Streamlined methods and compositions for rapid and sensitive molecular target detection in biological and non-biological specimens

Abstract

The present invention relates to the methods with compositions for detecting and visualizing molecular targets, such as biological or chemical molecules, proteins, or molecular structures in biological and non-biological specimens. That enables mono- or multi-labeling of one specimen and then visualization of the existence of various molecular targets in the same specimen simultaneously in a faster and cost-effective way while preserving the content and architecture of the specimen. The invention also provides methodologies that contain fewer steps using one composition or a kit described here, excluding the use of other solutions and / or steps used in conventional methods including antigen retrieving, clearing, and harvesting.

Description

[0001] STREAMLINED METHODS AND COMPOSITIONS FOR RAPID AND SENSITIVE MOLECULAR TARGET DETECTION IN BIOLOGICAL AND NON- BIOLOGICAL SPECIMENS

[0002] Technical Field

[0003] The present invention relates to methods and compositions for simultaneously identifying one or more molecular targets in various samples, including living organisms, biological specimens, cells, cell lines, spheroids, organoids, pathological specimens, and other non-biological specimens, in a fast and straightforward manner while preserving the specimen integrity and eliminating the need for some of separate additional treatments in conventional approaches, such as harvesting, antigen retrieval, permeabilizing, blocking, and clearing.

[0004] Prior Art

[0005] Methods for detecting various antigens are commonly used for visualization, localization, and / or quantification of target molecules. The target may be a peptide, polynucleotide sequence, gene, gene mutation, a genetic expression pattern, protein, antigen, or other substance, a particle, or debris of organic origin. There are various methods for detection of the desired targets, like but not limited to immunohistochemistry, immunocytochemistry, immunofluorescence microscopy, southern blot, northern blot, western blot, flow cytometry, and ELISA. Typically, those methods involve incubating a specimen containing the detectable target with a probe and then detecting the binding agent and target with a detectable label that may give off a color, a fluorescent signal, or radioactivity, for example. The target might be detected in situ or in isolation or through laboratory manipulation. One or many binding agent molecules may bind to each target, depending upon the specifics of the methodology used. The method of detecting a primary antibody that is bound to the antigen of interest in an experiment can be either direct or indirect. In some cases, the indirect method is preferred. For example, when the target is present in a low concentration, it is necessary to amplify the signal from the target-binding agent complex by adding one or more amplification layers to the system. If the binding agent is a primary antibody that recognizes the target, a secondary antibody that recognizes the primary antibody may be added. Such that many secondary antibodies bind to each primary antibody. In some cases, amplification methods are preferred. The secondary antibodies are attached to a detectable label such as a fluorophore or chromophore. Each target molecule may effectively be bound to multiple fluorophores or chromophores instead of only a few fluorophores or chromophores. Hence, the target will produce a stronger detection signal after amplification.

[0006] There are commercially available solutions enhancing immunodetection. Antibody dilution buffers, permeabilization agents, blocking buffers, clearing agents, and antibody enhancing solutions are developed to improve antigen detection success. Permeabilizing agents help reach out and detect the antigens located inside the cells. Blocking buffers have been created for blocking nonspecific signals. Dimethyl sulfoxide, urea and its derivatives, Focus Clear solution, and some recent more complex solutions are used for clearing the specimens, in other words, making the specimens transparent. A non-ionic synthetic polymer of sucrose, Ficoll, is used to reduce the non-specific binding of labeled probes to the membranes. All those approaches includes seperate steps and various solutions for each steps to be able to successfully visualize the target.

[0007] In addition to those, many protocols include another step called as antigen retrieval, to get or improve the labeling when necessary for the experiment. Heat- mediated epitope retrieval, citrate-solutions and proteolytic-induced epitope retrieval are schemes to achieve the goals related to antigen retrieval. That step may cause damage or loss of the samples, especially during the immunostaining of paraffin sections.

[0008] In conclusion, target detection methodologies currently suffer from long and complex workflows and compromised accuracy. Researchers need to utilize separate steps, like harvesting, antigen retrieval, blocking, permeabilizing, clearing etc., each requiring dedicated treatments and chemical agents. Each additional step increases the risk of target dissociation, leading to less accurate results. Additionally, using different solutions for primary and secondary antibodies adds unnecessary complexity, increasing the potential for errors. As a result, the current methodologies hinder scientific progress and limit the reliability of target detection data.

[0009] Some detection experiments tend to produce relatively diffuse-looking signals, especially if the specimen is allowed to rest for a period of time before analysis. For example, the one or more binding agents and / or detectable labels bound to a target may slowly diffuse away from the target or away from each other over time. In some cases, buffer changes that affect the binding affinity of the target, binding agent, and amplification layers can cause signal diffusion. For example, buffer changes after labeling may reduce the affinity between the target, binding agent, and detectable label, causing the various components to dissociate. In addition, simple diffusion over a period of time, such as several days, may also cause dissociation between the target, binding agent, and detectable label, rendering the signal diffuse.

[0010] Damage and loss of sensitive specimens with the complex and long processes cause data loss in addition to time and cost consumption when using current methods, kits and solutions.

[0011] Another problem associated with currently available detection procedures is that they are time-consuming. It usually takes 3-4 hours at minimum to process a specimen, from the step of labeling targets to the detection of the label. If the experiments contain the detection of more than one antigen, this duration extends to at least two days. This duration may extend to 1 week or longer for special specimens like organoids, spheroids, thick specimens, or zebrafish. Recently developed methodologies like the CARD method as described in United States patent application numbered US6593100 and another HRP-based amplification method allowing detection low abundance target molecules in IHC specimens (W002009036760) aimed to enhance antigenicity to get more accurate results. They both provide robust amplification, and the speed of the detection procedure is much faster than either traditional procedure.

[0012] The invention described in the patent numbered as WO 2004.077057 A1 aimed to bring standardization to the conventional immunohistochemistry, immuocytochemistry, and molecular cytogenetics by methods comprising standard protocols, a support medium for quantification and presence of a detectable entity in a sample. That is another approach, including harmful steps of other typical approaches and bringing solutions by getting sections from the specimens and staining.

[0013] Current technological developments do not fully solve the problems despite having a relatively good sensitivity to detecting target molecules in specimens. That burdens the method in cases of specimens improperly proceeding before the labeling or those with a low target expression level. Errors due to handling specimens through long and complex processes also increase.

[0014] Most of the available developing methodologies are still time-consuming and complicated with long steps. They are limited as they are mainly capable of detecting one target molecule at a time, allowing for labeling the specimen with primary and secondary antibodies. Researchers are often interested in more than one target molecule within a particular specimen. The user should often use primary and secondary antibodies sequentially if another target molecule is detected. Multiplexing, another recently developed technology, enables the simultaneous evaluation of several targets by using special equipment. The complexity, limited use of specific antibodies, and high-cost limit of multiplexing experiments in many laboratories make it challenging to use this high-tech methodology. Thus, it would be advantageous to have a method for simultaneously detecting multiple antigens within a specimen cost-effectively and simply.

[0015] Current target detecting methods for 3D structures, e.g. organoids, spheroids, living organisms, pose a significant challenge for researchers studying complex structures in microenvironments, like gels. Traditional techniques, often requiring 3 days to a week, necessitate harvesting these delicate specimens before staining. This harvesting process, however, disrupts the 3D architecture ~ spheroids lose their intricate structure, and organoids experience up to 30% loss. Embedding them in paraffin or resin blocks while allowing for sectioning and antibody staining is another common method for immunodetecting antigens. The harsh chemical treatments during that method destroy the molecules researchers aim to study and cause loss of antigenicity.

[0016] This prevents reaching crucial details within the 3D structure, leading to inaccurate results. The complex and lengthy steps associated with these methods significantly damage the delicate specimens. This translates to data loss, wasted time, and increased costs.

[0017] Our previous invention, which focused on immunofluorescence labeling and immunostaining of organoids and spheroids, was a significant step forward. However, it was limited to specific applications, labeling of organoids and spheroids in specific hydrogels; and was not suitable for other immune detection and visualization technologies. We also recognized the need to expand our method to identify targets in specimens within other environments, such as agarose, alginate capsule, scaffold, tissue engineering materials, extracellular matrix, paraffin, resin, and the like. Additionally, we addressed the limitation of the invention when the hydrogel's thickness increases, which had previously hindered our success.

[0018] In summary, the low success rate to preserve the delicate architecture of specimens during current complex and lengthy target-detecting methods lead to inaccurate and irreproducible data. This challenge hampers progress in the field. There is a longstanding, unfulfilled need to develop new target detection technologies that could maximize accuracy, eliminate the damage to the architecture of the specimen, minimize handling risks of precious specimens, provide large volume and high-quality data, and simplify the methods with easy and cost-effective approaches.

[0019] Brief Description of the Invention

[0020] The invention aims to bring a methodology combined with a composition for the negative aspects for rapid and sensitive molecular target detection stated above, constructed with the inspiration from the current state of art.

