JP2020523616A - Method of scale extrapolation for IHC antigen imaging - Google Patents

Abstract

Provided in this disclosure are methods of scale extrapolation of IHC antigen imaging. Provided in this disclosure are, in particular, methods for generating an antigen concentration scale from a known gradient density target series of secondary mammalian IgG sera and optionally antigen concentration. The main application of the method described above is to aid image analysis on slides using a target protein concentration scale. The above method is used to form a primary antigen concentration scale from a secondary protein concentration scale. In this case, the primary antigen concentration scale is applied to co-existing tissue sections to utilize abnormal tissue sections of detected cells such as cancer.

Description

[Cross-reference of related applications]
This application has priority over US Provisional Patent Application No. 62/520187 filed June 15, 2017 and US Provisional Patent Application No. 62/520319 filed June 15, 2017. Claimed, and each disclosure is incorporated herein by reference in its entirety.

The present invention relates to a method of scale extrapolation of IHC (immunohistochemical) antigen imaging. The invention particularly relates to a method for constructing an antigen concentration scale from a known gradient density target series of secondary mammalian IgG sera and optionally antigen concentration. The main application of the method described above is to aid image analysis on slides using a target protein concentration scale. The above method is used to form a primary antigen concentration scale from a secondary protein concentration scale. In this case, the primary antigen concentration scale is applied to co-existing tissue sections to utilize abnormal tissue sections of detected cells such as cancer.

Immunoassays are used when it is necessary to quantify an analyte of unknown concentration in a sample. To obtain the most accurate determination of unknown concentration, immunoassays should be based not only on standard assay development criteria (standard deviation or optimal signal window) but also on how well the immunoassay can predict the value of the unknown sample. Must be developed. First, it is necessary to establish the key success factors of the assay. Then there is a need to develop immunoassays that establish proof of concept. At the optimization stage, the quantifiable range of the immunoassay method is determined by calculating the accuracy profile in the matrix in which the experimental sample is measured. The analyte is then added to the matrix and spiked recovery is performed by determining the recovery of the analyte in the matrix. If the accuracy profile is within the desired operating range, the spiked recovery sample is assayed for several days to complete the immunoassay validation. If the accuracy profile limits are not within the desired operating range, further optimization of the immunoassay is required prior to validation.

The main application of the method disclosed herein is to aid image analysis on slides using a target protein concentration scale. The present invention discloses methods used to form a primary antigen concentration scale from a secondary protein concentration scale. The primary antigen concentration scale is then applied to co-existing tissue sections in order to utilize tissue sections for abnormalities of detected cells such as cancer.

In general, in one aspect of the invention, a method of scale extrapolation of IHC antigen imaging is provided.

In another aspect of the invention, the invention discloses a method of producing an antigen concentration scale from an antigen concentration and a known gradient density target series of secondary mammalian IgG sera.

In yet another aspect of the invention, the main use of the above method is to aid image analysis on slides using a target protein concentration scale.

In yet another aspect of the invention, the method is used to form a primary antigen concentration scale from a secondary protein concentration scale.

In yet another aspect of the invention, the primary antigen concentration scale is applied to subsequently co-located tissue sections to utilize tissue sections for abnormalities of detected cells such as cancer.

In the following description, other aspects of the invention are disclosed.

FIG. 6 shows a portion of a slide containing primary and secondary stained capture targets after IHC staining with identification of various targets. It is a figure which shows the influence of the antigen activation process with respect to one of the secondary protein arrays after IHC staining. FIG. 6 shows a slide with a secondary protein concentration scale that has co-located tissue sections and is subjected to IHC staining.

