Heat-induced antigen retrieval methods and compositions therefor

Description of equivalent WO 2006007841 A2

HEAT-INDUCED ANTIGEN RETRIEVAL METHODS AND COMPOSITIONS THEREFOR

Technical field The present invention relates to the field of immunohistochemistry. More specifically, this invention relates to methods and compositions for heat-induced antigen retrieval during immunohistochemistry.

Background of the invention Immunohistochemistry (IHC) is a method used for detecting the presence of a biological marker in a sample. IHC is especially valuable in disease diagnosis where expression of a biological marker may be indicative of the presence or the onset of a disease state. For example, identity of a particular type of cancer is often confirmed by the presence of a particular marker in a sample from a patient. Similarly, the progression of a disease can also be monitored by the presence of a particular biological marker, which is expressed only at a certain stage or stages of the disease.

The use of fixed, paraffin-embedded tissue sections for immunohistochemistry staining permits localization of a wide variety of antigens while retaining excellent morphologic details. However, most chemical fixatives produce denaturation or masking of many antigens. Specifically, the denaturing effects of tissue fixation often render target epitopes on antigens unavailable for binding by antibodies during IHC.

Antigen retrieval is a widely accepted method for heat-assisted retrieval of antigens in fixed paraffin-embedded tissues prior to IHC staining. The introduction of antigen retrieval by heating tissue sections in a microwave oven, pressure cooker, or other heating devices before immunostaining has been an important breakthrough in improving results of staining by IHC. Although, many antigens can be retrieved when heating is performed in distilled water, there are several antigens that require use of buffers during the heating process. However, thus far, no single chemical has been identified that makes antigen retrieval effective for all antigens. Many studies have shown an inverse relationship between the temperature and the duration of time that a sample is heated, such that low temperatures need prolonged incubation times for antigen retrieval compared to high temperatures that need shorter incubation times for antigen retrieval. Generally, higher temperatures of about 100⁰C can be achieved with water- based aqueous solutions. However, the duration of time needed for antigen retrieval is about 20-60 minutes and does not work for all antigens, resulting in poor staining for some antigens.

Pressurized devices are often more effective for antigen retrieval as they enable the temperatures to be even higher, i.e., about 120°C. Most pressurized devices, however, generally require about 40-50 minutes for effective antigen retrieval. At least a portion of this time is used for the cooling process after the antigen retrieval heating step has been completed. Because the heating step is performed under pressure, the contents of the pressure chamber must be allowed to cool sufficiently so that the internal pressure of the chamber decreases prior to opening the chamber, as this can result in explosive boiling of the liquid inside and would pose a significant risk to the user. Typically the pressure chamber is allowed to passively cool under ambient conditions for at least 25 - 30 minutes before the container can be opened. This long duration may pose a problem for certain users that require faster processing times. Furthermore, not every laboratory is willing to invest in the time and money necessary to set up a high pressure method of antigen retrieval. Because the water bath method of antigen retrieval is easier to perform, many laboratories still rely on this method, although results are frequently inferior to the pressure method. Also there are some antigens that are more sensitive to higher temperatures and become denatured when exposed to 12O⁰C for prolonged periods of time. These antigens could benefit from lower temperatures or shorter incubation times to prevent denaturation.

Therefore, there is a need for improved, easy-to-use, and cost effective methods of antigen retrieval that facilitate effective antigen retrieval in a shorter duration of time, while at the same time, avoid problems associated with current antigen retrieval methods, such as denaturation of antigens and poor staining during IHC for disease detection. It is thus highly desirable in the light of the aforementioned problems to develop compositions and methods for heat-induced antigen retrieval where effective antigen retrieval can be achieved in a short duration of time without the need for a pressurized system, which can also avoid problems associated with compositions and methods known in the art. In this respect, the present invention addresses this need and interest.

Summary of the invention

In view of the foregoing disadvantages known in the art of antigen retrieval with denaturation of antigens and poor staining during IHC for disease detection, the present invention provides new and improved methods and compositions to facilitate effective antigen retrieval in a shorter duration of time compared to known antigen retrieval methods and compositions.

One object of the present invention is to provide a composition for antigen retrieval comprising at least one chelating agent, at least one detergent and at least 50% by volume of at least one liquid that facilitates effective antigen retrieval.

In certain aspects, the invention provides compositions for antigen retrieval including certain chemicals which are liquids and which facilitate heat-induced antigen retrieval in duration of time, which is shorter than duration of time necessary for heat-induced antigen retrieval in absence of such chemicals.

In further embodiments of the composition the at least one liquid that facilitates antigen retrieval comprises a first and a second liquid.

In further embodiments of the composition the first liquid is propylene glycol.

In further embodiments of the composition the second liquid is ethylene glycol.

In further embodiments of the composition the propylene glycol is about 55% by volume of the total composition.

In further embodiments of the composition the ethylene glycol is about 25% by volume. In further embodiments of the composition the boiling point of the overall composition is at least 120^°C at sea level.

In further embodiments of the composition the boiling point of the overall composition is at least 131 ^°C at sea level. A further aspect of the present invention is a method of antigen retrieval. The method comprises the steps of a) providing at least one biological sample comprising at least one antigen, b) contacting at least one biological sample with at least one composition for antigen retrieval comprising at least one chelating agent, at least one detergent and at least 50% by volume of at least one liquid that facilitates antigen retrieval, c) heating the at least one antigen retrieval solution to an elevated temperature, and d) maintaining the elevated temperature of the at least one antigen retrieval solution for an duration of time to achieve effective antigen retrieval. In a further aspect, the invention also discloses another method for antigen retrieval, the method comprising the steps of a) providing at least one biological sample comprising at least one antigen, b) contacting the at least one biological sample with at least one composition for antigen retrieval comprising at least one chelating agent, at least one detergent and at least 50% by volume of at least one liquid with a viscosity of less than 10 centipoise but grater than 1 centipoise at 20^°C, c) heating the at least one antigen retrieval solution to an elevated temperature, and d) maintaining the elevated temperature of the at least one antigen retrieval solution for an duration of time to achieve effective antigen retrieval. In further embodiments the above methods are methods wherein the at least one liquid comprises a first and a second liquid.

In still even further embodiments, the first liquid is propylene glycol and the second liquid ethylene glycol.

In still even further embodiments the propylene glycol is present in an amount of about 55% by volume of total composition and the ethylene glycol is in an amount of about 25% by volume of total composition.