[0021] To solve the current problems in the art, the inventors of the present invention conducted diligent studies to reach new findings. They discovered that a combination of compositions and methodologies can quickly detect one or more targets simply, cost-effectively, and efficiently in various target detecting methods. Based on this thorough research, the present inventors arrived at the present invention, ensuring its reliability and effectiveness.

[0022] The main aim of the invention is to provide methods and compositions that preserve the architecture of the specimen while improving simultaneous detection of molecular targets and allow faster, more sensitive, and precise detection of molecular targets. The composition with the method help antibodies penetrates the specimen while blocking non-specific signals, creating sharp and bright images at any dept of the specimen. In addition, it clears the specimen with minimal change to the morphology or detection sensitivity with almost any fluorophore. Furthermore, it enhances signals, prevents background staining, minimizes signal and specimen loss, provides information for spatial biology. With that invention, separate steps harmful for specimens in conventional methods like harvesting, clearing, and antigen retrieval treatments are eliminated. The results with that methods provide large-volume and high-quality data when compared the traditional approches since the invention provides target detection of the entire specimen while still in its ideal environment, preserve the specimen and the information in 3D architecture while eliminating harmful steps in conventional methods.

[0023] The goals for the current invention are as follows:

[0024] • Protecting the integrity of specimens throughout the entire target detection process, delivering data while the specimens remain in their ideal natural state

[0025] • Creating a methodology combined with a composition that allows target detection in a specimen that is still in its supporting environment like hydrogel, agarose, alginate capsule, scaffold, tissue engineering materials, extracellular matrix, paraffin, resin, and the like

[0026] • Eliminating the need for multiple buffer changes to avoid dissociation between target, binding agent, and detectable label, rendering the signal diffuse, to prevent signal loss.

[0027] • Omitting separate current antigen retrievement, permeabilization, cleaning, and blocking steps and methods to decrease time and steps

[0028] • Optimization of immunolabeling and enhancing detection sensitivity

[0029] • Avoiding the use of the current clearing methodologies

[0030] • Avoiding the use of the current specimen harvesting methodologies

[0031] • Minimizing the number of specimen transferring, centrifuging, and pipetting

[0032] • Creating a composition combined with a methodology allows simultaneously labeling the specimens with multiple binding agents / antibodies

[0033] • Eliminating the need for multiple reagents, solutions, and kits during target detection

[0034] • Simplifying the current methodologies by creating single compositions or kits that can be used for the most of the steps including diluting the binding antigens / antibodies and washing, and the like.

[0035] In the invention, the methods with the compositions allow target detection within the specimen’s native environment, preserving its ideal physiological state. The invention ensures precise detection of multiple molecular targets simultaneously, with minimal impact on morphology. This translates to faster analysis throughput and superior data quality. In particular, the methods in the invention eliminate harvesting and the antigen retrieval steps in conventional methods. The methodology with the compositions brings the opportunity to omit the antigen retrieval steps in conventional methods. Additionally, the invention eliminates additional steps like antigen blocking and clearing in current technologies that may cause dissociation of antigens. The invention minimizes other handling steps like transferring, pipetting, centrifuging while eliminating those separate steps.

[0036] The method with the composition allows a faster, more sensitive, and precise detection of molecular targets simultaneously with no background staining. It helps antibodies penetrate the specimen while blocking non-specific signals, enabling sharp and bright images at any part of the specimen. It delivers ultrasharp resolution and allows users to peer deep into specimens in the intact three dimensional architecture of the specimen. At the same time, it minimizes shrinking, photobleaching, phototoxicity, and other potentially harmful outcomes that can irrevocably damage a specimen. Those cost-effective, time-saving, and simple methodologies associated with compositions maximize analysis throughput.

[0037] Those unexpected results with current approaches can be provided with the invention described here with the combinations of novel methods and compositions.

[0038] In one aspect, it makes possible to detect the target in a specimen that is still in its growing environment which might be any type of gel, agarose, alginate capsule, scaffold, tissue engineering materials, extracellular matrix, paraffin, resin, etc.

[0039] In one aspect, the present invention relates to a method for detecting targets, such as molecular targets, in various specimens supposedly comprising thereof. In particular, the invention relates to a method of detecting a target in a specimen comprising,

[0040] • Incubating a target or a specimen comprising thereof with one or more binding agents is / are capable of direct or indirect binding to the target within the described composition.

[0041] • Washing the specimen with the described composition.

[0042] In one embodiment, a target that may be detected by the method is a polypeptide, nucleic acid, carbohydrate, lipid or a derivative thereof, molecular complex, particle, eukaryotic or prokaryotic cell, or microorganism.

[0043] In one embodiment, a specimen may be a biological specimen, environmental specimen, or any other non-biological specimen.

[0044] In one embodiment, a target or specimen comprising thereof may be immobilized onto a solid support.

[0045] Provided herein are compositions and methods can be used for rapid and specific detection of various antigens in a specimen while still in its natural environment that brings the advantegous of protecting the whole architecture , providing three dimensional data and bringing accuracy to the information related to spatial biology.

[0046] The method in one embodiment is for immunohistochemical detection of a target. The method in another embodiment is for immunofluorescence detection of a target. In one embodiment, it may comprise a step of an enzyme-linked immunodetection. In another embodiment, it is the method used in flow cytometry experiments.

[0047] The method may be performed manually, semi-, or full-automatically. The method of the invention can be successfully plasticized with a number of different reporter molecules described herein, maintaining its advantageous features such as speed of detection, sensitivity, and specificity.

[0048] The structural and characteristic features and all advantages of the invention outlined in the the figures demonstrating results from the examples of some methods and compositions below and in the detailed description made by referring these figures will be understood clearly, therefore the evaluation might be made by taking these figures and detailed explanation into consideration. It should be noted that there might be loss of some details due to conversion of colorful images to black-white images.

[0049] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

[0050] Brief Description of Figures

[0051] Figure 1 , related to an example of the present invention, shows paraffin embedded sections treated with the composition according to the present methodology for immunohistochemistry of paraffin sections 1 a: EMA in tonsil from a patient, 1 b, CD23 in tonsil from a patient, 1c: CK19 in liver from a patient, 1d BCL2 in an adenoid from a patient.

[0052] Figure 2, related to still further another example of the present invention, shows liver cancer cells treated with the composition according to the present methodology for immunofluorescence to identify DNA in the nuclei and an one target, E-cadherin protein in the specimen.

[0053] Figure 3, related to still further another example of the present invention, shows liver cancer cells treated with the composition according to the present methodology for immunofluorescence to identify DNA in the nuclei and two targets, histone in the nuclei and Na-KATPase in the membrane of the specimen. Figure 4, related to still further another example of the present invention, shows a 3D growing liver cancer spheroid in an alginate capsule treated with the composition according to the present methodology for immunofluorescence microscopy to identify DNA in the nuclei and three targets, histone, betagalactosidase in the cytoplasm, and ZO-1 in the membrane of the specimen.

[0054] Figure 5, related to still further another example of the present invention, shows two lung organoids treated with the composition according to the present methodology for immunofluorescence to identify DNA in the nuclei, and two targets, albumin in the cytoplasm and Na-K ATP ase in the membrane of the specimen.

[0055] Figure 6, demonstrates the superior target detection accuracy of the invention compared to the traditional method. PDPN was visualized in paraffin embedded brain sections comparatively.

[0056] Detailed Description of the Invention

[0057] In this section, the preferred embodiment of the invention is clarified such that there is no limiting effect for the sake of better understanding the subject.

[0058] The invention encompasses the method and the compositions useful for detection of a wide variety of targets by using different modified or unmodified methodologies such as immunocytochemistry, immunohistochemistry, immunofluorescence microscopy, immunoelectron microscopy, western blot, flow cytometry, ELISA, etc. The main advantage of the methods and the compositions is that they bring quick, simple, efficient, practical and cost-effective solutions to the current technologies.

[0059] The invention is a method for identifying molecular targets, comprising: a) contacting a specimen with a multifunctional composition comprising:

[0060] (i) one or more reagents to penetrate the binding agents,

[0061] (ii) one or more reagents to prevent binding to non-specific sites, (iii) one or more reagents to minimize the refractive index differences between the components,

[0062] (iv) one or more reagents to reduce background or autofluorescence, and

[0063] (v) one or more diluting reagent ; b) incubating the specimen with the multifunctional composition, and c) detecting the target binding agents associated with the one or more molecular targets within the specimen; wherein the multifunctional composition:

[0064] (i) streamlines workflows by allowing all steps (permeabilization, blocking, washing, and detection) to be performed using a single solution,

[0065] (ii) eliminates the need for separate steps for permeabilization of the specimen and blocking, and

[0066] (iii) enhances accuracy for rapid and sensitive molecular target detection.

[0067] The method with the compositions eliminate the need for one or more of the seperate steps in traditional workflows, but not limited to:

[0068] • harvesting,

[0069] • antigen retrieval,

[0070] • permeabilization

[0071] • blocking,

[0072] • clearing.