The present invention may be understood more readily by reference to the following detailed description of the invention which forms a part of this disclosure. The present invention is not limited to the particular devices, methods, conditions, or parameters described and/or shown herein, and the terminology used herein is merely exemplary. It is to be understood that it is not intended to limit the claimed invention. Also, as used in the specification, including the claims appended hereto, the singular forms ('a','an', and'the') that do not specify a quantity include the plural forms and references to specific numerical values. Includes at least the specific numerical value, unless otherwise specified by the content. Where expressed in other embodiments herein, ranges may be expressed as from “about” or “approximately” another particular value. Also, unless stated otherwise, the dimensions and material properties referred to herein are exemplary rather than limiting, and are intended to provide a better understanding of the preferred practical sample embodiments. It should be understood that variations out of value may also fall within the scope of the invention depending on the particular application.

The invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following description or illustrated in the drawings. Other embodiments of the invention are possible and can be implemented or practiced in various ways. Also, the terms and terminology used herein are for the purpose of description and should not be construed in a limiting sense. Use of "including," "comprising," "having," "containing," "involving," and variations thereof includes additional items. ..

Immunohistochemical (IHC) staining is commonly used to assess the presence of specific antigenic sites in patient tissue sections. Subjective interpretations are applied to the staining densities on tissue sections to assign a diagnostic level of abnormal or cancerous conditions. In general, it is hypothesized that IHC treatments always function correctly and that tissue sections are labeled with visible chromogenic markers that identify them in the presence of abnormal or cancerous conditions. However, if antigen retrieval fails or the staining reagent is defective, no signature remains that identifies the artifact. For this reason, there is a large possibility that an effective diagnostic judgment cannot be entrusted to an experimental assistant or a pathologist. In other words, the physical form may not be sufficient to indicate an abnormal condition, the antigenic sites may not be labeled, and the slide may not provide more than that seen on hematoxylin and eosin (H&E) slides.

There are some passed in requirements needed to complete the calculation. The parameters you need to know are:
1. Antibody A: Dilution ratio, ID and host species (eg 25, ER, mouse)
2. Antibody B: Dilution and ID (example is 20, Ki-67). No host species are required because whatever antibody is used for antibody A, antibody B should be the opposite mouse/rabbit to antibody A.

It is known that the primary antibody is composed of processed host serum obtained from a host animal (mouse or rabbit) inoculated with the desired antigen fraction. The host then produces the serum protein, where the antigenic site contains the antibody reactant to the antigen antagonist. Subsequent contact of the antibody with a protein containing the target antigen results in the binding of the antigen and antibody. As a result, the antibody host species (mouse or rabbit) remains unreacted with the secondary staining kit.

The rationale for being able to use the IHC target to create an antigen density ruler for co-localized tissue sections follows the following set of items.

The density of adhesive binding sites on slides exceeds the area displacement of a single protein by at least two orders of magnitude.

The primary antibody and secondary protein have a known atomic mass (kDa), which can be converted to weight in nanograms.

The primary and secondary targets have well-defined circular attachment areas to which known aliquots of target material are applied. The protein deposit contains cross-linking couplers and therefore cannot sink beyond the depth of the protein within the pores of the slide coating. Therefore, the active surface protein density of the target can be calculated by knowing the atomic mass of the protein, the number of proteins of each protein type in the deposit, and the area of the target.

The applied concentration, aliquot and surface area of the primary antibody on the slide exposed to the reagent are known. It can reasonably be assumed that during the exposure time of the reagent, the majority of the floating antibody falls and is captured by the receptive antigenic sites. Only the antibody that has fallen directly above the antigen site will be captured, and the rest will be washed away by the buffer washing step. Thus, the adherent antibody concentration can be established when the concentration is greater than 25% above the cutoff and less than 25% of saturation.

a. The cutoff is defined as the density of target sites that are insufficient to capture the applied protein concentration.

b. Saturation is defined as the inability to capture all of the applied protein concentration.

Knowing the primary dilution allows one to choose the correct primary target density target and validate the primary concentration.

