

[0158] Therefore, a sample of polylysine gel matrix based on similar calculations would contain approximately 108 mM lysine side chains in the final gel. Assuming a formula weight of a single lysine side chain per 128 Da polylysine polymer, the final concentration of polylysine in the gel would be approximately 14 mg / mL. If commercially available polylysine contains a counterion salt, the weight per volume will be higher. For example, the formula weight of polylysine*HBr salt is 209 g / mol. The desired final polylysine concentration can be achieved by mixing an equal volume of a 216 mM polylysine solution (approximately 28 mg / mL pure, 45 mg / mL as HBr salt) with a concentrated formaldehyde stock (37%). Without wishing to be bound by theory, it is believed that the lysine side chains in the sample crosslink with each other within and / or between polylysine chains to form a gel.

[0159] Polylysine solutions at the above-mentioned concentration(s) are mixed with 37% formaldehyde (and, optionally, the antigen of interest), heated to 85°C for 10 minutes, and then tested for gel formation and antigen binding as described above. Polylysine of different polymer lengths is tested for gel formation upon formaldehyde treatment. In some embodiments, the polylysine comprises the L-enantiomer, the R-enantiomer, or a mixture of both the L- and R-enantiomers. Polylysine gels containing various concentrations of antigen are compared to negative controls, such as polylysine gels without antigen or polylysine gels containing nonspecific proteins or other antigens, and / or positive controls, such as the gels described in Examples 1-10 (e.g., 12.5% ​​BSA gels containing the same antigen). These gels are assayed for antigen binding, for example, on IHC slides or TMAs assayed by chromogenic or immunofluorescent binding assays as described in Examples 1-10.

[0160] The foregoing disclosure has been described in some detail by way of illustration and example, for purposes of clarity of understanding, but the descriptions and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. 1. A method for producing a solid antigen / carrier protein gel for immunohistochemistry (IHC) staining, comprising: (a) mixing a purified antigen with a liquid solution comprising a carrier protein selected from the group consisting of albumin protein, egg white protein or a mixture of egg white proteins, and gelatin to form an antigen / carrier protein liquid solution; (b) heating the antigen / carrier protein liquid solution to form the solid antigen / carrier protein gel.

2. 10. The method of claim 1, further comprising after (b): dehydrating the solid antigen / carrier protein gel; and embedding the dehydrated solid antigen / carrier protein gel in a paraffin block.

3. 3. The method of claim 2, further comprising, after embedding the dehydrated solid antigen / carrier protein gel in the paraffin block: transferring a core containing the antigen / carrier protein gel from the paraffin block to a recipient tissue microarray (TMA) block.

4. 10. The method of claim 1, further comprising after (b): incubating the solid antigen / carrier protein gel in a liquid embedding medium and freezing the solid antigen / carrier protein gel in the embedding medium.

5. 10. The method of claim 1, further comprising after (b): embedding the solid antigen / carrier protein gel in a plastic resin.

6. 6. The method of any one of claims 1 to 5, further comprising after (b): slicing the solid antigen / carrier protein gel into one or more solid antigen / carrier protein gel sections having a thickness of from about 30 nm to about 50 μm.

7. 7. The method of claim 6, wherein the solid antigen / carrier protein gel is sliced ​​into one or more solid antigen / carrier protein gel sections having a thickness of about 2 μm to about 30 μm.

8. 7. The method of claim 6, wherein the solid antigen / carrier protein gel is sectioned into one or more solid antigen / carrier protein gel slices having a thickness of about 30 nm to about 100 nm.

9. The method of any one of claims 1 to 8, further comprising including a fixative in the antigen / carrier protein liquid solution prior to (b).

10. The method of claim 8 , wherein the fixative comprises formaldehyde.

11. 11. The method of claim 10, wherein the antigen / carrier protein liquid solution comprises formaldehyde at a final concentration of at least about 1%.

12. 9. The method of claim 8, wherein the fixative comprises glutaraldehyde, Davidson's fixative, Bouin's fixative, half strength Karnovski's fixative, or a zinc salt.

13. 13. The method of any one of claims 8 to 12, further comprising subjecting the solid antigen / carrier protein gel to antigen retrieval.