[0073] Methodologies described in the invention reduces transferring, pipetting, centrifuging steps, and minimize damaging the specimen during the entire process. In some methodologies described here, the invention allows detection in a specimen while the specimen is still located in its natural or supporting environment like hydrogels, agarose, scaffolds, tissue engineering materials, paraffin, resin, etc.

[0074] With those advantages, the methods and compositions in this invention allow for minimizing damage to the specimens, decreasing working time, increasing assay sensitivity, and ultimately leading to more reliable and accurate molecular target identification.

[0075] The invention is a method for identifying molecular targets with a composition suitable for the detection of molecular targets comprising the following;

[0076] • one or more reagents to penetrate the binding agents,

[0077] • one or more reagents to prevent binding to non-specific sites,

[0078] • one or more reagents to minimize the refractive index differences between the components,

[0079] • one or more reagents to reduce background and autofluorescence,

[0080] • one or more diluting reagents.

[0081] The compositions of the compunds may include various combinations of the following active agents according to its purpose of use, but are not limited to

[0082] • Reagent to penetrate the binding agents into the specimen (e.g., Triton X (Registered Trademark) series such as Triton X - 100 and Triton X - 140; Tween (Registered Trademark) series such as Tween - 20, Tween - 40, Tween - 60, and Tween - 80, surfactant, methanol, acetone, saponin, and NP - 40 (product name))

[0083] • Reagent to prevent non-specific binding in the specimen (e.g., Bovine Serum Albumin, Goat Serum, Donkey serum, Tween 20, Nonfat dry milk, BLOTTO, blotting grade blocker, gelatin, casein, dimethysulfoxide, hydrogen peroxide, Ficoll PM70 (product name), Polyvinyl pyrrolidone, Tris buffered saline, and Percoll (product name)

[0084] • Reagent to minimize the refractive index differences between tissue components (e.g., Glycine, Glycerol, Heparin, Benzyl alcohol, Benzyl benzoate, Dichloromethane, Dibenzylther, Sucrose, Diatrizoic acid, Formamide, Fructose, Chloroform, Xylene, Toluene, Thioglycerol, Urea and Urea derivatives, 2,2-thiodiethanol, Dimethyl sulfoxide)

[0085] • Reagent to reduce background and autofluorescence (e.g., Tween 20, SDS, Nonidet P-40 (NP-40), sodium borohydride, Thiocarbohydrazide, Sudan Black B, Ammonium ethanol, Hydrogen Peroxide, Methanol) • Reagent to dilute the composition (e.g., Water, Distilled water, Double distilled water, Phosphate Buffered Saline, Dulbecco’s Phosphate Buffered Saline, Ca and Mg free Dulbecco’s Phosphate Buffered Saline, Glycerol, Cell culture media, Tris-buffered saline, cell culture supplements, dilution solutions with gels, any buffer that resist changes in pH

[0086] The compositions of the compounds may include various combinations of the following accessory agents according to its purpose of use, but are not limited to

[0087] • Drying inhibition reagent (e.g., glycerol, carboxy vinyl polymer, hydroxypropyl methylcellulose, propylene glycol, and macrogol)

[0088] • Antimicrobial reagent (e.g., Ampicillin, Amphotericin, Kanamycin sulfate, Neomycin sulfate, Na-Azide, Penicillin, Glutamine, Polymixin B, Streptomycin, Gentamicin sulfate)

[0089] • Antifungal reagent (e.g., the polyene macrolides (e.g. amphotericin B), the azoles (e.g. the imidazoles ketoconazole and miconazole and the triazoles itraconazole and fluconazole) and the allylamines (e.g. terbinafine))

[0090] • Water-soluble macromolecular compound (e.g., polyethylene glycol, polyvinyl pyrolidone, sucrose, Ficoll PM70 (product name) and Percoll (product name)

[0091] • Epitope-unmasking reagent (e.g., Trypsin, Pepsin, Hyaluronidase,

[0092] Proteinase-K, Hydrogen peroxidase, Formic acid, Urea, Sodium Citrate, EDTA, Tris-EDTA)

[0093] • Immersion medium to provide required refractive index (e.g., glycerin, mineral oil, neatsfoot oil, olive oil, palm oil)

[0094] • Cell separating and organelle isolation reagent (e.g., non-ionic synthetic polymer of sucrose-Ficoll, Percoll (product name)

[0095] • Reagent to dissolve the surrounding habitat like scaffold, hydrogel, paraffin, resin, tisue engineering material, etc (e.g. xylene, toluene, isopropyl alcohol, acidified chloral hydrate glycerol solution, chloroform, methyl benzoat, methyl salicylat, citrus fruit oils, mineral spirits, naphtha, propylene oxide, turpentin, trypsine, and any other digestive enzyme)

[0096] Accessory agents may be added to the invention to add value for examinations of specific specimens; such as prevention of drying, prevention of contamination, preservation of composition, seperation of specific components in the specimen, and dissolving the surrounding habitat without effecting 3D architecture of the entire specimen.

[0097] The composition may comprise one or more active agents from each subgroup listed above and optional one or more accessory agents, or some combination of these.

[0098] The invention is a method for identifying molecular targets with a composition suitable for the detection of molecular targets comprising;

[0099] • Incubating a target or a specimen comprising thereof with one or more binding agents is / are capable of direct or indirect binding to the target within the described composition.

[0100] • Washing the target or the specimen with the described composition.

[0101] In some embodiments, the method of the invention further comprises inhibition of drying.

[0102] In some embodiments, the method of the invention further comprises changing refractive index of the medium.

[0103] In some embodiments, the method of the invention further comprises one or more treatment of the specimen with a reagent to dissolve the surrounding habitat or treating with a chemical from a group involving xylol, thylenol, acetone, trypsine, and / or a digestive enzyme suitable for the surrounding habitat In some embodiments, the method of the invention further comprises one or more treatment of the specimen from the group comprising heating, cooling, trimming, and the like.

[0104] In some embodiments, the method of the invention further eliminates the need for separate steps for antigen retrieval treatment

[0105] In some embodiments, the method of the invention further eliminates the need for separate steps for clearing the specimen

[0106] For example, in some of the compositions, chemical agents may be added to enable a binding agent to access the target molecule in the specimen. Drying inhibition agents are used when more extended-term stability of the specimen is needed for future investigations. Immersion mediums are used to increase the resolving power of a microscope. That is essential for some microscopy techniques like light sheet microscopy. Sodium borohydride can be used to decrease formalin-induced autofluorescence. Polyethylene glycol prevents crystal formation in emulsions. PVP is a stabilizing agent, possesses disinfectant and lubricant properties, and is used in various compositions. Sucrose is used as a medium for the sedimentation of particles in the specimen or cellular organelles. It also prevents ice crystal formation in thick samples and decreases cell deformation. Sodium borohydride neutralizes Schiff bases by reducing aminealdehyde compounds into non-fluorescent salts. Ficoll and Percoll are used to separate a sample (e.g., blood, semen) into its components (e.g., blood cells, sperm). Heating or cooling is used to facilitate the process in some embodiments.

[0107] In some embodiments, reagents to penetrate the binding agents into the specimen are used at a concentration between about 0,01 % (v / v) to about 50%(v / v).

[0108] In some embodiments, reagents to prevent non-specific binding in the specimen are used at a concentration between about 0,01 % (w / v) to about 50%(w / v). In some embodiments, reagents to minimize the refractive index differences between tissue components are used at a concentration between about 0,01 % (w / v) to about 50%(w / v).

[0109] In some embodiments, reagents to prevent binding to non-specific sites are used at a concentration between about 0,01 % (v / v) to about 50%(v / v).

[0110] In some embodiments, background and autofluorescence reducing reagents are used at a concentration between about 0,01 % (v / v) to about 10%(v / v).

[0111] In some embodiments, epitope-unmasking reagents are used at a concentration between about 0,01 % (w / v) to about 30%(w / v).

[0112] In some embodiments, drying inhibition reagents are used at a concentration between about 1 % (v / v) to about 50%(v / v).

[0113] In some embodiments, antimicrobial reagents are used at a concentration between about 0,1 % (v / v) to about 10%(v / v).

[0114] In some embodiments, antifungal reagents are used at a concentration between about 0,1 % (v / v) to about 10%(v / v).

[0115] In some embodiments, water-soluble macromolecular compounds are used at a concentration between about 0,1 % (v / v) to about 30%(v / v).

[0116] In some embodiments, immersion medium is used at a concentration between about 0,01 % (v / v) to about 30%(v / v).

[0117] In some embodiments, one of cell separating and organelle isolation reagents is used at a concentration between about 1 % (v / v) to about 80%(v / v). As used herein, the term "about" or "approximately" means that the value presented can be varied by + / -10%. The value can also be read as the exact value and so the term "about" can be omitted. For example, the term "about 100" encompasses 90-110 and also 100.