Each secondary and primary target is ((mouse or rabbit)+(donkey+crosslinker+fungal inhibitor)) or ((KLH with antigen A or KLH with antigen B)+(unconjugated KLH+crosslinker). Agent+fungal inhibitor))). Each dot has the same amount of total protein, but the mixing ratio must be adjusted slightly, as the atomic masses may differ between the proteins that make up a particular target. For example:
A. Mouse IgG=155 kDa
B. Rabbit IgG=150 kDa
C. Donkey IgG = 160 kDa
D. KLH subunit conjugated to an antigenic peptide chain (where the subunits are KLH1 and KLH2)=350 kDa & 390 kDa

The 2D secondary target series ranges from 10% to 100% according to the 20 log (dilution ratio) profile, where the dilution ratio ranges from 1:1 to 1000:1. A single 2D/3D target is used to measure the staining density Δ between 2D base and 3D particles. Δ can be applied to the rest of the 2D array to create a color density scale that fits well with the 3D behavior seen in or on tissue sections.

The secondary 100% 2D/3D and 2D targets confirm that the two deposits are matched for 2D staining density. This confirms that the 3D particle component does not consume 100% protein material sufficiently to cause a shift of the 2D component.

The secondary staining employs a 1-20 fold enzyme gain function that is a function of the staining reagent construction. Thus, as the gain increases, the lower concentration of secondary target shifts to saturation, while when the gain decreases to 1, only the high concentration of the secondary target is visibly stained.

When the secondary target array is not covalently immobilized, damage is seen in the deposits from the antigen retrieval process that IHC slides undergo. Although this provides a measure of AR effects, it is not useful for generating an antigen density scale that can be applied to tissues, as AR effects on tissues are always unknown. Therefore, the antigen density scale can only reflect what is left on or within the tissue section. As such, two antigen-stimulated targets are provided for the QC application of assessing the AR process.

Due to the considerable size difference between the secondary and primary target proteins, the density of protein concentration is established by the primary protein. Accepting the assumption that the KLH subunits KLH1 and KLH2 have a 50:50 distribution, their average value of 370 kDa can be used to set the primary target dilution.

Using an average primary antibody atomic mass of 150 kDa, the weight of a single antibody was 150 kDa (1.6605×10 ⁻¹² ) which corresponds to a weight of 249×10 ⁻¹² ng. The amount of primary reagent applied can be elucidated when a single area of the slide is the only part exposed. Therefore, using a closed capillary gap with internal dimensions of less than 20.3 mm square by 0.14 mm high, the volume is 57.2 μl. The ratio of 1 mm diameter to the target zone representing one area of the target dot results in 0.1 μl of applied primary antibody reagent.

The primary antibody reagent is diluted from the concentrate to the range of 1 μg/ml to 100 μg/ml. Thus, for an applied primary dilution of 1 μg/ml to 100 μg/ml, the target is exposed to 0.1 μg to 1 μg of antibody. Considering that the weight of the antibody is nominally 249×10 ⁻¹² ng, the target maximum protein exposure range of 1 mm is 41.06 to 4106 antibodies.

To ensure 100% capture capacity, the primary target should have a safety factor of 100-1000 fold. If the 1000× option is chosen, the primary target should contain 4×10 ⁶ antigenic sites. The KHL subunit is larger than the applied antibody, but the increment is not sufficient for the number of capture antibodies to vary by more than 1:1. Each KLH subunit has an average atomic mass of 370 kDa, which corresponds to a weight of 614.4×10 ⁻¹² ng.

The volume of a protein molecule can be very simply and reliably estimated from the molecular weight of the protein and the average protein partial specific volume (partial specific volume=volume/molecular weight). The average experimentally determined partial specific volume for soluble globular protein is about 0.73 cm ³ /g. This value varies from protein to protein, but the range is rather narrow. Convert the equation to a protein volume of approximately (1.212×10 ³ ×MW) nm ³ . Thus, for the KLH subunit, the individual volume is 448.44 nm ³ . When the protein is modeled as a sphere, the diameter of the sphere is 0.132×MW ^1/3 (nm). For the KLH subunit this is 9.436 nm.