In still even further embodiments the elevated temperature is about 130^°C, such as about 131⁰C, 132°C, 133°C, 134⁰C or even about 135°C.

Detailed description of the invention

Compositions for antigen retrieval The inventors have discovered that the addition of certain chemicals to antigen retrieval solutions may enable effective antigen retrieval in a shorter duration of time relative to antigen retrieval in absence of such chemicals, without the need of a pressurized system. In certain aspects, the invention provides compositions for antigen retrieval including certain chemicals which are liquids and which facilitate heat-induced antigen retrieval in duration of time, which is shorter than duration of time necessary for heat-induced antigen .retrieval in absence of such_. chemicals.

In some embodiments, a composition for antigen retrieval comprises at least one chelating agent, at least one detergent and at least 50%, by volume of at least one liquid. In some further embodiments, the at least one liquid has a viscosity of at least 15 centipoise at 2O⁰C.

In some other embodiments a composition for antigen retrieval comprises as least one chelating agent, at least one detergent and about 50%, 60%, 70%, 80% or even 90% by volume of at least one liquid that facilitates heat induced antigen retrieval. Examples of liquids that facilitate heat-induced antigen retrieval in a shorter duration of time, include, but are not limited to, glycols and glycerols. Examples of glycols are ethylene glycol (1 , 2-ethanediol) and propylene glycol (1 , 2-propanediol). In some embodiments, compositions of the invention include at least one antigen retrieval solution comprising at least 50%, 60%, 70%, 80% or even 90% by volume of ethylene glycol. In other embodiments compositions of the invention include at least one antigen retrieval solution comprising at least 50%, 60%, 70%, 80% or even 90% propylene glycol. In even further embodiments, the antigen retrieval solution comprises a first and a second liquid. Said first liquid may in some embodiments comprise at least 50%, 60%, or even 70% of the total volume of the antigen retrieval solution and said second liquid comprise at least 10%, such as 10, 20, 25, 30, 35 or even 40% of the total volume of the antigen retrieval solution. In other embodiments, compositions of the invention include at least one antigen retrieval solution comprising at least 50% by volume of glycerol. In other embodiments, compositions of the invention include at least one antigen retrieval solution comprising at least 70% by volume of ethylene glycol, or at least 70% by volume of glycerol.

One particular embodiment of the composition according to the invention comprises about 55% by volume of propylene glycol as a first liquid and about 25% by volume of ethylene glycol as a second liquid.

In certain embodiments, compositions of the invention include at least one chelating agent. Examples of chelating agents include, but are not limited to, citric acid, sodium citrate and EDTA. In certain embodiments, compositions of the invention include at least one detergent. Detergents, which can be used in the methods of the invention, include, but are not limited to, Tween 20, NP40, Triton X, Saponin and Brij.

The invention further discloses a composition for antigen retrieval comprising at least one chelating agent, at least one detergent and at least 50% by volume of at least one liquid that facilitates antigen retrieval.

In further embodiments of the composition the at least one chelating agent is chosen from citric acid, sodium citrate and EDTA.

In further embodiments of the composition the at least one detergent is chosen from Tween 20, NP40, Saponin, and Brij. In further embodiments of the composition the at least one liquid that facilitates antigen retrieval is a glycol.

In further embodiments of the composition the glycol is ethylene glycol. In further embodiments of the composition the glycol is polypropylene glycol. In further embodiments of the composition the at least one liquid that facilitates antigen retrieval is a glycerol.

In further embodiments of the composition the at least one liquid that facilitates antigen retrieval comprises a first and a second liquid.

In further embodiments of the composition the first liquid is propylene glycol. In further embodiments of the composition the second liquid is ethylene glycol.

In further embodiments of the composition the propylene glycol is about 55% by volume of the total composition. In further embodiments of the composition the ethylene glycol is about 25% by volume.

In further embodiments of the composition the boiling point of the overall composition is at least 120⁰C at sea level. In further embodiments of the composition the boiling point of the overall composition is at least 131⁰C at sea level.

In further embodiments of the composition the viscosity of the overall composition is between 15-235 centipoise at 20^°C.

In further embodiments of the composition the viscosity of the overall composition is between 1-10 centipoise at 2O⁰C.

The invention also discloses a composition for antigen retrieval comprising at least one chelating agent, at least one detergent and 50% by volume of at least one liquid having a viscosity of between 15 centipoise and 1000 centipoise at 2O⁰C. In further embodiments the viscosity of the overall composition is between 15-235 centipoise at 2O⁰C.

In further embodiments the at least one chelating agent is chosen from citric acid, sodium citrate and EDTA.

In further embodiments the at least one detergent is chosen from Tween 20, NP40, Triton X, Saponin and Brij.

In further embodiments the liquid is a glycol. In further embodiments the glycol is ethylene glycol. In further embodiments the glycol is propylene glycol. In further embodiments the liquid is glycerol. The invention further discloses a composition for antigen retrieval comprising at least one viscous liquid chosen from glycerol and a glycol, wherein the viscosity of the overall composition is between 15 and 235 centipoise at 20⁰C. In further embodiments the glycol is ethylene glycol. In further embodiments the glycol is propylene glycol. In further embodiments the comprising further comprises at least one chelating agent and at least one detergent.

In further embodiments the at least one detergent is chosen from Tween 20, NP40, Triton X, Saponin and Brij. In further embodiments the at least one chelating agent is chosen from citric acid, sodium citrate and EDTA.

Methods for antigen retrieval The invention also provides methods of heat-induced antigen retrieval

In one aspect the invention discloses a method of antigen retrieval, the method comprises the steps of a) providing at least one biological sample comprising at least one antigen, b) contacting at least one biological sample with at least one composition for antigen retrieval comprising at least one chelating agent, at least one detergent and at least 50% by volume of at least one liquid that facilitates antigen retrieval, c) heating the at least one antigen retrieval solution to an elevated temperature, and d) maintaining the elevated temperature of the at least one antigen retrieval solution for an duration of time to achieve effective antigen retrieval.

In further embodiments of the method the composition is any of the compositions according to the invention.

In a further aspect, the invention also discloses a method for antigen retrieval, the method comprising the steps of a) providing at least one biological sample comprising at least one antigen, b) contacting the at least one biological sample with at least one composition for antigen retrieval comprising at least one chelating agent, at least one detergent and at least 50% by volume of at least one liquid with a viscosity of less than 10 centipoise but grater than 1 centipoise at 20^°C, c) heating the at least one antigen retrieval solution to an elevated temperature, and d) maintaining the elevated temperature of the at least one antigen retrieval solution for an duration of time to achieve effective antigen retrieval.