[0118] For example, in a preferred embodiment, the composition comprises bovine serum albumin (e.g. about 10% -w / v), Heparin (e.g. about 10% -w / v), Non fat dry milk (e.g. about 10%-v / v), Triton-X (e.g. about 10%-v / v), Na-Azide (e.g. about 5%) and propylene glycol (e.g. about 5%) in a diluting reagent.

[0119] In another preferred embodiment, the composition comprises goat serum albumin (e.g. about 10%-w / v), gelatin (e.g. about 10%-w / v), Na-citrate (e.g. about 10% -w / v), Tween-20 (e.g. about 10% -v / v), Tween-80 (e.g. about 10% -v / v), Penicillin (e.g. about 1 %) and propylene glycol (e.g. about 5%) in a diluting reagent.

[0120] In another preferred embodiment further comprises optionally percoll (e.g., about 40%)

[0121] The composition is in a form suitable for a detection methodology to a specimen. The reagents may be present in the composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0122] The relative amounts of the reagents in a composition of the invention will vary, depending upon the methodology, specimen type, specimen size, and condition of the specimen treated and further depending upon the route by which the composition is to be administered. For example, when the size of the specimen increases, like sections from vibratome, the percentage and the number of the reagents for penetration in the composition increase. The compounds in the composition increase comparatively if the cells are treated and embedded in paraffin. The compositions for the methodology in immunofluorescence microscopy and immunohistochemistry may differ. As used herein, for examination under a light-sheet microscope after labeling, a compound that contains a refractive index providing immersion medium might be added. Durability of a composition comprising the active agents, for example, maybe extended by adding an accessory antifungal and / or antimicrobial agents, or drying inhibition agent. Similarly, sodium azide inhibits horseradish peroxide and a different biocide, like gentamicin sulfate, might be preferred in another combination. The composition or kit according to the present invention may contain an autofluorescence reductant agent, if necessary. As the surfactant, a mixture of two or more kinds may be used, if necessary.

[0123] The composition according to the present invention may further contain a water- soluble macromolecular compound, which does not substantially intrude into cells and / or does not cause denaturation, if necessary.

[0124] Since undistilled water may cause inactivation of the specific enzymes in some embodiments, double distilled water may be preferred for diluting. The agents above may be diluted in other diluting agents, like culture media, glycerol, etc.

[0125] The composition may vary depending on the assay and antibody type since some components like detergents may affect the binding of some antibodies. For example, for investigation of the proteins located in the cell membrane, permeabilization agents might be used in a low concentration.

[0126] The compositions that are useful in the methods of the invention may in the form of a single solution, single powder or a kit including components in solution or powder form.

[0127] The compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for any methodology described herein. Other compositions suitable may include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, an emulsion, or a kit.

[0128] Compositions in this invention may include, but are not limited to, inert diluting agents, granulating and disintegrating agents, binding agents, and lubricating agents.

[0129] The compositions with the methods may include, but are not limited to, reagents that might provide long term storage of primary or secondary antibodies for further uses.

[0130] According to the immune detection methodology, the specimen, the purpose and the targets; the invention might be prepared as only one composition or a kit. For example, it might be a kit according to the present invention containing several compositions for immunofluorescence microscopy:

[0131] 1 . A composition for target detection while diluting the primary and secondary binding agents and reducing the background and autoflurosence.

[0132] 2. A composition to provide specific refractive index to visualize the specimen under light-sheet microscope.

[0133] The composition can be used for immune detection and visualization technologies including the followings but not limited to:

[0134] • Immunohistochemistry

[0135] • Immunocytochemistry

[0136] • Fluoroimmunoassay (FIA) for immunofluorescence microscopy and others

[0137] • Western Blot

[0138] • Southern Blot

[0139] • Northern Blot

[0140] • Flow cytometry

[0141] • FACES

[0142] • Enzyme Immunoassays (EIA) or Enzyme-Linked Immunosorbent Assay (ELISA) • Insitu Hybridization (ISH)

[0143] • Immunoprecipitation (IP)

[0144] • Immunochromatographic assays

[0145] • Immunogold labeling assays for electron microscopy and others

[0146] • High-performance liquid chromatography (HPLC)

[0147] • Immunomagnetic separation and Electrochemiluminescence (ECL)

[0148] • TRF (Time-resolved fluorescence)

[0149] • Lateral flow immunochromatographic assay (handheld assay-HHA)

[0150] • Light shield microscopy

[0151] • Superresolution microscopy

[0152] • Correlative light electron microscopy

[0153] • Radioimmunoassay (RIA)

[0154] • Mass Cytometry (CyTOF)

[0155] • Flow cytometry

[0156] • Counting Immunoassay (CIA)

[0157] • Chemiluminescenceimmunoassay(CLIA)

[0158] • Proximity Ligation Assay (PLA)

[0159] Simple workflow is easily adapted to different techniques mentioned above, but not limited to.

[0160] The invention comprise not only a method that is designed according to the nature of specimen but also a composition as described above, the antigen(s) needs to be detected and the composition that is created for a specific visualization technology.

[0161] The composition and the methodology provide detection of cellular and microbial antigens in the following types of specimens but not limited to:

[0162] Cells

[0163] Cell debris

[0164] 2D growing cells and cell lines • Vibratome sections of fresh or fixed specimens

[0165] • Compression sections of fresh or fixed specimens

[0166] • Thick sections for light shield microscopy

[0167] • Frozen sections

[0168] • Paraffin -embedded specimens and sections

[0169] • Agarose-embedded specimens and sections

[0170] • 3D growing specimens like organoids, spheroids, embryos, oocytes, cells, etc

[0171] • Living organisms (e.g., zebrafish, bacteria, fungi)

[0172] • Fluids (e.g., serum, biological fluids, blood)

[0173] • Whole-mount specimens like 3D growing organisms or cells in gels

[0174] • Pathological specimens

[0175] • Non-biological specimens

[0176] The composition and the methodology may further provide detection of cellular and microbial antigens in the specimens described above while they are still in their growing habitat, e.g. a gel, alginate capsule, etc. , or in an environment like paraffin or a resin.

[0177] The capability of detecting cellular and microbial antigens in the variety of specimens has played an important role in understanding the cellular and molecular events. This invention will not only be of great use to many researchers in the biological and medical field, but it also has the potential to impact clinical practices as it can be used for diagnosis and clinical histopathologic analyses, like microbial infections and innate and adaptive host immune responses.

[0178] The method in the invention eliminates harmful steps in typical approches like harvesting and antigen retrievement while preserving the spatial biology of the specimens. It allows target detection in the specimen that is still in its natural or growing environment like extracellular matrix, hydrogel, agarose, scaffold, etc. For example, the 2D or 3D growing cells, co-culture specimens, organoids, or spheroids can be labeled while the they are stil in their growing environment, like hydrogel or agarose, but not limited to. Protection of the structure of those sensitive structures with the methodology in the invention provides improved results with more acurate data. The method in the invention includes fewer steps when compared with the traditional workflows since it eliminates seperate harvesting, antigen retrieving, blocking, permeabilizing, and / or clearing steps. That also provides omission of transferring, centrifuging, and pipetting stages between those separate steps in the conventional methods. As a result, the duration of the immune detection experiment significantly shortens with the technology when compared with the traditional workflow. The user can dilute more than one primary or secondary antibody in the same composition and detect several targets simultaneously. Accordingly, the method also provides for flexibility of the detection procedure, and for reproducible detection of targets in a huge variety of specimens immobilized onto a solid support.

[0179] The method is very advantageous for immunochemical detection of targets in challenging specimens such as histological specimens, as it increases accuracy in specific immunochemical labeling of molecular targets in preserved entire specimens and thus facilitates interpretation and quantification of the specimen content.

[0180] The methodology may need to be performed at a certain temperature. According to the nature of the target, the specimen, and the composition, the heating or cooling treatments vary. The duration of each steps also may vary.

[0181] The methodology can be slightly modified according to the use of any special instrument or equipment.

[0182] The amount of an antibody used for detection of a target in a specimen is very much dependent on the antibody, target, or specimen. Therefore, this amount should be individually defined in every particular case, which is a routine procedure known to a skilled in the art. Possibility of multiple amplification of a signal associated with the target, i.e. first in step of deposition of a reporter of the invention, and then in step of labeling the deposited reporter, makes the detection by the present method also less dependent on the affinity of a primary binding agent, i.e. affinity to the target, because even a weak binding of the binding agent to a target may be detected.

[0183] Amplification of a specific signal, i.e., target associated, may be further increased by repeating steps and increasing the amount of primary and or secondary antibody concentration in the composition, or modifying the ingredients amount in the composition. Duration of the steps may vary according to the specimen size, and the concentration of the ingredients in the composition. For example, for 1 mm section 1.5 hr in the composition containing the primary antibody, 1 hr in the composition containing secondary antibody, and 15 min for each washing steps are recommended for immunostaining the proteins located in the cytoplasm of the cells. For example, for 8 mm section 4 hr in the composition containing primary antibody, 2 hr in the composition containing secondary antibody, and 15 min for each washing steps are recommended for labeling the same proteins. When the permeabilization agent increased in the composition, all steps might be shorter. The location of the target for immune detection also effects the duration of different steps. For example, detection of a target in the nuclei of the cell takes longer than the detection of a target in the cytoplasm. If a target on the cell membrane is investigated, the duration of the steps in the composition decreases.