At a target diameter of 1 mm, a monolayer of KLH subunits requires 11.237×10 ²⁷ proteins. With an active target density of 4×10 ⁶ proteins, the minimum dilution would be 1:2.8×10 ²¹ . Practically any dilution factor close to 1:1000 is effective since its active protein concentration governs the evaluation of the primary antibody. Therefore, the target density is limited only by its low concentration lower limit.

Secondary target arrays are in serial dilution increments of 1:1 to 1000:1. The linear gradient of dilution occurs as -20 log (dilution ratio) (hereinafter referred to as dBd). For the stated dilution range of 1:1 to 1000:1, the half-log range is 0 dBd to -60 dBd. If a dilution step of -3 dBd is chosen, the secondary target dilution will be 0 dBd, -3 dBd, -6 dBd, -9 dBd, -12 dBd, -15 dBd, -18 dBd, -21 dBd.

Staining may undergo saturation or cutoff depending on the concentration of primary antibody and the enzyme gain of the secondary staining kit. Saturation is when the density of enzyme sites exceeds the ability to precipitate the dye from the chromogen. In other words, the color of the stain will be as dark as feasible. The cut-off occurs when the concentration of primary antibody and the enzyme gain of the secondary staining kit are too low, resulting in insufficient pigment precipitation. These two factors shift the darkness of the secondary columns to saturation (100%) or cutoff (0%). Based on FIG. 2, this movement is seen as the number of visible targets. As the secondary enzyme gain increases, 100% dot density shifts to the 0% position side. Typical enzyme gains are 1,2,4,5,8,10,15 and 20. These can be restated as a shift of the secondary array towards the 0% position:
20-fold shift of all targets to -26 dBd;
15-fold shift of all targets to -23.52;
10-fold shift of all targets to -20;
5-fold shifts all targets to -13.98;
4-fold shifts all targets to --12.04;
2-fold shifts all targets to -6.02;
1x 2D Only 100% dots are close to black.

When the primary target array is present, the increase in secondary enzyme gain shifts the staining density towards the lower primary density dots. The same applies when the primary antibody concentration increases. The antigen retrieval process degrades both primary and secondary targets to some extent, which reverses the shift to the cutoff side. If 3 or more dots disappear at the end of IHC staining, the slide is considered to have undergone excessive antigen activation time, temperature, or both, and the presence of excessive antigen is lost on the tissue, limiting diagnostic interpretation. Becomes This determination is independent of the efficacy of the primary antibody as it has already been shown to impair secondary staining. There is no way in the antibody stage to overcome this damage level.

Usually, AR damage that shifts the secondary array to 100% position by 3 dots or more is considered excessive, and it is necessary to repeat the slide using a secondary staining kit of higher enzyme gain or a higher concentration of antibody. is there.

Therefore, the color density of the primary antigen target is the total antibody concentration times the enzyme gain of the secondary staining kit. On the other hand, the secondary target density is the enzyme gain multiplied by only the secondary target protein concentration.

Depending on the digital imaging system, changes in illumination shift the dynamic range of the image to compression (darker) or saturation (brighter). These changes shift the antigen color scale, but not the numerical scale of antigen density. Therefore, the numerical scale is independent of the illuminance, and the color scale is dependent on the illuminance.

In QC mode, coexisting targets provide feedback for the IHC process, as shown in FIG. Four columns of the secondary array within the nominal value, above the nominal value, significantly above the nominal value and excessively above the nominal value (5%, 10%, 30% and 40% respectively) It is shown by the difference in the degree of antigen activation performed. The antigen activation process seeks to unmask the antigenic site by reversing the Schiff base bond between formaldehyde and protein. The rate at which the antigen is exposed depends largely on the reaction temperature. As the temperature rises, the possibility of nucleated boiling arises. Nucleate boiling causes physical damage to both tissue and protein deposits. Ideally, antigen-stimulating activity is uniform across slides, but in practice this is not the case and regions with higher or lower antigen-stimulating activity depending on the method and environment used. Occurs. Assuming uniform antigen-stimulating activity, the following can be used to indicate that the slide can be used for diagnostic judgment.