In further embodiments the above methods are methods wherein the at least one liquid comprises a first and a second liquid.

In still even further embodiments, the first liquid is propylene glycol and the second liquid ethylene glycol.

In still even further embodiments the propylene glycol is present in an amount of about 55% by volume of total composition and the ethylene glycol is in an amount of about 25% by volume of total composition.

In still even further embodiments the elevated temperature is at least about 100⁰C.

In still even further embodiments the elevated temperature is at least about 120⁰C or at least about 125°C. In still even further embodiments the elevated temperature is about 130^°C, such as about 131⁰C, 132°C, 133°C, 134°C or even about 135°C.

In still even further embodiments the heating is performed prior to the contacting step.

In still even further embodiments the heating is performed after the contacting step.

In still even further embodiments the heating step comprises use of a pressure device.

In still even further embodiments the heating step comprises use of a non- pressure device. In still even further embodiments the elevated temperature is substantially equal to the boiling point of the total composition for antigen retrieval.

In still even further embodiments the methods further comprises the step of verifying effective antigen retrieval by contacting at least one biological sample with at least one antibody which binds the at least one antigen thereby verifying effective antigen retrieval. Said antibody will then substantially not bind other antigens under the specified conditions of use, herein being immunohistochemistry. "Substantially not bind" as used herein means that the antibody will score below detection limit of the performed assay under conditions of use according to known scoring standards.

In certain embodiments of the invention, a method according to the invention includes the steps of: providing at least one biological sample comprising an antigen; contacting the at least one biological sample with at least one antigen retrieval solution comprising at least 50% of the total volume of a first liquid and at least 10% of the total volume of a second liquid; heating the at least one antigen retrieval solution to an elevated temperature; and maintaining the elevated temperature of the at least one antigen retrieval solution for an effective duration of time.

In one embodiment, the first liquid is a chemical, addition of which results in antigen retrieval in a duration of time shorter than the duration without the chemical.

In certain embodiments, the first liquid is a glycol, such as, for example, ethylene glycol and propylene glycol.

In other embodiments, the first liquid is a glycerol. In some embodiments, the second liquid is an aqueous liquid, such as water.

In other embodiments, the second liquid is an antigen retrieval solution, which does not include the first liquid. In some embodiments the first liquid comprises about 55% by total volume of antigen retrieval solution of propylene glycol and the second liquid about 25% by total volume of ethylene glycol.

In certain aspects of the invention, methods of antigen retrieval include the steps of providing at least one biological sample comprising an antigen; contacting the at least one biological sample with at least one antigen retrieval solution comprising at least 20%, such as 20, 25, 30, 50, 70 or even 80%, ethylene glycol; heating the at least one antigen retrieval solution to an elevated temperature; and maintaining the elevated temperature of the antigen retrieval solution for a duration of time necessary for antigen retrieval. In yet other aspects, methods of antigen retrieval include the steps of: providing at least one biological sample comprising an antigen; contacting the at least one biological sample with at least one antigen retrieval solution comprising a liquid with a viscosity of less than 235 centipoise at 20⁰C; heating the at least one antigen retrieval solution to an elevated temperature; and maintaining the elevated temperature of the at least one antigen retrieval solution for a duration of time necessary for antigen retrieval.

Viscosity generally refers to the measurement of a fluid's internal resistance to flow. Viscosity of a liquid, as used herein, can be measured by one or more methods known in the art and can be designated in units of centipoise or poise or any other acceptable measurements. Viscosity can be easily measured using one of many viscosimeters known in the art. Examples of viscosimeters include, but are not limited to, the Saybolt universal and the Saybolt Furol viscosimeters, which measure viscosity of liquids by allowing a measured volume of a liquid to flow through an orifice of specified dimensions and measuring the time in seconds that it took to get through the orifice. The Irany, Zahn and Redwood viscosimeters operate on the same principal. Other examples of viscosimeters include the Brookfield viscosimeter which includes a disc which is rotated in the liquid to be tested and the drag is noted and read directly in centipoise. Viscosities found herein were obtained from published sources, for example Handbook of Chemistry and Physics or The Merck Index.

In one embodiment, the elevated temperature is at least 100⁰C to enable effective heat-induced antigen retrieval. In certain embodiments, the elevated temperature is substantially equal to the boiling point of the antigen retrieval solution after the chemical(s) is/are added, i.e. the overall boiling temperature of the composition for antigen retrieval.

In some embodiments, the elevated temperature is about 120⁰C. In some further embodiments the elevated temperature is about 125⁰C. In still some further embodiments the elevated temperature is about 130⁰C, or even 131⁰C, 132°C, 133°C, 134°C or 135°C.

In certain embodiments of the methods of the invention, the heating step is performed prior to the contacting step. However, the heating step can also be performed after the contacting step. The heating step, for example, can be performed using a pressurized device or a non-pressurized device. Pressurized devices include, but are not limited to, pressure cookers and autoclaves. Non-pressurized devices include, but are not limited to, water baths, steamers, ovens, hot plates and heating chambers. In some embodiments, a microwave oven is used during the heating step.

In certain embodiments, methods of the invention further comprise the step of contacting the biological sample with an antibody, which binds the antigen. In some embodiments, a heating device according to the invention is comprised of a heating chamber constructed from Teflon coated aluminum. The chamber is designed to accommodate a single rack of 24 microscope slides. The rack with slides is lowered into the liquid reservoir of the heating chamber. The liquid reservoir contains about 200 ml of antigen retrieval solution, or any other suitable volume that applies for that particular liquid reservoir, at ambient temperature. The chamber is constructed with a heating blanket surrounding the aluminum reservoir. Current is applied to the heating chamber and after about 12 minutes, or any other suitable length of time, the antigen retrieval solution reaches a temperature of about 12O⁰C, 125°C or even 130, 131 or 132°C. Slides are maintained at about 120°C-13O⁰C for about eight minutes and are then transferred without the need for cooling to a second container which contains water at an ambient temperature. Using such a device or similar, the total time of antigen retrieval is only about 20 minutes.

In another embodiment, a heating device includes a lid that can be used to create a sealed chamber. The sealed chamber then becomes a pressure device, and the pressure becomes an additional means of controlling the internal temperature within the heating chamber. By applying both heat and pressure simultaneously, the internal temperature of the heating chamber can be controlled regardless of the composition of the antigen retrieval solution. This would allow the heating device to accept any antigen retrieval solution regardless of its formulation, including the commonly used antigen retrieval solutions that are aqueous in nature. In order to achieve high temperature antigen retrieval, a heating device would operate without pressure when used in conjunction with antigen retrieval solutions described herein, or in the pressurized mode when used with known antigen retrieval solutions that are aqueous.