[0184] To detect very low levels of antigen, which may be present at low concentrations in vivo or in environmental specimens, it is crucial to preserve the specimen by decreasing the labeling steps. The invention increases the possibility of the detection of a target at low level since the step number decreases significantly when compared with the traditional workflows and the specimen may remain intact while using the immunohistochemistry, immunocytochemistry, immunofluorescence microscopy methods in the invention. With this methodology, expression of different antigens can be determined and quantified by using different assays on the same specimens like correlative light electron microscopy.

[0185] As used herein, each of the following terms has its meaning associated with it in this section.

[0186] Specimen

[0187] The term specimen means an amount of a material that shows what the rest of the material is or should be like, e.g., a specimen of biological, non-biological, chemical, environmental material, e.g., a specimen of body tissue, a specimen of food, a soil specimen.

[0188] In one embodiment, the specimen is a biological specimen.

[0189] A specimen may be exemplified by:

[0190] 1. a specimen comprising suspended cells and / or cells debris, e.g., blood specimen, suspension of cloned cells, body tissue homogenate, etc.;

[0191] 2. a specimen comprising of intact or damaged cells of an animal body, a body tissue, Smear or fluid, or a specimen of a tumor, e.g., a biopsy specimen: It may be a fresh tissue specimen or preserved tissue specimen, e.g., a formalin-fixed paraffin-embedded tissue specimen; or a fresh tissue section prepared using a vibratome

[0192] 3. a specimen comprising 3D growing tissue or cell or others, in a specific environment, like Matrigel, Geltrex, Cultrex, or others.

[0193] 4. a specimen comprising 3D growing living organisms and scaffolds

[0194] 5. a specimen comprising 3D growing living organisms in a bioresorbable material

[0195] 6. a specimen comprising a living organism, e.g., a specimen of a medium comprising an animal, plant, bacterium, fungi, etc.;

[0196] 7. a specimen comprising viral particles, debris thereof, or viral products, e.g., a body Smear comprising viral nucleic acids, proteins, peptides, etc.;

[0197] 8. a specimen comprising a cell organelle(s): 9. a specimen comprising natural or recombinant biological molecules, e.g., blood plasma specimen, conditioned cell culture media, etc.

[0198] 10. a chemical specimen includes but is not limited to specimens of libraries of chemical compounds, e.g., peptide libraries.

[0199] 11. environmental specimens include but are not limited to soil, water, or air specimens and food specimens.

[0200] 12. a specimen comprising non-biological specimens

[0201] The specimen may, in one embodiment, be immobilized onto a solid support, e.g., a body tissue specimen or cells immobilized on a glass or plastic slide; a cell-free specimen comprising biological molecules immobilized onto a nitrocellulose membrane, a specimen embedded in a biomaterial like gel, agar, etc.

[0202] The term solid support means a piece of any solid water-insoluble material, e.g., a nitrocellulose membrane, glass slide, coverslip, etc. The support may, in one embodiment, be a one-molecular layer thick membrane or be a multimolecular layered piece of a material, e.g., plastic or glass. The target in this embodiment is immobilized on a surface of the support.

[0203] In another embodiment, the solid support may be a three-dimensional structure, e.g., a gel block or a mesh of fibers. In this embodiment, the target is immobilized within the structure. In one embodiment, the solid support is a cellular membrane, e.g., the plasma membrane. The term immobilized means that a specimen or target is not movable on or within the support or is movable to a minimal degree.

[0204] Examples of supports suitable for immobilizing the specimens include but are not limited to synthetic polymer supports, such as polystyrene, polypropylene, substituted polystyrene, e.g., aminated or carboxylated polystyrene; polyacrylamides; polyamides; polyvinylchloride; glass; agarose; nitrocellulose: nylon; polyvinylidene difluoride; surface-modified nylon, personal, hydrogels, biodegradable materials, bioresorbable materials, scaffolds, etc. 1

[0205] The invention relates to a solid support that is chemically inert under conditions described herein, i.e., the chosen support may not have any significant influence on the results of detection by the method. Accordingly, any such inert support suitable for immobilizing a specimen or target fitting the chosen assay format, e.g., for IHC, ELISA, blotting, etc., may be selected.

[0206] In one embodiment, a specimen may be itself solid, e.g., a specimen of formalin- fixed solid tissue (i.e., not a blood specimen) and / or paraffin-embedded tissue specimen, e.g., a formalin-fixed paraffin -embedded specimen of a solid tumor, a specimen of skin, liver, breast, lung, etc. In this embodiment, the specimen itself may be accounted as Solid Support comprising an immobilized target.

[0207] In other embodiment, a specimen may be itself solid, e.g., a specimen of nonfixed solid tissue (i.e., vibratome sections). In this embodiment, the specimen itself may be accounted as solid support comprising an immobilized target.

[0208] The term target means an object of interest (supposedly present in the specimen) that can be characterized by particular physical and functional features. In the context of the invention, the term target relates to the whole pool of substantially similar entities of that object present in the specimen or every single individual unit of that object. The term substantially similar in the present context means that all entities of the pool possess the feature that makes them recognizable as the target.

[0209] The method described herein relates to visualizing a plurality of single units of a molecular target, i.e., the whole pool of target molecules in the specimen, or visualizing single individual units, e.g., single molecules, of this target. In the field of medical diagnostic biological objects such as molecules, molecular complexes, structures, particles, or organisms are often associated with characteristic features of a cell type, tissue, cellular structure, physiological condition, etc. and are termed biological markers to designate a therapeutic target or distinctive molecular feature of a particular disease.

[0210] In some embodiments of the invention, the term target is used interchangeably with the term biological marker. It relates to a molecule, molecular complex, structure, or particle characteristic of a particular cell type, tissue, physiologic condition, etc. Non-limited examples of such biological markers include but are not limited to specific nucleotide sequences, proteins, or other biological molecules, e.g., carbohydrates or lipids, chromosomal or membrane structures, viruses, bacteria, microorganisms, etc.

[0211] In one embodiment, the target is a polypeptide, nucleic acid, carbohydrate, lipid or a derivative thereof, molecular complex, particle, eukaryotic or prokaryotic cell, or microorganism.

[0212] Among targets of chemical and environmental specimens may be different pollutants, toxins, warfare substances, members of molecular libraries, industrial toxic waste, etc.

[0213] The target may be a part of a cell structure, e.g., a plasma membrane protein. In this embodiment, the cellular structure where the target is immobilized may be considered as a type of solid support within the context of the present invention.

[0214] Antigen

[0215] Antigen is used herein to refer to a substance that results in a detectable response when placed in contact with a binding agent. An antigen may be a lipid, peptide, protein, carbohydrate, nucleic acid, or combinations and variations thereof.

[0216] Incubation

[0217] The term incubating means that a specimen or target or a complex of a target with a binding agent is maintained in a medium for a period of time, e.g., in a multifunctional composition, e.g., in a composition comprising a particular reagent that specifically interacts with a target, e.g., a binding agent that is capable of directly or indirectly binding to the target, etc. The period of time may vary from 10 seconds to 3 min or continues for a more extended period of time, for example, 5-10 min, 10-20 min, 20-40 min, 40-60 min, and 1-2 hours or longer, e.g., overnight. The incubating may be performed in different temperature conditions depending on different embodiments, e.g., the type of target molecule to be detected, the type of binding agent and / or reporter used for the detection, etc. The term incubating in some embodiments may be interchangeably used with the term washing, which is usually used in conditions when a specimen is incubated in a medium that lacks a specific binding agent and serves to remove particular agents from the specimen.

[0218] The invention in most embodiments relates to incubation times within a range of 10 seconds to 24 hours.

[0219] Incubation Time

[0220] The term incubation time means, in the present context, a solution comprising particular compounds where a specimen is maintained during a certain period of time to allow a desirable reaction between the particular compounds of the composition the specimen taking place. This time may vary depending on the embodiment, from approximately 3 seconds to approximately 24 hour, e.g., around 10 seconds, 20 seconds, 30 seconds, 1 minute, 2 minutes, etc., for example, 3-10 minutes, 10-20 minutes, 20-40 minutes, 40-60 minutes, 1 -2 hours or longer, e.g., overnight. In one embodiment, the incubating time at all steps of the detection procedure may have the same duration.

[0221] The term binding agent designates a molecule capable of direct or indirect binding to a target, wherein the term directly means that the binding agent has affinity to the target and is capable of specifically recognizing and interacting with the target and binding to it. The term indirectly means that the binding agent does not have a specific affinity to the target but has such affinity to a substance associated with the target and can specifically bind to this Substance. The binding agent capable of directly binding to a target is termed first binding agent. The binding agent capable of indirect binding to a target is termed second binding agent. The first binding agent is typically used to contact the specimen. It may comprise any molecule that will specifically bind to the target supposedly present in the specimen. The second binding agent may be, e.g., any molecule that binds the first binding agent.