If the AR is minimal or excessive, the secondary array may not be able to reflect the failure. However, two AR targets suggest excessive failure conditions.

Low AR is seen as less than constant 2D/3D and above constant 2D, both targets are black. The secondary array is fully visible with no residual AR shift of the target.

Low AR activity can result from the following situations in the IHC stain device:
AR heating device is not working or is set to much lower than 80°C;
AR buffer has a neutral pH of 7 rather than pH 6 or 9;
Exposure time is too short.

High AR is seen as less than constant 2D/3D, significantly whitened, and more than constant 2D target is less than 50% black. Most of the secondary array also becomes white.

High AR activity can result from the following situations in the IHC stainer:
The heating device operates at temperatures above 95°C;
Excessive exposure time.

Errors in chromogenic precipitation can occur under two circumstances.

At high concentrations of the secondary target, the staining intensity drops rather than at maximum darkness. In secondary arrays, there is always a need to increase the site density. If not, the chromogen precipitation depleted the capacity of the secondary reagent kit. The solution is to increase the dilution of the primary antibody (similar to decreasing the antibody concentration).

Chromogenic reagents are degraded due to activation (often caused by DAB). The solution is to use a new DAB mixture.

Viewing a microscope slide with a conventional microscope is subjective in terms of illumination level. Whole slide imaging (WSI) uses full white and black holes in the scanner to establish white balance and contrast. This does not apply to manual microscopes. FIG. 3 shows the effect on the image when the illumination level is too dark (-5% less than optimal), optimal (+0) and excessively bright (such as +10% or +15%). When the light level is below optimal, compaction of dyeing density is seen. With respect to the stage of the cancer, this may make the diagnosis one step higher than it should be. When the light level is above the optimum, whitening of the image is seen. With respect to the stage of the cancer, this can cause the diagnosis to be one step lower than it should be. An antigen color density and numerical ruler can be generated from the primary and secondary targets and overlaid on the WSI image. While numerical scale is an independent matter, color density is a dependent matter. When applying the color and numerical ruler of antigen density to WSI, the numerical scale remains fixed as the user raises or lowers the illumination level. On the other hand, the color density scale shifts according to the change of the illumination level. The advantage is that the user has the option of raising or lowering the apparent illumination without losing the numerical relationship with the color density so that the features on the histology are "best seen". This is also practical when changing the magnification.

There are two formats in which an antigen density ruler can be created. Type A is based on the assumption that the primary antibody is always applied with less than 10% excess antibody to the tissue antigenic site. Type B uses a gradient density array of primary antigens.

Type A: Antigen Ruler Based on Secondary Only This format uses only secondary target arrays. Confirmed information incorporated into the 2D barcode includes (a) primary antibody data: dilution of the antibody in host species and -dBd, and (b) secondary enzyme gain.

The secondary gradient density target array is composed of known concentrations of proteins with a -3 dBd decrement between targets. The maximum concentration is chosen according to the lowest dilution used for the primary antibody. Most users dilute to a constant intermediate concentration of 1 μg/ml using the concentration reference values provided by the antibody reagent manufacturer. Then all other dilutions are made as needed to accommodate different tissue types. Generally, the second set of primary antibody dilutions ranges from 1:1 to 1000:1.