14. 14. The method of claim 13, wherein subjecting the solid antigen / carrier protein gel to antigen retrieval comprises heating the solid antigen / carrier protein gel in a liquid solution.

15. (b) including a fixative in said antigen / carrier protein liquid solution; (b) followed by dehydrating the solid antigen / carrier protein gel; embedding the dehydrated solid antigen / carrier protein gel in a paraffin block; sectioning the paraffin block containing the embedded antigen / carrier protein gel into one or more sections having a thickness of about 30 nm to about 50 μm; subjecting the one or more sections of the embedded solid antigen / carrier protein gel to antigen retrieval; 10. The method of claim 1, further comprising blocking the one or more sections of the embedded solid antigen / carrier protein gel after antigen retrieval.

16. (b) followed by incubating the solid antigen / carrier protein gel in a liquid embedding medium; freezing the solid antigen / carrier protein gel in the embedding medium; slicing the frozen antigen / carrier protein gel into one or more sections having a thickness of about 30 nm to about 50 μm; 10. The method of claim 1, further comprising blocking the one or more sections of the frozen solid antigen / carrier protein gel.

17. The method of any one of claims 1 to 16, wherein the antigen is a polypeptide antigen.

18. 18. The method of claim 17, wherein the antigen comprises an N-terminal tyrosine, a C-terminal cysteine, or both.

19. 20. The method of claim 18, further comprising, prior to (b), cross-linking the antigen to the carrier protein using a cysteine-reactive reagent.

20. The method of any one of claims 1 to 16, wherein the antigen comprises a non-polypeptide antigen.

21. The method of any one of claims 1 to 20, wherein the carrier protein is a serum albumin protein.

22. 22. The method of claim 21, wherein the serum albumin protein is bovine, caprine, equine, or human serum albumin.

23. 21. The method of any one of claims 1 to 20, wherein the carrier protein is an egg white protein or a mixture of egg white proteins.

24. 24. The method of any one of claims 21 to 23, wherein the antigen / carrier protein liquid solution produced in (a) comprises the carrier protein at a concentration of 2% (w / v) or greater.

25. 25. The method of claim 24, wherein the antigen / carrier protein liquid solution produced in (a) comprises a final concentration of the carrier protein of about 25% (w / v) or less.

26. 21. The method of any one of claims 1 to 20, wherein the carrier protein is gelatin, and the method further comprises, after (b), cooling the heated antigen / carrier protein liquid solution to form the solid antigen / carrier protein gel.

27. After the method has cooled the heated antigen / carrier protein liquid solution, incubating the solid antigen / carrier protein gel with a fixative to form a fixed antigen / carrier protein gel; 27. The method of claim 26, further comprising dehydrating the immobilized antigen / carrier protein gel.

28. 28. The method of claim 26 or claim 27, wherein the antigen / carrier protein liquid solution produced in (a) comprises the carrier protein at a concentration of about 0.5% (w / v) or greater.

29. 29. The method of any one of claims 1 to 28, wherein the antigen / carrier protein liquid solution is heated to at least about 65°C in (b).

30. 30. The method of claim 29, wherein the antigen / carrier protein solution in (b) is heated to at least about 65°C for at least 6 minutes.

31. A solid antigen / carrier protein gel produced by the method of any one of claims 1 to 30.

32. 31. A tissue microarray (TMA) comprising at least a first solid antigen / carrier protein gel produced by the method of any one of claims 1 to 30, and a second solid antigen / carrier protein gel produced by the method of any one of claims 1 to 30.

33. 1. A solid antigen / carrier protein gel for immunohistochemistry (IHC) staining, comprising a purified antigen cross-linked to a carrier protein selected from the group consisting of albumin protein, egg white protein or a mixture of egg white proteins, and gelatin.

34. 34. The solid gel of claim 33, wherein the solid gel has a thickness of about 30 nm to about 50 μm.

35. 35. The solid gel of claim 34, wherein the solid gel has a thickness of about 2 μm to about 30 μm.

36. 35. The solid gel of claim 34, wherein the solid gel has a thickness of about 30 nm to about 100 nm.

37. 37. The solid gel of any one of claims 33 to 36, wherein the solid gel is frozen in an embedding medium.