The invention further discloses a method of antigen retrieval, the method comprising: a) providing at least one biological sample comprising at least one antigen; b) contacting at least one biological sample with at least one antigen retrieval solution comprising at least 50% of the total volume of a first liquid and at least 10% of the total volume of a second liquid, wherein the combined liquids have a viscosity of at least 15 centipoise but not more than 200 centipoise at 20⁰C; c) heating the at least one antigen retrieval solution to an elevated temperature; and d) maintaining the elevated temperature of the at least one antigen retrieval solution for an effective duration of time,

In further embodiments the first liquid is a glycol. In further embodiments the glycol is ethylene glycol.

In further embodiments the glycol is propylene glycol.

In further embodiments the first liquid is glycerol.

In further embodiments the second liquid is water.

In further embodiments the elevated temperature is at least 100⁰C. In further embodiments the elevated temperature is at least 120°C.

In further embodiments the heating step is performed prior to the contacting step.

In further embodiments the heating step is performed after the contacting step. In further embodiments the heating step comprises use of a pressure device.

In further embodiments the pressure device is chosen from a pressure cooker and an autoclave.

In further embodiments the heating step comprises use of a non-pressure device.

In further embodiments the non-pressure device is chosen from water bath, steamer, oven, hot plate and a heating chamber.

In further embodiments the oven is a microwave oven.

In further embodiments the at least one antigen retrieval solution comprises at least one chelating agent.

In further embodiments the at least one chelating agent is chosen from citric acid, sodium citrate and EDTA. In further embodiments the at least one antigen retrieval solution comprises at least one detergent.

In further embodiments the at least one detergent is chosen from Tween 20, NP40, Triton X, Saponin and Brij. . In further embodiments the elevated temperature is substantially equal to the boiling point of the first liquid.

In further embodiments the method comprises the step of contacting at least one biological sample with at least one antibody which binds the at least antigen. . The invention further discloses a method of antigen retrieval, the method comprises the steps of: a) providing at least one biological sample comprising at least one antigen; b) contacting the at least one biological sample with at least one antigen retrieval solution comprising at least 50% ethylene glycol; c) heating the at least one antigen retrieval solution to an elevated temperature; and d) maintaining the temperature of the at least one antigen retrieval solution at the elevated temperature for a duration of time necessary for antigen retrieval.

In further embodiments the elevated temperature is at least 100⁰C. In further embodiments the heating step is performed prior to the contacting step.

In further embodiments the heating step is performed after the contacting step.

In further embodiments the heating step comprises use of a pressure device.

In further embodiments the pressure device is chosen from an autoclave and a pressure cooker. In further embodiments the heating step comprises use of a non-pressure device.

In further embodiments the non-pressure device is chosen from water bath, steamer, oven, hot plate, and heating chamber. In further embodiments the oven is a microwave oven.

In further embodiments the at least one antigen retrieval solution comprises at least one chelating agent.

In further embodiments the at least one chelating agent is chosen from citric acid, sodium citrate and EDTA.

In further embodiments the at least one antigen retrieval solution comprises at least one detergent.

In further embodiments the at least one detergent is chosen from Tween. 20, NP40, Triton X, Saponin and Brij. . In further embodiments the elevated temperature is substantially equal to the boiling point of the antigen retrieval solution.

In further embodiments the method further comprises the step of contacting the at least one biological sample with at least one antibody, which binds the at least one antigen. . The invention further discloses a method of antigen retrieval, the method comprising: a) providing at least one biological sample comprising at least one antigen; b) contacting the at least one biological sample with at least one antigen retrieval solution comprising at least 50% by volume of a liquid with a viscosity of less than 10 centipoise but greater than 1 centipoise at 20⁰C; c) heating the antigen retrieval solution to an elevated temperature; and d) maintaining the temperature of the at least one antigen retrieval solution at the elevated temperature for a duration of time necessary for antigen retrieval.

Definitions and further embodiments

In order that the present invention be more readily understood, certain terms are defined. Additional definitions are set forth throughout the detailed description.

"Antigen" and "biological marker" as used interchangeably herein, refer to any molecule present in a biological sample, such as a protein, including a glycoprotein or lipoprotein, phosphoprotein, methylated protein, or a protein fragment, e.g., a peptide or a polypeptide, a nucleic acid, e.g., DNA, RNA, a lipid, a glyco-lipid, a sugar, a polysaccharide, a starch. In some embodiments, an antigen is a molecule, which can bind an antibody. The biological marker may be expressed on the surface of the biological sample, e.g., membrane bound. Alternatively, the biological marker may be contained in the interior of the biological sample, i.e., within the cell membrane, e.g., within the cytoplasm, within the nucleus, within an intracellular compartment or organelle.

"Antigen retrieval" as used herein, refers to the process of applying heat to a biological sample in order to induce certain chemical and physical changes in a biological marker such that a biological marker is rendered capable of physically combining with an antibody. The mechanism by which antigen retrieval induces these changes is not well understood but may involve the loosening or breaking of the molecular crosslinkages that are formed during sample preparation. For example fixing a biological sample in formalin is known to create inter- and intra- molecular crosslinkages that can mask the biological marker from an antibody rendering it undetectable by IHC.

For example, in certain instances, biological samples are treated with an antigen retrieval solution subsequent to fixation to achieve antigen retrieval. Factors that affect antigen retrieval include, but are not limited to, temperature, pH of solution, concentration of salt and metal ions in the antigen retrieval solution. Antigen retrieval may be verified as being effective, for example, by recognition of an antigen of interest by an antibody, which binds the antigen. Not all antigens require antigen retrieval in order to combine with an antibody. Therefore, some antigens can be detected in IHC procedures without the application of antigen retrieval methods. However, a high percentage of antigens do benefit by this procedure, such that it has become standard practice in many IHC procedures. It is up to the investigator to determine whether the antigen of interest requires antigen retrieval. This is easily accomplished by dividing a biological sample under investigation into two smaller samples. One sample is tested with antigen retrieval and the second sample is tested without antigen retrieval. If the sample with antigen retrieval tests positive and the sample without antigen retrieval tests negative, the requirement for antigen retrieval has been verified. For future tests of the same antigen in similar biological samples, it can be assumed that antigen retrieval is required. Compositions of the invention can be formulated using any antigen retrieval solution, for example, commercially available solutions for antigen retrieval S1699 (Dakocytomation), High pH S3307 (Dakocytomation). The S3307 solution contains 1mM EDTA at pH 9.9 when diluted to its working concentration and the S1699 solution contains 1OmM sodium citrate when diluted to its working concentration.