[0222] According to the invention, the detection system may comprise any binding agents, e.g., a first, second, third, fourth, etc., binding agents. These binding agents may be used to recognize a label comprised of a deposited reporter molecule. Such binding agents are particularly advantageous when it is desirable to enhance a signal associated with the target, e.g., in the case of a relatively low amount of the target in a specimen. According to the invention, the first, second, and further binding agents may be the members of a specific binding pair.

[0223] A number of different specific binding pairs are known in the art. These are the pairs of two different molecules with mutual affinity for each other and capable of specific binding to each other. Members of specific binding pairs suitable for use in practicing the invention may be of the immune or non-immune type. Non- immune specific binding pairs include systems where the specific binding to each other components share a mutual affinity for each other, but they are not antibodies. Exemplary non-immune binding pairs are biotin-avidin or biotinstreptavidin, folic acid-folate binding protein, complementary nucleic acids, receptor-ligand, etc. The invention also includes non-immune binding pairs, which form a covalent bond with each other. Exemplary covalent binding pairs include sulfhydryl reactive groups such as maleimides and haloacetyl derivatives and amine-reactive groups such as isothiocyanates, succinimidyl esters, sulfonyl halides, and coupler dyes such as 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethyl-amino)benzoic acid (DMAB), etc. Immune-specific binding pairs may be exemplified by antibody / antibody systems, hapten / anti-hapten antibodies, or antigen / antibody systems. In one embodiment, the immune specific binding pair may be an antibody / antibody binding pair comprising two or more antibody molecules having affinity to each other, for example, a primary antibody and secondary antibody pair, wherein the primary antibody represents a first binding agent, and the secondary antibody represents a second binding agent, or an antibody system comprising 3 or 4, or more antibody members. In other embodiments, the immune-specific binding pair may be represented hapten / anti-hapten system. For example, the first binding agent may be represented by a molecule comprising a hapten, e.g., a hapten labeled primary antibody, and the second binding agent may be represented by an antihapten antibody.

[0224] The term hapten designates a small molecule that can be considered an isolated epitope to which an antibody can be made; although the hapten alone will not induce an immune response if injected into an animal, it must be conjugated to a carrier (usually a protein). As haptens are small molecules, multiple copies of a hapten may be attached to a large molecule, e.g., a polymer molecule, such as protein, nucleotide sequence, dextran, etc. In addition, haptens may serve as convenient label molecules for assay formats that are necessary or advantageous to amplify a signal. Thus, the multiple bound copies of a hapten provide for enhanced sensitivity, e.g., increased signal strength. Non-limited examples of Suitable haptens include FITC, DNP myc Digoxigenin, nitrotyrosine biotin, avidin, Streptavidin, and anti-dye antibodies tetramethylrhodamine, Texas Red, dansyl, Alexa Fluor488, BODIPY FL, lucifer yellow and Alexa Fluor 405 / Cascade Blue fluorophores, probes, or those described in US20080305497.

[0225] A binding agent might comprise a nucleic acid or nucleic acid analog molecule, e.g., a DNA molecule, an RNA molecule, a peptide nucleic acid (PNA), locked nucleic acids (LNA), but not limited to. The binding agent, in some embodiments, may comprise at least one sequence that specifically hybridizes to a target sequence in a biological specimen, e.g., a nucleic acid sequence.

[0226] In some embodiments, the binding agents may hybridize to a target sequence in a specimen under moderately stringent conditions. Moderate stringency, as used herein, include conditions that can be readily determined by those having ordinary skill in the art based on, for example, the length of the DNA.

[0227] In one embodiment, the binding agent is represented by an antibody derivative, preferably the antigen-binding domain Fab. In some embodiments, the binding agents represented by the Fab regions of primary and / or secondary antibodies that are conjugated with at least one moiety of HRP may be preferred before the corresponding whole antibody binding agents. Such binding agents are more compact molecules than the entire antibody binding agents. That is advantageous for obtaining a more condensed reporter deposition near the target site. That may be beneficial for the precision of detection of the target. That also may be of a particular advantage when the invention method is used for immunohistochemical detection of target molecules.

[0228] In some embodiments, Fab molecules might be conjugated with a moiety of HRP or comprise a Fab region and HRP. The advantage of using HRP conjugated Fab binding agents is that these binding agents are relatively small in size and therefore have better assess to hidden or masked targets in biological specimens that are typically difficult to access when larger antibody-based binding agents are used.

[0229] Antibody

[0230] The term antibody designates an immunoglobulin or a part thereof and includes any polypeptide comprising an antigen-binding site regardless of the source, method of production, and other characteristics. The term includes, for example, polyclonal, monoclonal, monospecific, polyspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and CDR-grafted antibodies. A part of an antibody can include any fragment that can still bind antigen, such as a Fab, F(ab). A primary antibody, as used herein, refers to an antibody that specifically binds to a target molecule of a specimen. In certain embodiments, the primary antibody may be polymerized. Secondary antibody, as used herein, refers to an antibody that has an antigen-binding domain that specifically binds to the primary antibody, or a hapten deposited in the target site, or hapten linked directly or indirectly to a primary antibody or another binding agent. Tertiary antibody, as used herein, refers to an antibody that has an antigen-binding domain that specifically binds to a secondary antibody or a hapten linked to a secondary antibody or a hapten linked to polymer conjugated to a secondary antibody, or hapten deposited in the target site. Sometimes an antibody may function both as a secondary and a tertiary antibody.

[0231] Antibodies used in the invention, including primary antibodies, secondary antibodies and tertiary antibodies, may be derived from any mammal species, e.g., a rat, a mouse, a goat, a guinea pig, a donkey, a rabbit, horse, llama, camel, or any avian species e.g., chicken, duck. Derived from any mammal or avian species, as used herein, means that at least a part of the nucleic acid sequence encoding a particular antibody originated from the genomic sequence of a specific mammal, e.g., a rat, a mouse, a goat, or a rabbit or a specific bird e.g., chicken, duck. The antibody may be of any isotype, e.g., IgG, IgM, IgA, lg|D, IgE or any subclass, e.g., lgG1 , lgG2, lgG3. lgG4.

[0232] In certain embodiments, the primary antibody contains an antigen-binding region that can specifically bind to a biological marker expressed by cells comprising a biological specimen. The marker may be expressed on the cell surface or within the cell membrane, i.e. , on the interior of the cell, e.g., within the cytoplasm, within the nucleus, within the endoplasmic reticulum. In some embodiments, the biological marker is secreted from the cell and thus is present in solution, e.g., in cell culture media, in blood or plasma. In certain embodiments, the secondary antibody contains an antigen-binding region which specifically binds to the primary antibody, e.g., the constant region of the primary antibody. In certain embodiments, the secondary antibody is conjugated to a polymer. In some embodiments, the polymer is conjugated with 2-20 secondary antibodies, such as 5-15 secondary antibodies. In other embodiments, the polymer is conjugated with 1-10 secondary antibodies, such as 2, 3, 4, 5, 6, 7, 8, or 9 secondary antibodies.

[0233] In certain embodiments, the tertiary antibody contains an antigen-binding region which specifically binds to the secondary antibody, e.g., a constant region of the secondary antibody, or a hapten linked to the secondary antibody or a polymer conjugated to the secondary antibody. In certain embodiments, the tertiary antibody is conjugated to a polymer. In some embodiments, the polymer is conjugated with US 8,999,639 B2111 -20 tertiary antibodies. In other embodiments, the polymer is conjugated with 1 -5 tertiary antibodies, such as 2, 3, or 4 tertiary antibodies.

[0234] The antibodies that may be used in the methods and compositions of the invention include monoclonal and polyclonal antibodies, engineered antibodies including chimeric, CDR grafted and artificially selected antibodies produced using phage display or alternative techniques, and the like.

[0235] Antibodies may be produced recombinantly or synthetically. Nucleic acids encoding antibodies or humanized immunoglobulins (U.S. Pat. No. 5,585,089, Jones et al. 1986, Nature 332:323) may be used. The antibodies may be altered antibodies, e.g. an antibody 30 comprising an effector protein Such as a toxin or a label, e.g., a detectable substance.

[0236] In one embodiment of the invention, the antibody is represented by the Fab region of an antibody. In another embodiment, the binding agents may be members of a non-immune specific binding pair, such as a complementary nucleotide sequence pair, or a pair of two nucleic acid analog molecules having mutual affinity.

[0237] A binding agent of the invention may be directly or indirectly conjugated with one or more peroxidase moieties, (the term moiety in the present context means a whole molecule of a peroxidase, or a portion of said molecule capable of peroxidase enzymatic activity).

[0238] Non-limiting examples of binding agents which comprise an enzyme with peroxidase activity may be a primary or secondary antibody molecule or a derivative thereof, e.g. a Fab, conjugated with one or more moieties of the full- length HRP, and nucleic acid binding agents conjugated with HRP. Such binding agents may bind directly or indirectly to the target molecules and form thereby complexes each comprising one or more molecules of binding agents that comprise an enzyme with peroxidase activity.