38. 37. The solid gel of any one of claims 33 to 36, wherein the solid gel is embedded in paraffin.

39. 37. The solid gel of any one of claims 33 to 36, wherein the solid gel is embedded in a plastic resin.

40. 40. The solid gel of any one of claims 33 to 39, wherein the solid gel is immobilized on a solid substrate.

41. 41. The solid gel of any one of claims 33 to 40, wherein the solid gel is fixed in a fixing agent.

42. 42. The solid gel of claim 41, wherein the fixative comprises formaldehyde.

43. 43. The solid gel of claim 42, wherein the fixative comprises formaldehyde at a concentration of at least about 1%.

44. 42. The solid gel of claim 41, wherein the fixative comprises glutaraldehyde, Davidson's fixative, Bouin's fixative, half strength Karnovski's fixative, or a zinc salt.

45. 45. The solid gel of any one of claims 33 to 44, wherein the solid gel has been subjected to antigen retrieval.

46. 46. ​​The solid gel of any one of claims 33 to 45, wherein the antigen is a polypeptide antigen.

47. 47. The solid gel of claim 46, wherein the antigen comprises an N-terminal tyrosine, a C-terminal cysteine, or both.

48. 48. A solid gel according to claim 47, wherein the N-terminal tyrosine and / or C-terminal cysteine ​​is cross-linked to the carrier protein.

49. 46. ​​The solid gel of any one of claims 33 to 45, wherein the antigen comprises a non-polypeptide antigen.

50. 50. The solid gel of any one of claims 33 to 49, wherein the carrier protein is a serum albumin protein.

51. 51. The solid gel of claim 50, wherein the serum albumin protein is bovine, caprine, equine, or human serum albumin.

52. 50. The solid gel of any one of claims 33 to 49, wherein the carrier protein is egg white protein or a mixture of egg white proteins.

53. 53. The solid gel of any one of claims 50 to 52, wherein the solid gel comprises the carrier protein at a concentration of 2% or greater.

54. 54. The solid gel of claim 53, wherein the solid gel comprises the carrier protein at a concentration of about 25% or less.

55. 50. The solid gel of any one of claims 33 to 49, wherein the carrier protein is gelatin.

56. 56. The solid gel of claim 55, wherein the solid gel comprises the carrier protein at a concentration of 0.5% or greater.

57. 57. The solid gel of any one of claims 33 to 56, wherein the solid gel comprises the antigen at a concentration of at least about 25 nM.

58. 1. A tissue microarray (TMA) comprising at least first and second solid antigen / carrier protein gels, wherein both the first and second solid antigen / carrier protein gels comprise purified antigens cross-linked to a carrier protein selected from the group consisting of albumin protein, egg white protein or a mixture of egg white proteins, and gelatin.

59. 59. The TMA of claim 58, wherein the first solid antigen / carrier protein gel comprises a first purified antigen and the second solid antigen / carrier protein gel comprises a second purified antigen different from the first purified antigen.

60. 59. The TMA of claim 58, wherein the first solid antigen / carrier protein gel comprises a first purified antigen at a first concentration and the second solid antigen / carrier protein gel comprises the first purified antigen at a second concentration different from the first concentration.

61. 1. A method for immunohistochemical (IHC) staining of an antigen, comprising: providing a solid antigen / carrier protein gel according to any one of claims 33 to 57 or produced by the method of any one of claims 1 to 30, said solid antigen / carrier protein gel containing said antigen; Providing a sample; contacting the solid antigen / carrier protein gel and the sample with a primary antibody that specifically binds to the antigen; after contacting the solid antigen / carrier protein gel and the sample with the primary antibody, contacting the solid antigen / carrier protein gel and the sample with a secondary antibody that specifically binds to the primary antibody, wherein a detectable moiety is conjugated to the secondary antibody; detecting the detectable moiety signal from the solid antigen / carrier protein gel; and detecting a signal of the detectable moiety from the sample, wherein detection of a signal from the sample compared to the signal detected from the solid antigen / carrier protein gel indicates the presence of the antigen in the sample.

62. 62. The method of claim 61, wherein the sample is a tissue sample.