There are many commercially available antigen retrieval solutions. Generally these can be categorized according to their pH. For example, there are low pH (pH 2-4), mid-pH (pH 6-7), and high pH (8-10) solutions. Typically these solutions also contain a chelating agent such as citric acid or EDTA.

"Sample" and "biological sample" as used interchangeably herein, refer to a sample derived from any living organism, e.g., animal, plant, bacteria. It may be comprised of eukaryotic cells or prokaryotic cells. It may be comprised of a tissue sample derived from an organ. The sample may be derived either from a diseased or a normal tissue. The sample may also be comprised of a cell smear. In some embodiments, the sample is derived from a cancerous tissue. A biological sample, as used herein, may contain compounds which are added to the cell/ tissue derived from a living organism, such as, for example, preservatives, anticoagulants, fixatives, nutrients, antibiotics, or the like. A biological sample may be treated in any suitable manner prior to its use in the methods of the invention, as needed for detecting the desired antigen in the sample.

"Duration of time" as used herein, refers to any duration of time necessary for antigen retrieval. Duration of time necessary for antigen retrieval can be easily determined using one or more assays provided herein. For example, the duration of time necessary for retrieval of a particular antigen can be determined by evaluating staining for that antigen using methods of the invention. Duration of time required for antigen retrieval using methods of invention is shorter than duration of time required with the known pressurized and non-pressurized devices. The efficacy of an antigen retrieval method can be determined by taking the finished biological sample and viewing it under the microscope in order to judge its staining characteristics. An acceptably stained biological sample will demonstrate strong staining of the biological marker of interest. Staining intensity can be visually scored by an observer examining the stained biological sample under a microscope. Staining intensity is rated on a scale of 0 - 4 where 0 is no staining, 1 is weak staining, 2 is moderate staining, 3 is strong staining, and 4 is very strong staining.

"Elevated Temperature" as used herein, generally refers to a temperature of a composition of the invention, which is necessary for effective antigen retrieval. In some embodiments, an elevated temperature is at least 100⁰C, such as about 100, 110, 120, 125, and 130⁰C, 131⁰C, 132⁰C, 133°C, 134°C, 135°C. For the purposes of antigen retrieval, in certain instances, an elevated temperature may be substantially equal to the boiling point of a composition used for antigen retrieval. Boiling point of a composition is generally defined as a temperature at which the composition changes its state from liquid to gas, i.e. the temperature at which the vapor pressure of a given liquid reaches atmospheric pressure and thus starts to boil. Boiling point may also be defined as the temperature at which the liquid and vapor (gas) phases of a composition can exist in equilibrium. "Boiling point" as used herein is referred to at sea level. The boiling point changes at different heights above (or below) the sea level. By adding substances to a liquid its boiling point may be elevated, or increased. A substantially equal elevated temperature may either be identical to the boiling point of a composition used for antigen retrieval or be greater or less than the boiling point of the composition. For example, a substantially equal elevated temperature includes a temperature greater or less than the boiling point of the composition by at least 1 degree Celsius (⁰C), 2 degrees Celsius, 3 degrees Celsius, 4 degrees Celsius, 5 degrees Celsius, or between 5 and 10 degrees Celsius.

In certain embodiments, an elevated temperature is substantially equal to the boiling point of an antigen retrieval solution following addition of a substance, such as, a glycol, for example ethylene glycol.

In other embodiments, an elevated temperature is substantially equal to the overall boiling point of the antigen retrieval solution.

Both an elevated temperature necessary for effective antigen retrieval as well as a duration for which the temperature remains elevated can be determined by one or more assays provided herein.

Sample processing In one embodiment, typical IHC sample processing is used. IHC typically require a series of sample processing steps conducted on the biological sample prior to the assay. Generally, a biological sample is fixed and then dehydrated through an ascending series of alcohol, infiltrated and embedded with paraffin or other sectioning media so that the sample may be sectioned. The sections are subsequently attached to a planar surface such as a glass slide and used in the methods of the invention. Typically, sections of a biological sample are mounted on a slide using a suitable slide adhesive. Examples of adhesives include, but are not limited to, silane, gelatin, poly-L-lysine, and the like. However, methods of the invention can also be used on samples including cells, which are in suspension rather than being attached to a surface.

Biological samples are usually fixed prior to use in an IHC assay. Many methods of fixation are known in the art. Examples include, but are not limited to, alcohol-fixation and formalin-fixation. Many fixatives are known in the art that can be used for preserving cells and tissues and protect them from the rigors of the processing and staining techniques. Examples of fixatives include, but are not limited to, formalin, methanol, ethanol, acetone, 2-propanol, acrolein, glutaraldehyde, glyoxal, osmium tetroxide, paraformaldehyde, mercuric chloride, trichloroacetic acid, Bouin's, B-5, and tungstic acid. It may be necessary to unmask or retrieve a biological marker prior to its detection by staining. Antigen retrieval allows a biological marker to be available for interacting with an antibody, which binds the biological marker.

The present invention provides compositions for antigen retrieval comprising chemicals that reduce the duration of time required for heat-induced antigen retrieval relative to the duration of time in the absence of such chemicals. Antigen retrieval in a shorter duration of time may preserve the integrity of the biological sample and avoid denaturation of antigens being detected. This may be especially useful for accurate disease diagnosis using the presence or absence of an antigen as an indicator for the presence or absence of a disease in a patient. Furthermore, in the absence of such chemicals, in some instances, it may not even be possible to achieve the desired result despite the increase in the duration of time, or the required duration of time may be impractical. Further, compositions of the invention may enable effective antigen retrieval using a non-pressurized device. However, pressurized systems may also be used with the compositions of the invention.

In one embodiment, chemicals added to an antigen retrieval solution are chemicals that raise, or elevate, the boiling point of the antigen retrieval solution. Examples of such chemicals include, but are not limited to, glycols and glycerols.

In some embodiments, a chemical is ethylene glycol at a concentration of 50%, 60%, 70%, 80% or even 90% by volume of an antigen retrieval solution.