[0239] In one embodiment, the binding agent is a conjugate comprising one, or two or more peroxidase moieties that are directly linked to the binding agent, e.g., an antibody molecule linked to one or more HRP moieties.

[0240] The Detectable Label

[0241] The detectable label, may be any substance which can be visually detected, e.g. a fluorescent or luminescent substance, or any substance that can be detected by using some detecting means, e.g., a radioactive label, a member of a specific binding pair, e.g. a nucleic acid sequence, hapten, etc.

[0242] In one preferred embodiment the label is a hapten- e.g. 2,4-dinitrophenol, digoxigenin, fluorescein, Texas Red, tetramethylrhodamine, nitrotyrosine, acetylaminoflurene, mercury trinitrophenol, estradiol, bromodeoxyuridine, dimethylaminonaphthalene sulfonate (dansyl), custom-made probe. In another preferred embodiment, the label is a fluorescent substance.

[0243] In another preferred embodiment, the label is a member of a specific binding pair- e.g. a hapten, biotin, streptavidin, complementary natural and non-natural oligonucleotide sequences, Zinc fingers binding domain pairs.

[0244] In some embodiments the detectable label may be an enzyme-like alkaline phosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase, beta-N- acetylglucosaminidase, R-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose oxidase.

[0245] The number of detectable labels per target may vary. In some embodiments 1 to 3, for example 1 , 2 or 3 labels per target may be preferred. In a preferred embodiment such target comprises one detectable label. In one embodiment a target comprising two to four residues Y of the formula (II) or two to four tyrosine and one single label may be preferred.

[0246] In some embodiments, the target may comprise more than 3 labels, such as 4 to 150 labels per reporter molecule.

[0247] Compound

[0248] As used herein, the term compound means reagents in a composition listed in this invention, which is to be administered to the specimen for detection of target by using any methodology described. The formulations of the compositions described herein may be prepared by any method known or hereafter developed in the art.

[0249] The relative amounts of compounds will vary depending upon the technology used, specimen type, specimen size, and condition of the specimen treated. Some reagents add value like extending the deadline of the product, or protecting the composition against bacterial or fungal infections, decreasing autofluorescence, changing refractive index, seperating certain structures or cell types. Other reagents are used to reduce viscosity, enhance solubility, increase the size, make thinner, less concentrated by admixture to optimize the methodology and improve detection of the antibodies. The reagents might be polar or non-polar, can be organic or inorganic. More than one reagent in each group can be used to optimize the formula of the composition.

[0250] Those combinations may vary according to the specimen, experiment type, binding antigens, targets, immun detection methods, in some embodiments, the composition comprise reagents from each groups while other embodiments may comprise certain group of reagents according to the needs of the experiments.

[0251] EXAMPLES FOR METHODS AND COMPOSITIONS

[0252] The following examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0253] Method 1 : Immunohistochemistry of Wholemount Organoids / Spheroids in

[0254] 1 . Warm-up the composition -1 (C-1 ) to 3713. C-1 includes reagents to penetrate the binding agents, reagents to prevent non-specific binding, reagents to prevent binding to non-specific sites, epitope-unmasking reagents, drying inhibition reagents and antimicrobial reagents within diluting reagents.

[0255] 2. Incubate specimen with 3% hydrogen peroxide (H2O2) in 200 uL dH2O for 5 minutes at 37°C.

[0256] 3.Aspirate hydrogen peroxide solution, wash in dH2O, 5 min at 37°C.

[0257] 4. Aspirate dH2O, incubate with 100 uL composition -1 (C-1 ), at 3713, 2X, each 10 min.

[0258] 5. Aspirate C-1 , incubate with the 100 uL primary antibody diluted in the C-1 for 1 -2 hours at 3713

[0259] 6. Aspirate C-1 containing primary antibodies, 3X, 5 min, at 37°C. 7. Aspirate 100 uL C-1 , and incubate with a biotinylated secondary antibody for 10 min at 37°C.

[0260] 8. Aspirate C-1 containing secondary antibodies, 3X, 5 min, at 37°C.

[0261] 9. Aspirate C-1 , incubate with 100 uL Horseradish Peroxidase Labeled Streptavidin, 10 min, at 37°C.

[0262] 10. Aspirate HEP labeled streptavidin, incubate with 100 uL C-1 , 3X, 5 min, at 3713.

[0263] 11. Aspirate C-1 , incubate with 100 uL DAB / AEC Chromogen solution mixture, 5- 10 min, at 37°C.

[0264] 12. Monitor the intensity of the staining under a light microscope.

[0265] 13. Wash with distilled water, 3X, 2 min.

[0266] 14. Optional: Incubate with 100 uL Mayer's Hematoxylin for nuclear counterstaining, 5 min.

[0267] 15. Aspirate Hematoxylin, wash in dH2O, 5 min.

[0268] 16. Aspirate distilled water, cover hydrogel drop with 100 uL glycerol as the mounting media

[0269] Method 2: Immunohistochemistry of paraffin embedded sections:

[0270] 1 . Warm-up the composition -2 (C-2) to 37°C. That composition includes reagents to penetrate the binding agents, reagents to prevent non-specific binding, reagents to prevent binding to non-specific sites, epitope-unmasking reagents, immersion medium, drying inhibition reagents and antimicrobial reagents within diluting reagents.

[0271] 2. Incubate specimen with 3% hydrogen peroxide (H2O2) in 200 uL dH2O for 5 minutes at 37°C.

[0272] 3.Aspirate hydrogen peroxide solution, wash in dH2O, 5 min at 37°C.

[0273] 4. Aspirate dH2O, incubate with 100 uL C-2, at 37°C, 2X, each 10 min.

[0274] 5. Aspirate C-2, incubate with the 100 uL primary antibody diluted in the C-2 for 1 -2 hours at 3713

[0275] 6. Aspirate C-2 containing primary antibodies, 3X, 5 min, at 37°C. 7. Aspirate 100 uL C-2, and incubate with a biotinylated secondary antibody in C- 2 for 10 min at 37°C.

[0276] 8. Aspirate C-2 containing secondary antibodies, 3X, 5 min, at 37°C.

[0277] 9. Aspirate C-2, incubate with 100 uL Horseradish Peroxidase Labeled Streptavidin, 10 min, at 37°C.

[0278] 10. Aspirate HEP labeled streptavidin, incubate with 100 uL C-2, 3X, 5 min, at 3713.

[0279] 11. Aspirate C-2, incubate with 100 uL DAB / AEC Chromogen solution mixture, 5- 10 min, at 37°C.

[0280] 12. Monitor the intensity of the staining under a light microscope.

[0281] 13. Wash with distilled water, 3X, 2 min.

[0282] 14. Optional: Incubate with 100 uL Mayer's Hematoxylin for nuclear counterstaining, 15 min.

[0283] 15. Aspirate Hematoxylin, wash in dH2O, 5 min.

[0284] 16. Aspirate distilled water, cover section with 100 uL glycerol as the mounting media

[0285] Method 3: Immunofluorescence Labeling of Whole Mount Spheroids

[0286] 1. Warm up the following materials to 37°C: phosphate buffered saline, the composition 3 (C-3) which includes reagents to penetrate the binding agents, reagents to prevent non-specific binding, reagents to prevent binding to nonspecific sites, epitope-unmasking reagents, immersion medium, drying inhibition reagents and antimicrobial reagents within diluting reagents, Primary Antibody Solution in C-3, and Secondary Antibody Solution in C-3, Nuclear stain, Glycerol.

[0287] 2. Aspirate cell culture medium, fix with 200 uL 4%PFA, 15-30 min, at 4°C.

[0288] 3. Aspirate fixative, wash in C-3, 3X, 10 min, at 37°C.

[0289] 4. Aspirate C-3 that surrounds hydrogel drop and incubate hydrogel drop with 100 uL primary antibody solution in C-3, 30-60 min, at 37°C.

[0290] 5. Aspirate primary antibody solution, wash in C-3, 3X, 10 min, at 37°C.

[0291] 6. Aspirate C-3, incubate with 100 uL secondary antibody solution in C-3, 30-60 min, at 37°C in the dark. 7. Aspirate secondary antibody solution, wash with PBS, 3X, 10 min, at 37°C in the dark.

[0292] 8. Aspirate PBS and incubate with 100 uL nuclear -DNA stain containing mounting medium or glycerol at 37°C in the dark.

[0293] NOTES:

[0294] 1 to 3 primary antibodies can be diluted in C-3 simultaneously. Similarly, 1 to 3 matching secondary antibodies suitable for the experiment can be diluted simultaneously in C-3.