To accommodate the range of secondary enzyme gains, the secondary array must consist of a wider range of dilutions. Therefore, at -3 dBd steps, the lowest dilution of the secondary array starts at 1000:1 or -60 dBd (represented by SdBd). In this case, the maximum value of the 8-dot series is −0 dBd or 1:1. Secondary proteins are degraded by the action of antigen activation (represented by ARdBd). Each dot out of eight in the secondary array represents an increment of -3 dBd. The disappearance due to antigen activation (no longer visible) for the disappearance of the two targets is +6 dBd. This means that the secondary array is (-S+AR)dBd or (+6dBd to -54dBd) for the 2D target. Here, it is necessary to take into account the antibody concentration and the secondary enzyme gain. The antibody concentration is AdBd, but the enzyme gain is EdBd. This results in a secondary array of (-S+AR-E)dBd and a tissue of (+AR-E+A)dBd. The next factor to apply is 100% 2D vs. 3D difference. The staining difference between 100% 2D/3D target and 3D objects at 100% 2D represents the chromogenic precipitation constant of the secondary stain, which is used to assign color density to the numerical scale and is assigned to DdBd. The difference in color density is applied to each 2D target in the array. Therefore, the 2D array exists with a dye color density of (+AR-E+A+D) dBd.

For example, when the enzyme gain is 10 times, E becomes −20 dBd. In this case, the 2D secondary array is -14, -17, -20, -23, -26, -29, blank, blank (dBd). Two dots near 0% are blank because they have been damaged by the antigen retrieval process so that they cannot be recovered by staining. For example, when the 2D/3D color density difference is 10 times, D becomes +20 dBd, and the 3D secondary array becomes −34, −37, −40, −43, −46, −49, blank, blank (dBd). Become. It is assumed that the primary antibody reagent finds a suitable antigenic site in the primary target and yields 100%. It is also assumed that there are far more than two antigenic peptide chains per KLH protein, but only one antibody per KLH protein can effectively bind and stain. Any additional antibody that finds a suitable antigen on the same KLH protein prevents the completion of secondary staining due to overlapping occupancy. Therefore, the number of antigenic sites that can be detected per primary antigen-carrying protein is 1. Since the primary target contains the same number of proteins per micron as the secondary, to adjust the secondary color density to the numerical antigen density, a primary dilution from 500 μg/ml antibody master was added to the secondary array data. Apply. By monitoring the secondary target, one selects a target with an intermediate color density. Intermediate color density is defined as the 50% point between maximum black and maximum white. This point corresponds to 1.5 dBd in the 3 dBd range. In this case, this point serves as an anchor that establishes the antigen density ruler. Using the final target range above, the midpoint would be -41.5 dBd.

Dilute secondary protein to 10 μg/ml master diluent. Each array is a mouse or rabbit blend mixed with donkey IgG protein. It is assumed that the proteins all have different atomic masses, but below all are 150 kDa, the total number of proteins per target dot is constant and the mixing ratio is not constant. For the moment, only the reactive protein concentration is considered. At 150 kDa, the molecular weight MW of the individual proteins is 249.07×10 ⁻¹² ng. Standard target dots are 1 mm in diameter. If the deposit to be printed is 1 μm thick, the concentration of deposit is 10 μg/ml and 31.5×10 ⁶ proteins are deposited. In this case, the region with a diameter of 1 μm has 31.5 proteins. Antigen density can be established by assuming that one protein corresponds to one antigenic site. The secondary array uses the same number of proteins per deposit, but the ratio of mouse or rabbit to donkey changes as the concentration of mouse or rabbit is reduced. 100% targets were all mice or rabbits, corresponding to a 0 dBd point on the ruler.

In the secondary, the tissue is stained only if the primary antibody binds to an antigenic site on the tissue. This is not particularly dependent on the concentration of antibody applied, except that sufficient antibody concentration must be provided to bind the available antigenic sites. Therefore, the measured value of antigen density on the tissue remains constant, but the value must be corrected for damage due to antigen activation and secondary enzyme gain. In this case, it is necessary to adjust the color density for the numerical measurement value.