63. 1. A method for immunohistochemical (IHC) staining of a control antigen, comprising: providing first and second solid antigen / carrier protein gels, each of the first and second solid antigen / carrier protein gels according to any one of claims 33 to 57 or produced by the method of any one of claims 1 to 30, wherein the first solid antigen / carrier protein gel comprises the antigen at a first concentration and the second solid antigen / carrier protein gel comprises the antigen at a second concentration higher than the first concentration; contacting the first and second solid antigen / carrier protein gels with a primary antibody that specifically binds to the antigen; after contacting the first and second solid antigen / carrier protein gels with the primary antibody, contacting the first and second solid antigen / carrier protein gels with a secondary antibody that specifically binds to the primary antibody, wherein a detectable moiety is conjugated to the secondary antibody; detecting a first signal of the detectable moiety from the first solid antigen / carrier protein gel; and detecting a second signal of the detectable moiety from the second solid antigen / carrier protein gel, wherein detection of a second signal greater than the first signal indicates control IHC staining of the antigen.

64. 64. The method of claim 63, wherein the first concentration is 0 nM and detection of the absence of a first signal indicates control IHC staining of the antigen.

65. Providing a sample; contacting the sample with the primary antibody; after contacting the sample with the primary antibody, contacting the sample with the secondary antibody; detecting a third signal of the detectable moiety from the sample; 65. The method of claim 63 or claim 64, further comprising comparing the third signal with the first and second signals, wherein the amount of the third signal compared to the amount of the first and second signals indicates the abundance of the antigen in the sample compared to the amount of the antigen in the first and second solid antigen / carrier protein gels.

66. 1. A method for immunohistochemistry (IHC) staining with a control secondary antibody, comprising: providing first and second solid antigen / carrier protein gels, each of said first and second solid antigen / carrier protein gels according to any one of claims 33 to 57 or produced by the method of any one of claims 1 to 30, wherein said first solid antigen / carrier protein gel comprises a first antibody having a first isotype and said second solid antigen / carrier protein gel comprises a second antibody having a second isotype different from said first isotype; contacting the first and second solid antigen / carrier protein gels with a secondary antibody that specifically binds to the first isotype, wherein a detectable moiety is conjugated to the secondary antibody; detecting the detectable moiety signal from the first solid antigen / carrier protein gel; detecting the absence of the signal of the detectable moiety from the second solid antigen / carrier protein gel, wherein detection of the absence of the signal associated with the first solid antigen / carrier protein gel and the signal associated with the second solid antigen / carrier protein gel indicates staining with a control secondary antibody.

67. 67. The method of any one of claims 61 to 66, wherein the detectable moiety comprises an enzyme, and detecting the signal of the detectable moiety comprises exposing the detectable moiety to a chromogenic substrate for the enzyme, and detecting a signal from the chromogenic substrate upon reaction with the enzyme.

68. 67. The method of any one of claims 61 to 66, wherein the detectable moiety comprises a fluorophore, a metal particle, a metal ion, a radioisotope, a nucleic acid, an electrochemiluminescent reporter, or a quantum dot.

69. 1. A kit for immunohistochemistry (IHC) staining of a control antigen, comprising: (a) a first solid gel comprising a purified antigen cross-linked to a carrier protein selected from the group consisting of albumin protein, egg white protein or a mixture of egg white proteins, and gelatin, wherein the purified antigen is present in the first solid gel at a first concentration; (b) a second solid gel comprising the purified antigen cross-linked to the carrier protein, wherein the purified antigen is present in the second solid gel at a second concentration different from the first concentration.

70. 70. The kit of claim 69, wherein the first and second solid gels are each sliced ​​to a thickness of about 30 nm to about 50 μm.

71. 71. The kit of claim 69 or claim 70, wherein the first and second solid gels are immobilized on one or more solid substrates.

72. 72. The kit of any one of claims 69-71, further comprising (c) a third solid gel comprising the carrier protein, the third solid gel being free of the purified antigen.

73. 73. The kit of claim 72, wherein the third solid gel is sliced ​​to a thickness of about 30 nm to about 50 μm.

74. 74. The kit of claim 72 or claim 73, wherein the third solid gel is immobilized on a solid substrate.

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