[0295] Method 4: Immunofluorescence Labeling of Whole Mount Organoids / Spheroids / Living Organisms in Alginate or Scaffolds

[0296] 1. Warm up the following materials to 37°C: phosphate buffered saline, the composition 4 (C-4) which includes reagents to penetrate the binding agents, reagents to prevent non-specific binding, reagents to prevent binding to nonspecific sites, epitope-unmasking reagents, immersion medium, drying inhibition reagents and antimicrobial reagents within diluting reagents, Primary Antibody Solution in C-3, and Secondary Antibody Solution in C-3, nuclear stain, glycerol.

[0297] 2. Aspirate cell culture medium, fix with 200 uL 4%PFA, 15-30 min, at 37°C.

[0298] 3. Aspirate fixative, wash in C-3, 3X, 15 min, at 37°C.

[0299] 4. Aspirate C-3 that surrounds hydrogel drop and incubate hydrogel drop with 100 uL primary antibody solution in C-3, 30-90 min, at 37°C.

[0300] 5. Aspirate primary antibody solution, wash in C-3, 3X, 15 min, at 37°C.

[0301] 6. Aspirate C-3, incubate with 100 uL secondary antibody solution in C-3, 30-90 min, at 37°C in the dark.

[0302] 7. Aspirate secondary antibody solution, wash with PBS, 3X, 15 min, at 37°C in the dark.

[0303] 8. Aspirate PBS and incubate with 100 uL nuclear -DNA stain containing mounting medium or glycerol at 37°C in the dark.

[0304] NOTES:

[0305] 1 to 3 primary antibodies can be diluted in C-3 simultaneously. Similarly, 1 to 3 matching secondary antibodies suitable for the experiment can be diluted simultaneously in C-3. Method 5: Immunofluorescence Labeling of Whole Mount Organoids in Hydrogel

[0306] 1. Warm up the following materials to 37°C: phosphate buffered saline, the composition 5 (C-5) reagent to penetrate the binding agents, reagent to prevent non-specific binding, reagent to prevent binding to non-specific sites, epitopeunmasking reagent, immersion medium, drying inhibition reagent and antimicrobial reagent in diluting reagents, Primary Antibody Solution in C-3, and Secondary Antibody Solution in C-3, nuclear stain, glycerol.

[0307] 2. Aspirate cell culture medium, leave the dish at 413, 2- 5 min.

[0308] 2. Fix with 200 uL 4%PFA, 15-30 min, at 37°C.

[0309] 3. Aspirate fixative, wash in C-3, 3X, 10 min, at 37°C.

[0310] 4. Aspirate C-3 that surrounds hydrogel drop and incubate hydrogel drop with 100 uL primary antibody solution in C-3, 30-60 min, at 37°C.

[0311] 5. Aspirate primary antibody solution, wash in C-3, 3X, 10 min, at 37°C.

[0312] 6. Aspirate C-3, incubate with 100 uL secondary antibody solution in C-3, 30-60 min, at 37°C in the dark.

[0313] 7. Aspirate secondary antibody solution, wash with PBS, 3X, 10 min, at 37°C in the dark.

[0314] 8. Aspirate PBS and incubate with 100 uL nuclear -DNA stain containing mounting medium or glycerol at 37°C in the dark.

[0315] NOTES:

[0316] 1 to 3 primary antibodies can be diluted in compositions simultaneously. Similarly, 1 to 3 matching secondary antibodies suitable for the experiment can be diluted simultaneously in compositions.

[0317] Compositions and combinations in C-1 , C-2, C-3, C-4, and C-5 may vary acording to the specimen, the surrounding environment of the specimen, and the location of the target molecules

Claims

CLAIMS1. A method for identifying molecular targets that eliminates the need for separate steps for permeabilization of the specimen and blocking and / or the need for separate steps for harvesting and isolation and / or the need for separate steps for antigen retrieval treatment and / or the need for separate step for clearing the specimen, streamlining workflows and enhancing accuracy for rapid and sensitive molecular target detection , procedures characterized by comprising: a) Contacting a specimen with a multifunctional composition comprising(i) one or more reagents to penetrate the binding agents,(ii) one or more reagents to prevent binding to non-specific sites,(iii) one or more reagents to minimize the refractive index differences between the components,(iv)one or more reagents to reduce background or autofluorescence, and(v) one or more diluting reagent; b) Incubating the specimen with the multifunctional composition, and c) Detecting the target binding agents associated with the one or more molecular targets within the specimen.

2. The method according to any preceding claim, characterized by comprising; the steps of contacting a target or a specimen comprising thereof with a composition according to claims 1 , wherein the composition comprises one or more binding agents capable of direct or indirect binding to the target.

3. The method with a composition according to any preceding claim, wherein one or more reagent to penetrate the binding agents is selected from the group comprising Triton X series such as Triton X - 100 and Triton X - 140; Tween series such as Tween - 20, Saponin, Methanol, Acetone, and any other penetrating reagent.

4. The method with a composition according to any preceding claim, wherein one or more reagent to prevent binding to non-specific sites which is selected from the group comprising Bovine Serum Albumin, Goat Serum, Donkey serum, Tween 20, Nonfat dry milk, blotting grade blocker, gelatin, casein, dimethysulfoxide, Ficoll PM70, Percoll, Polyvinyl pyrrolidone, Tris buffered saline, and any other reagent to prevent binding to non-specific sites.

5. The method for identifying molecular targets with a composition according to any preceding claim, wherein one or more background and autofluorescence reducing reagent is selected from the group comprising Tween 20, SDS, Nonidet P-40 (NP-40), sodium borohydride, Thiocarbohydrazide, Sudan Black B, Eriochrome black T, and any other reagent to reduce background or autofluorescence.

6. The method of any preceding claim for identifying molecular targets, wherein the composition further comprises one or more refractive index changing reagents selected from the group consisting of Glycine, Glycerol, Heparin, Benzyl alcohol, Benzyl benzoate, Dichloromethane, Dibenzylther, Sucrose, Diatrizoic acid, Formamide, Fructose, Chloroform, Xylene, Toluene, Thioglycerol, Urea and Urea derivatives, 2,2-thiodiethanol, Dimethyl sulfoxide, glycerin, mineral oil, neatsfoot oil, olive oil, palm oil, and any other reagent to change refractive index.

7. The method for identifying molecular targets with a composition according to any preceding claim, wherein one or more diluting agent is selected from the group comprising water, distilled water, double distilled water, phosphate buffered saline, Dulbecco’s Phosphate Buffered Saline, Ca and Mg free Dulbecco’s Phosphate Buffered Saline, glycerol, cell culture media, Glycerol, Tris-buffered saline, a buffer that resist changes in Ph, and any other reagent to dilute the composition.

8. The method with a composition according to any preceding claim; wherein one or more epitope-unmasking reagent is added to the composition from the group comprising Trypsin, Pepsin, Hyaluronidase, Proteinase-K, Sodium Citrate, EDTA, Tris-EDTA, and any other reagent for unmasking epitopes.

9. The method with a composition according to any preceding claim, wherein one or more drying inhibition agent, is added to the composition from the group comprising glycerol, carboxy vinyl polymer, hydroxypropyl methylcellulose, propylene glycol, and macrogol, and any other reagent for inhibition drying.

10. The method with a composition according to any preceding claim, wherein one or more water-soluble macromolecular compound, is added to the composition from the group comprising polyethylene glycol, polyvinyl pyrolidone, sucrose, Ficoll PM70 (product name) and Percoll (product name), and any other reagent as water soluble macromolecular compound, and any other reagent as a water soluble molecular compound.

11. The method with a composition according to any preceding claim, wherein one or more immersion medium, is added to the composition from the group comprising glycerin, mineral oil, neatsfoot oil, olive oil, palm oil, and any other reagent as an immersion medium.

12. The method with a composition according to any preceding claim, wherein the method further comprises cell seperation or organelle isolation.

13. The method with a composition according to any preceding claim, wherein one or more cell separating and organelle isolation reagent, isadded to the composition from the group comprising non-ionic synthetic polymer of sucrose-Ficoll, Percoll, and any other reagent seperation of cells or isolation of organelles.

14. The method with a composition according to any preceding claim, wherein the reagents may be present in the composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion.

15. The method with a composition according to any preceding claim, wherein the composition in the form of a liquid or powder.

16. The method with a composition according to any preceding claim, the composition in the form of a single solution, single powder or a kit including components in solution or powder form.

17. The method with a composition according to any preceding claim, wherein the method further comprises one or more treatment from the group comprising heating, cooling, oxidizing, treating with ultrasound, or trimming.

18. The method with a composition according to any preceding claim, wherein the method further comprises coating, covering, or embedding the specimen.

19. The method with a composition according to any preceding claim, wherein the method further comprises treatment of the habitat that surrounds the specimen from a group reagent involving xylol, thylenol, acetone, trypsine, a digestive enzyme, a lipid removing agent, and / or any other dissolving agent.

20. The method with a composition according to any preceding claim, wherein the method further comprises treatment of the habitat that surrounds the specimen from a group reagent involving xylol, thylenol, acetone, trypsine, a digestive enzyme, a lipid removing agent, and / or any other dissolving agent.

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