In other embodiments, a chemical is glycerol at a concentration of 50%, 60%, 70%, 80% or even 90% by volume of an antigen retrieval solution.

In other embodiments compositions of the invention include at least one antigen retrieval solution comprising at least 50% propylene glycol.

In other embodiments compositions of the invention include at least one antigen retrieval solution comprising at least 20% by volume of ethylene glycol, such as e.g. about 20, 25, 30, 40, 50, 60, 70, 80, 90 or even 95% ethylene glycol.

In even further embodiments, the antigen retrieval solution comprises a first and a second liquid. Said first liquid may in some embodiments comprise at least 50% of the total volume of the antigen retrieval solution and said second liquid comprise at least 10%, such as 10, 20, 25, 30, 35, or even 40%, of the total volume of the antigen retrieval solution.

In other embodiments, compositions of the invention include at least one antigen retrieval solution comprising at least 50% by volume of glycerol.

In other embodiments, compositions of the invention include at least one antigen retrieval solution comprising at least 70% by volume of ethylene glycol, or at least 70% by volume of glycerol.

One particular embodiment of the composition according to the invention comprises about 55% by volume of propylene glycol as a first liquid and about 25% by volume of ethylene glycol as a second liquid. Compositions of the invention can be used on a variety of biological samples, including but not limited to, human cell preparations, frozen tissue sections and paraffin embedded tissue sections. Examples of cell preparations include, but are not limited to, smears of peripheral blood, bone marrow, and other bodily fluids containing a large number of hematopoietic cells. Compositions of the invention may include additional compounds, such as, for example, detergents and/or chelating agents. Examples of chelating agents include, but are not limited to, EDTA, citric acid and sodium citrate. Examples of detergents include, but are not limited to, Tween 20, NP40, Triton X, Saponin, and Brij.

Compositions of the invention can be used at any pH that is useful for effective antigen retrieval.

In some embodiments, a pH of a composition of the invention is between about 2.0 and about 9.0. In other embodiments, a pH of a composition of the invention is between about 2.0 and about 6.0. However, a skilled artisan can easily test compositions of the invention with varying pHs to determine which pH is most effective for antigen retrieval.

Compositions of the invention may be especially useful for antigen retrieval for use in laboratories located at higher elevations where boiling points are generally depressed.

This invention also provides methods of antigen retrieval using the compositions of the invention.

Generally, methods of the invention include, for example, contacting a biological sample with an antigen retrieval solution of the invention and heating the solution to an elevated temperature for duration of time necessary for antigen retrieval.

Both pressurized as well as non-pressurized devices can be used in the methods of the invention. The following examples provide illustrative embodiments of the invention.

One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit and scope of the present invention. Such modifications and variations are encompassed within the scope of the invention. The examples do not in any way limit the invention.

Kits and uses The invention further discloses use of a composition for antigen retrieval comprising at least one chelating agent, at least one detergent and at least 50% by volume of at least one liquid that facilitates antigen retrieval.

In further embodiments of said uses, the composition is any composition according to the invention.

The invention further provides a kit for antigen retrieval comprising a) a composition for antigen retrieval comprising at least one chelating agent, at least one detergent and at least-50% by volume of at least one liquid that facilitates antigen retrieval, b) optionally instructions for using said antigen retrieval composition. In further embodiments, the kit provides a composition according to the invention.

In further embodiments, the kit may provide several compositions according to the invention such as at least 2, 3, 4, 5, 6, 7, 8, 9, or even 10 compositions for antigen retrieval, and, optionally, instructions for using the at least two compositions according to the invention.

Examples

All antibodies used in these studies were obtained from DakoCytomation (Carpenteria, CA). Various combinations of enzyme blocking reagents were prepared from citric acid (Sigma Chemical Co., St. Louis, MO), acetic acid (Sigma) and hydrogen peroxide (Sigma).

Example 1: Tissue Fixation

Human tissues were fixed in 10% neutral buffered formalin (NBF) using 20 ml_, of 10 mM NaH₂PO₄/ Na₂HPO₄, (Merck, Whitehouse Station, NJ) and 0.145 M NaCI (Merck, Whitehouse Station, NJ), pH 7,0. The samples were incubated overnight (18 hours) in a ventilated laboratory hood at room temperature.

Example 2: Sample Dehydration and paraffin embedding

The tissue samples were dehydrated by sequential incubation with 70% ethanol two times for 45 minutes, 100% ethanol two times for 45 minutes, xylene two times for 45 minutes, and then transferred to melted paraffin (melting point 56- 58⁰C) (Merck, Whitehouse Station, NJ). Paraffin-infiltrated samples were transferred to fresh warm paraffin and incubated for an additional 60 minutes before being embedded with paraffin in a cast (Sekura, Japan). The samples were cooled to form the final paraffin blocks. The marked paraffin blocks containing the embedded tissue samples were stored at 2-8⁰C in the dark before being cut, mounted, deparaffinated and stained.

Example 3: Cutting, mounting and deparaffination of embedded samples The paraffin blocks were mounted in a microtome (0355 model RM2065,

Feather S35 knives, set at 4.0 micrometer) (Leica, Bannockburn, IL). The first few mm were cut and discarded. Paraffin sections were then cut, at room temperature, into sections 4-micrometers thick and collected. The paraffin sections were gently stretched on a 37°C hot water bath before being collected and mounted onto marked microscope glass slides (Superfrost plus) (Fisher, Medford, MA).

The slides were then dried overnight at ambient temperature. The slides were deparaffinized by incubating two times in Histoclear for 2-5 minutes, two times in 100% ethanol for 2-5 minutes, two times in 70% ethanol for 2-5 minutes and once in tris buffered saline (TBS) (50 mM Tris-HCI, 150 mM NaCI, pH 7.6) (Crystal Chem Inc., Downers Grove IL) for 5 minutes.

After mounting on slides, the tissue samples were antigen retrieved (AR) using any one of the methods described below.

Example 4: Compositions for Antigen Retrieval

Seven different compositions were used for antigen retrieval. Ethylene glycol (Sigma, St. Louis, MO) and glycerol (JT Baker, Phillipsburg, NJ) were added in varying concentrations to commercially available antigen retrieval solutions - TRS S1699 and high pH S3307 (Dakocytomation, Carpinteria, CA). The seven compositions were formulated as described below for use in various antigen retrieval methods:

1. TRS-S1699-diluted 10 fold to achieve a final citrate concentration of 1X (1OmM) at pH 6.1 , (no ethylene glycol nor glycerol added). 2. High pH S3307-diluted 10 fold to achieve a final EDTA concentration of 1X (1 mM) at pH 9.9, with no ethylene glycol or glycerol added.