In the previous example, the enzyme gain was 10-fold and antigen activation caused the disappearance of two dots from the secondary array. The enzyme gain is -20 dBd and the loss due to antigen activation is +6 dBd. The result is -14 dBd. In this case, the dilution is converted as follows:

Type B: Ruler Based on Primary Antigen This format uses both primary and secondary target arrays. Confirmed information incorporated into the 2D barcode includes (a) primary antibody data: dilution of antibody in host species and dB dilution, and (b) secondary enzyme gain. Lot code data contains information about the primary target combination used.

If there is a primary target sequence, there will be 3 dots, where the densest dots will have the same 100% density as the secondary array, but the dots will be spaced -6 dBd apart. In practice, the primary and secondary arrays have the same dilution gradient. The primary targets are −0 dBd, −6 dBd, −12 dBd and are represented by PdBd. It is reasonable to expect that antigen retrieval causes damage and is nearly identical to the secondary array. The primary array is subject to the secondary staining and therefore the same enzyme gain function. Thus, the primary array will be (-A+AR-E)dBd, where the primary target density is controlled by the dilution of the primary antibody. The only requirement is that P will always be greater than A. With a 10-fold enzyme gain=−20 dBd and +6 dBd of antigen-stimulated elimination, the primary array is −20 dBd, −26 dBd, −32 dBd. The disappearance due to antigen retrieval does not act as much as it would blank them to the primary target, based on their effect on the secondary array. The secondary array is sufficient to make the antigen density ruler, but it is important to make sure that the primary dilution was applied correctly. Therefore, the primary target functions in its capacity.

This application incorporates in its entirety references to the definition of slides with a target protein concentration scale disclosed in US Provisional Patent Application No. 62/520319, entitled "Process Record Slide for Immunohistochemical Staining". The above slides with target protein concentration scale, also defined in US Provisional Patent Application No. 62/520319, consist of at least two secondary protein target arrays and optionally one or more primary antigen target arrays.

In one embodiment of the invention, the secondary protein target array described above forms a mouse IgG and dummy that form a gradient density series of 5 members or more going from maximum density to minimum density in a 20 log (dilution ratio) curve. (Dummy) IgG formed as two rows of other rabbit IgG mixed with serum protein, where the dilution ratio can range from 1:1 to 1000:1.

In another embodiment, in the final process step, the identified antigenic sites are colored by chromogenic precipitation. This allows the mouse and rabbit target arrays to reflect the 20 log (dilution) curve of the chromogenic precipitate of the secondary staining kit.

In another embodiment, a preferred solution of the method of forming the primary antigen density scale is that the target mixture is successfully composed and they adhere on a slide with the target protein concentration scale, between the adhesive and the target substance. It is assumed that it has a covalent bond of.

In another embodiment, curve fitting between datasets can be easily done by computer algorithms, assuming that the target array has been successfully applied and that both primary and secondary staining reagents work reasonably well. In another embodiment, the primary staining is IHC-approved using a mouse or rabbit host protein that is not further conjugated to a fluorescent marker or integrated with an enzymatic site (such as HRP or AP). It can be selected from any antibody. In another embodiment, the secondary staining is selected from secondary staining with enzyme gain of 1 to 25 times, each of which is independently independent in mouse and rabbit and each uses different color chromogens. However, the present invention is not limited to these.

It is appropriate to note that in another embodiment of the invention, the absolute performance results on one slide may not be the same as another slide performed at another time. This is because the secondary staining kits perform differently from lot to lot like primary conjugated primary antibodies. However, for the performance of any one of the slides with the target protein concentration scale, the antigen scale is effective and is roughly equivalent to another run with different staining reagents.

In another embodiment, the primary antigen concentration scale is applied to subsequently co-located tissue sections in order to utilize abnormal cell sections of detected cells such as cancer.

In another embodiment, the slide with the target protein concentration scale is as described below.

A slide comprising a detection zone and a control zone,
The detection zone is the space where tissue sections or loose cells are applied for treatment by immunohistochemistry (IHC) and subsequent testing,
The control zone comprises one or more sets of primary and/or secondary target arrays,
The secondary target array may be one or more (eg 1 to 50, 5 to 45, 10 to 40, 15 to 35 or 20 to 30, especially 1, 2, 3 4, 5, 6, 7, 8, 9 or 10 secondary target loading dots (dots are circular, oval, square, diamond, etc. any regular or irregular shape) Each secondary target loading dot is a mixture of a host protein (eg IgG) and a dummy protein (eg IgG) immobilized on the slide in a certain ratio.

The term "dummy protein" means a protein that does not react with the secondary antibody and is used in admixture with the host protein to obtain a gradient dilution. A preferred dummy protein is donkey protein (IgG) or equine protein (IgG).

The term "host protein" means a protein (especially IgG) having the same origin as the primary antibody such as mouse, rat, rabbit, donkey, horse and goat protein (IgG).

An example of a slide is shown in FIG. 1 and detailed target identification is shown in FIG.

Various embodiments are described herein as examples. It will be apparent to those skilled in the art that various modifications can be made and other embodiments can be made without departing from the broader scope of the invention presented herein. These and other changes to the exemplary embodiments are intended to be covered by the present invention.

Claims (10)

A method of extrapolation of IHC imaging in which an antigen concentration scale is generated from a known gradient density target series of antigen concentration and a secondary mammalian IgG, the method comprising:
(A) exposing the microscope slide to a primary staining reagent composed of antibodies,
(B) capturing the antibody on its compatible antigen target;
(C) binding the antibody of step (a) to the secondary target array using a secondary staining reagent kit for antigen identification,
(D) coloring the identified antigen of step (c) by chromogenic precipitation;
(E) a step of calculating/comparing color intensity,
Wherein the microscope slide comprises a detection zone and a control zone,
The detection zone is a space where tissue sections or free cells are applied for treatment by immunohistochemistry (IHC) and subsequent testing,
The method wherein the control zone comprises two secondary mammalian IgG arrays fixed to the slide in a certain ratio and optionally one or more sets of antigen arrays. The method according to claim 1, wherein the antibody is a mouse IgG or rabbit IgG type antibody. 3. The method of claim 1 or 2, wherein all antigens and secondary mammalian IgG loading dots can be any regular or irregular shape such as circles, ellipses, squares, diamonds and the like. The antibody is further selected from IHC-approved antibodies that use mouse or rabbit host proteins that are not conjugated to fluorescent markers or integrated with enzymatic sites (such as HRP or AP). Item 4. The method according to any one of Items 1 to 3. The secondary staining reagent can be selected from secondary staining having enzyme gains of 1 to 25 times, each of which is independently independent for mouse and rabbit and each uses different color chromogens. Item 5. The method according to any one of Items 1 to 4. 6. The method of any one of claims 1-5, wherein the method can be performed on an adhesive coated microscope slide having a target array with minimally a gradient density array of mouse and rabbit IgG proteins co-localized with tissue sections. The method described in. 7. The method of any one of claims 1-6, wherein all array targets are members on a 20 log (dilution ratio) curve and the dilution ratio can range from 1:1 to 1000:1. After compensating for the variation of the reactivity of the staining reagent itself, the extended antigen scale was changed from three points on the 20 log (dilution ratio) profile to a larger number of points of the secondary protein series by the 20 log (dilution ratio) profile. The method according to any one of claims 1 to 7, wherein the projection is performed on the extended scale used. The method according to any one of claims 1 to 8, wherein each antigen scale can be applied to the measurement of the same antigen density in coexisting tissue sections by an objective scale. 10. The method of any one of claims 1-9, wherein the primary antigen concentration scale is applied to subsequently coexisting tissue sections to utilize abnormal cell sections of detected cells such as cancer. ..