3. 10% TRS-S 1699 and 70% glycerol in water.

4. 10% TRS-S 1699 and 90% glycerol by volume. 5. 70% ethylene glycol in water.

6. 10% TRS-S 1699 and 70% ethylene glycol in water.

7. 10% High pH S3307 and 70% ethylene glycol in water.

Each of these antigen retrieval solutions was subsequently used for antigen retrieval using the various methods described herein.

Example 5: A Non-Pressure Water Bath Device for Antigen Retrieval

A water bath capable of maintaining the temperature of about 95°-97°C was used. A staining bucket (Sakura, Torrence, CA) filled with about 220 ml of an antigen retrieval solution was pre-heated in the water bath to the final temperature of approximately 95°-97°C. Slide racks containing up to 24 slides were then placed into the heated solution. The length of exposure to heat was 20 minutes for all antibodies except for Ki-67 and TdT where the exposure time was 40 minutes. In all cases the length of exposure to heat was determined from the manufacturer's recommendations for each antibody.

The staining buckets and slides were removed from the water bath and allowed to cool at ambient temperature for an additional 20 minutes. During this cooling phase the antigen retrieval solution reached a temperature of approximately 55°C. Slides were then transferred to a second staining bucket containing water at ambient temperature.

Total time of antigen retrieval was 40 minutes except for Ki-67 and TdT where the total time was 60 minutes.

Example 6: A Pressure Device for Antigen Retrieval Slides were loaded into staining buckets containing about 220 ml of an antigen retrieval solution at ambient temperature and placed into a pressure device (Digital Decloaking Chamber; BioCare Medical, Walnut Creek, CA). Heat was applied and the final operating temperature (120°-124°C) was achieved in approximately 20 minutes. The temperature was maintained for another 5 minutes, and then slides were allowed to cool for approximately 25 minutes in the sealed pressure chamber. After the cooling period the temperature of the antigen retrieval solution was about 85°C, and slides were removed and placed into a second staining bucket containing water at ambient temperature.

Total time of antigen retrieval was approximately 50 minutes.

Example 7: An Open Bath Heating Chamber for Antigen Retrieval

An open bath-heating chamber was used for antigen retrieval. The heating chamber was designed to accommodate a single rack of 24 microscope slides. The rack with slides was lowered into the liquid reservoir containing about 200 ml of an antigen retrieval solution at an ambient temperature. The chamber was constructed with a heating blanket surrounding the aluminum reservoir.

Current was applied to the heating chamber, and after approximately 12 minutes, the antigen retrieval solution had reached its operating temperature of about 12O⁰C. Slides were maintained at approximately 120°C for about eight minutes and then removed, without cooling, to a second container with water at ambient temperature.

Total time of antigen retrieval was 20 minutes.

Example 8: Effect of Heat on antigen retrieval solutions

Various antigen retrieval solutions described in Example 4 were heated in an open bath-heating chamber for approximately 60 minutes. A solution of 70% ethylene glycol lost about 16% of its volume after heating the solution at about 12O⁰C for about 60 minutes, whereas a solution of 70% glycerol lost about 25% of its volume.

By comparison, the water-based TRS S1699 antigen retrieval solution heated to 97°C for 60 minutes lost 48% of its original volume.

These results showed that 70% solutions of either ethylene glycol or glycerol retained sufficient volume at 12O⁰C for their use as potential antigen retrieval solutions. Example 9: Antigen Retrieval Using Ethylene Glycol Containing Antigen Retrieval Solutions

Ethylene glycol, either alone, or in combination with antigen retrieval solutions containing detergents and/or chelating agents ((TRS S 1699 which includes citrate at a final working concentration of 1OmM or High pH S3307 which includes EDTA at a final working concentration of 1 mM) (Dakocytomation)) was used for antigen retrieval by heating for about 20 minutes at approximately 120⁰C in an open bath heating chamber.

Human tissue samples derived from tonsil, thymoma and breast carcinoma were treated with different antigen retrieval solutions, as summarized in Table 1. Specifically, tonsil tissue was treated either with ethylene glycol in water, with TRS-S 1699 and ethylene glycol or with high pH S3307 and ethylene glycol. Thymoma tissue was treated either with ethylene glycol alone or with high pH S3307 and ethylene glycol. Breast carcinoma tissue was treated either with ethylene glycol alone or with TRS-S1699 and ethylene glycol. Subsequent to antigen retrieval, the tissues were stained via immunohistochemistry using antibodies against specific antigens. Tonsil tissue was stained for the detection of Bcl-2, CD5, and Ki-67 antigen, thymoma tissue was stained for detection of TdT antigen, and breast carcinoma was stained for the detection of the estrogen receptor antigen. After antigen retrieval the IHC staining procedure consisted of the following steps:

1. Incubation in 3% H₂O2 for 5 minutes to block endogenous peroxidase

2. Rinse in Tris buffer 3. Application of primary antibody for 30 minutes

4. Rinse in Tris buffer 5. Application of Envision (DakoCytomation) + conjugate (polymer conjugate containing secondary antibodies and peroxidase enzyme) for 30 minutes

6. Rinse in Tris buffer 7. Application of diaminobenzidine/ H₂O₂ substrate for 5 minutes

8. Rinse in distilled water

9. Counterstain in Meyer's hematoxylin for 1 minutes

10. Mount with Faramount (DakoCytomation) and apply a glass cqver slip.

The efficacy of an antigen retrieval method can be determined by taking the finished biological sample and viewing it under the microscope in order to judge its staining characteristics. An acceptably stained biological sample will demonstrate strong staining of the biological marker of interest.

Staining intensity can be visually scored by an observer examining the stained biological sample under a microscope. Staining intensity is rated on a scale of 0 - 4 where 0 is no staining, 1 is weak staining, 2 is moderate staining, 3 is strong staining, and 4 is very strong staining.

As depicted in Table 1 , staining results from treatment with antigen retrieval solutions containing 70% ethylene glycol and chelating agents were superior to staining results with 70% ethylene glycol in water. Staining with the negative control reagents failed to produce any staining in the tissues studied.

Table 1

<img file="WO2006007841A2_D0001.tif" /><img file="WO2006007841A2_D0002.tif" />

In the next set of experiments, TRS-S1699 containing 70% glycerol, 90% glycerol, or 70% ethylene glycol were used for antigen retrieval. Antigen retrieval was performed in an open bath-heating chamber for about 20 minutes at 120⁰C. Table 2 depicts a comparison of these solutions in a typical IHC assay. These results showed that solutions of both ethylene glycol and glycerol were effective antigen retrieval solutions for the antibodies tested (ID5, 8G7G3 and MIB 1), however, ethylene glycol produced superior results in terms of intensity and number of sites stained compared to glycerol.

Negative control reagents failed to produce any staining. Staining results were an average of duplicate determinations.

Table 2.

<img file="WO2006007841A2_D0003.tif" />

EXAMPLE 10: Effect of temperature on antigen retrieval using ethylene glycol.

Antigen retrieval was performed at two different temperatures-97°C and 120⁰C in an open heating chamber, in order to determine whether the enhanced staining observed with ethylene glycol containing TRS-S1699 was a function of elevated temperature of the retrieval process (12O⁰C) or a function of the composition of the solution. Results from this comparison are shown in Table 3. For each of the three antibodies evaluated, the antigen retrieval was more effective at the higher temperature. No staining was observed in the negative controls. Table 3

<img file="WO2006007841A2_D0004.tif" />

Example 11 : lmmunohistochemistry using different antibodies following antigen retrieval

Following antigen retrieval using three different antigen retrieval devices- water bath, pressure device and open bath heating chamber, 40 different antibodies, selected to represent a broad spectrum of antigens were used for staining a wide variety of tissues. Antigen retrieval in a non-pressure device was performed by heating the

TRS-S1699 solution in a water bath at 97°C for 20 (except for Ki-67 and Tdt where the heating time was 40 minutes).

Antigen retrieval in a pressure device was performed by heating the TRS- S1699 solution at 120⁰C for 50 minutes, as previously described herein. An ethylene glycol containing TRS-S1699 solution was used with an open bath-heating chamber by heating at 120⁰C for 20 minutes.

As depicted in Table 4, in every case the open bath chamber device used for antigen retrieval using a temperature of 120⁰C achieved comparable or superior results compared to the pressure device. Both methods were in fact superior to a water bath at 97⁰C. The low viscosity of the ethylene glycol allowed for easy handling and rinsing of slides and easy movement of fluids through tubing of the heating chamber device. Furthermore, the entire process from beginning to end was less than 25 minutes including slide loading and unloading. When compared to a standard water bath method, the open bath heating chamber method produced superior results in terms of intensity and number of sites stained. Results were comparable to a pressure device method; however, the open bath method with ethylene glycol had the added advantage of faster throughput (20 vs. 50 minutes).

Table 4.

Antibody (Clone) Tissue Antigen Retrieval Device

Water bath Pressure device

Ooen bath chamber

ER (1D5) Breast carcinoma 1.5 1.5 2.75

PR (PgR 636) Breast carcinoma 2.5 2.75 3.0

Alk-1 (ALK1) ALCL 2.0 2.75 3.25

CEA (P) Colon carcinoma 2.5 2.75 3.25

CD 30 (Ber-H2) Hodgkin's disease 2.25 2.5 2.75

P53 (DO-7) Lung carcinoma 2.25 2.5 2.75

Ttf (8G7G3) Lung carcinoma 2.25 2.5

2.5^e

C-kit (P) GIST 2.75 3.0 3.5

Melan-A (A103) Melanoma 3.0 3.5 3.5

CD 15 (C3D-1) Hodgkin's disease 2.5 2.75 3.0

Bcl-2 (124) Tonsil 2.0 1.5 2.0

Tdt (P) Thymoma 3.0 3.0 3.0

Thyroglobulin (P) Thyroid 3.0 3.0 3.25

CD 79a (JCB117) Tonsil 2.25 2.75 2.75 Calretinin (P) Mesothelioma 2.0 2.5

2.5^e

CD 1a (010) Skin 2.0 2.75

2.75^e

E-Cad (NCH-38) Breast carcinoma 3.0 3.0 3.25

CK 7 (OV-TL 12/30) Breast carcinoma 1.5 2.0 2.75

g CK 20 (Ks 20.8) Colon carcinoma 2.75 3.5 3.5

CK (mnf 116) Lung carcinoma 2.0 2.5 3.0

CK hmw (34/8E12) Skin 2.5 2.75 2.75

CD 68 (PG M1 ) Tonsil 2.75 3.0

3.0^e

FVIII (P) Tonsil 1.75 3.0

3.0^e rj Kappa Light Chains (P) Tonsil 1.75 2.75 2.5

Lambda Light Chains (P) Tonsil 2.25 3.5 3.0

Example 12: Composition for antigen retrieval using two liquids

The following composition is an example of an antigen retrieval solution 5 using two liquids that will give a boiling temperature of 131-132^°C at sea level.

The composition is compared to a one liquid composition with a lower boiling point

The composition was formulated as described below for use in various antigen retrieval solution methods. 0

Composition 1 - high boiling point

10% TRS-S1699 (DakoCytomation) (final citrate concentration 1OmM at pH 6.1 ) 24.9% ethylene glycol 55.1 % propylene glycol 5 10% deionised water Boiling point: 131-132^°C.

Composition 2 - low boiling point

This composition corresponds to composition no 6 in Example 4. Boiling point: 122-123⁰C.

The composition is used as antigen retrieval solution in an open water bath at 121⁰C.

Antibodies tested

Primary Antibody: anti-ER (anti-estrogen receptor), or anti-PR (progesterone receptor)

Detection system: EnVision+ (DakoCytomation)

Chromogen: DAB+ (DakoCytomation)

Tissue: Human breast carcinoma, pre-screned to be positive for either ER or PR.

Staining intensity is rated on a scale of 0 - 4 where 0 is no staining, 1 is weak staining, 2 is moderate staining, 3 is strong staining, and 4 is very strong staining.

Results are shown in Table 5 (anti-ER) and Table 6 (anti-PR).

Table 5 - Staining intensities obtained with two formulations

<img file="WO2006007841A2_D0005.tif" /> human breast carcinoma

The results show in table 5 gives that the high boiling temperature composition (1 ) will give the same or even slightly higher staining intensities compares to the low boiling temperature composition (2). Table 6 - Staining intensities obtained with two formulations

<img file="WO2006007841A2_D0006.tif" />

The results in Table 6 shows that the high boiling temperature formulation will give about the same staining intensities, or slightly lower, compared to the low boiling temperature composition.

The observed differences are within the normal acceptance criteria of ±0.25 grade.