WO2018228577A1 - Method for ihc antigen imaging scale extrapolation - Google Patents

Method for ihc antigen imaging scale extrapolation Download PDF

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WO2018228577A1
WO2018228577A1 PCT/CN2018/091689 CN2018091689W WO2018228577A1 WO 2018228577 A1 WO2018228577 A1 WO 2018228577A1 CN 2018091689 W CN2018091689 W CN 2018091689W WO 2018228577 A1 WO2018228577 A1 WO 2018228577A1
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antigen
primary
antibody
scale
target
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PCT/CN2018/091689
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French (fr)
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Frederick Knute Husher
Jee Jong Shum
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Sunstone Scientific Limited.
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Priority to EP18816945.2A priority Critical patent/EP3639031A4/en
Priority to CN201880038946.3A priority patent/CN110753846B/en
Priority to KR1020207001301A priority patent/KR102342988B1/en
Priority to JP2020519175A priority patent/JP7440411B2/en
Publication of WO2018228577A1 publication Critical patent/WO2018228577A1/en
Priority to US16/715,715 priority patent/US11662564B2/en
Priority to US18/302,495 priority patent/US20230280578A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/44—Sample treatment involving radiation, e.g. heat
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2496/00—Reference solutions for assays of biological material

Definitions

  • the present invention relates to a method for IHC (immunohistochemical) antigen imaging scale extrapolation.
  • the present invention particularly relates to a method by which an antigen concentration scale is developed from a known gradient density target series of secondary mammal IgG blood serums and optionally antigen concentrations.
  • the primary application of the aforementioned method is to support image analysis on the slide with a target protein concentration scale.
  • the method is used to form a primary antigen concentration scale from a secondary protein concentration scale.
  • the primary antigen concentration scale is then applied to the co-resident tissue section to access the tissue section for detected cellular defects, such as cancer.
  • Immunoassays are used when an unknown concentration of an analyte within a sample needs to be quantified. To obtain the most accurate determination of the unknown concentration, an immunoassay must be developed based not only on the usual assay development criteria (standard deviation or optimal signal window) but also on how well the immunoassay can predict the value of an unknown sample. First, one needs to establish the assay critical success factors. Then the immunoassay needs to be developed, which establishes proof of concept. During the optimization phase, the quantifiable range of the immunoassay method is determined by calculating a precision profile in the matrix in which the experimental samples will be measured.
  • a spiked recovery is then performed by spiking the analyte into the matrix and determining the percent recovery of the analyte in the matrix. If the precision profile is within the desired working range, then assaying spiked recovery samples over several days completes the validation of the immunoassay. If the precision profile limits are not within the desired working range, further optimization of the immunoassay is required prior to validation.
  • the primary application of the method disclosed in the instant application is to support image analysis on the slide with a target protein concentration scale.
  • the present invention discloses a method which is used to form a primary antigen concentration scale from a secondary protein concentration scale.
  • the primary antigen concentration scale is then applied to the co-resident tissue section to access the tissue section for detected cellular defects, such as cancer.
  • Generally, in one aspect of the present invention provides a method for IHC antigen imaging scale extrapolation.
  • the invention discloses a method by which an antigen concentration scale is developed from a known gradient density target series of antigen concentrations and secondary mammal IgG blood serums.
  • the primary application of the aforementioned method is to support image analysis on the slide with a target protein concentration scale.
  • the method is used to form a primary antigen concentration scale from a secondary protein concentration scale.
  • the primary antigen concentration scale is then applied to the co-resident tissue section to access the tissue section for detected cellular defects, such as cancer.
  • Immunohistochemical staining in general, used to assess the presence of specific antigen sites in a patient tissue section. Subjective interpretation is applied against the stain density on the tissue section to assign the diagnostic level of an abnormal or cancerous condition.
  • IHC Immunohistochemical
  • failure of the antigen retrieval or stain reagents leaves no signature identifying artefacts.
  • the physical morphology may not be enough to signal an abnormal condition, but without the antigen sites being marked, the slide offers nothing more than would be found on a Hematoxylin and Eosin (H&E) slide.
  • H&E Hematoxylin and Eosin
  • Antibody A Dilution ratio, ID, and host species (example would be 25, ER, Mouse)
  • Antibody B Dilution ratio and ID (example would be 20, Ki-67) . The reason that the host species is not needed as whatever the Antibody A uses Antibody B must be the opposite Mouse/Rabbit.
  • the primary antibody is composed of processed host blood serum obtained from the host animal (Mouse or Rabbit) that was inoculated by the desired antigen fraction.
  • the host then produces blood serum proteins where the antigen sites now contain the antibody reactant to the antigen antagonist.
  • the antibody is subsequently brought in contact with a protein that contains target antigen the antigen and antibody bind together.
  • the result is that the host species of the antibody (Mouse or Rabbit) is left free to react with the secondary stain kit.
  • the foundation upon which the IHC targets can be used to develop antigen density ruler against the co-resident tissue section follows the following sequence:
  • the slide’s adhesive binding site density exceeds the area displacement of a single protein by at least two orders of magnitude.
  • the primary antibody and secondary proteins have known atomic masses, kDa, which can be converted into weight as nanograms.
  • the primary and secondary targets have well defined and round deposition areas upon which the known dispensed volume of target material is applied. Since the protein deposits incorporate a cross-linking coupler they are not able to sink into the porosity of the slide coating more than a protein deep. Thus, knowing the protein’s atomic mass, the number of proteins of each protein type in the deposit, and the target’s area the target’s active surface protein density can be computed.
  • the applied concentration, dispensed volume, and surface area on slide exposed to the reagent of primary antibody are known. It can be reasonably assumed that during the exposure time of the reagent that most of the suspended antibodies will have fallen down and been captured by receptive antigen sites. Only those that fall directly over antigen sites will become captured and the balance washed away by a buffer wash step. Thus, the deposited antibody concentration can be established if the concentration is greater than 25%above cutoff and less than 25%from saturation.
  • Cutoff is defined as insufficient target site density to capture the applied the protein concentration.
  • Saturation is defined as the inability to capture all of the applied protein concentration.
  • the correct primary target density target can be chosen and the primary concentration can be validated.
  • Each secondary and primary target is a mix blend of [ (Mouse or Rabbit) + (Donkey + crosslinker + fungal inhibitor) ] or [ (KLH with antigen A or KLH with antigen B) + (unconjugated KLH + crosslinker + fungal inhibitor) ] .
  • Each dot has the same volume of total proteins, but the mix ratio must be adjusted slightly as the atomic masses may be different between the proteins composing a specific target. For example,
  • subunits are KLH1 and KLH2
  • the 2D secondary target series ranges between 10 to 100%following a 20log (dilution) profile, wherein the dilution ranges between 1: 1 and 1,000: 1.
  • a single 2D/3D target is used to measure the stain density delta between a 2D base and the 3D particles. The delta can be applied to the balance of the 2D array to produce a color density scale that is a good match to the 3D behavior seen in or on the tissue section.
  • the secondary 100%2D/3D and 2D targets verify that the two deposits are matching in regards to the 2D stain density. This is a verification that the 3D particle component did not consume enough of the 100%protein material to cause shifting of the 2D component.
  • the secondary stain incorporates an enzyme gain function between 1 and 20x that is a function of the construction of the stain reagent. Therefore, as the gain rises the lower concentration secondary target will shift into saturation whereas when the gain drops to one only the high concentration secondary targets will be visibly stained.
  • the secondary target array is not covalently fixed there will be damage to the deposit from the antigen retrieval process that an IHC slide experiences. While that provides a measure of AR impact it is not useful in generating an antigen density scale that can be applied to the tissue as it will always be unknown what the AR impact to the tissue will be. Therefore, the antigen density scale can only reflect what is left on or within the tissue section. That is why two antigen retrieval targets are provided for the QC usage of evaluating the AR process.
  • the weight of a single antibody is 150kDa (1.6605x10 ⁇ -12) which equates to a weight of 249x10 ⁇ -12 ⁇ g. If we elect to have a single area of the slide as the only part exposed then we can develop the amount of applied primary reagent. Therefore, with a closed capillary gap within inside dimensions of 20.3mmsq x 0.14mm high the volume is 57.2 ⁇ l. Ratio for a target zone of 1mm diameter, representing the area of one of the target dots, which yields 0.1 ⁇ l of the applied primary antibody reagent.
  • the primary antibody reagent is diluted from its concentrate to a range between 1 to 100 ⁇ g/ml. Therefore, for the applied primary dilution of 1-100 ⁇ g/ml the target is exposed to 0.1 to 1 ⁇ g of antibodies. Given the weight of the antibody is nominally 249x10-12 ⁇ g the 1mm target maximum protein exposure range will be 41.06 to 4106 antibodies.
  • the primary target should have a safety factor of 100 to 1000x. Choosing the 1000x option then the primary target needs to contain 4x10 ⁇ 6 antigen sites. While the KHL subunits are bigger than the applied antibodies, the increase is not enough to change the number of captured antibodies beyond 1: 1.
  • Each KLH subunit has an average atomic mass of 370kDa which equates to a weight of 614.4x10 ⁇ -12 ⁇ g.
  • the volume of a protein molecule can be approximated very simply and reliably from the molecular weight of the protein and an average protein partial specific volume.
  • Partial specific volume volume /molecular weight.
  • the average of experimentally determined partial specific volumes for soluble, globular proteins is ⁇ 0.73 cm 3 /g. This value varies from protein to protein, but the range is rather narrow.
  • the equation reduces down to a protein volume of ⁇ (1.212 x10 ⁇ 3 x MW) ⁇ m 3 .
  • the individual volume is 448.44 ⁇ m 3 .
  • the diameter of the sphere become 0.132 *MW ⁇ 1/3 in ⁇ m.
  • the target diameter of 1mm a monolayer of the KLH subunits requires 11.237*10 ⁇ 27 proteins.
  • the minimum dilution ratio becomes 1: 2.8x10 ⁇ 21.
  • any dilution approaching 1: 1000 is workable as the evaluation of the primary antibody is dominated by its active protein concentration.
  • the target density is only limited by its low concentration floor value.
  • the secondary target arrays are stepped dilution increments between 1: 1 and 1,000: 1.
  • a linear slope for the dilution occurs as -20log (dilution) , hereafter call dBd.
  • dBd dilution
  • the semi-log range is 0 to -60dBd.
  • the staining can experience saturation or cutoff as a function of the concentration of the primary antibody and the enzyme gain of the secondary stain kit.
  • Saturation is when the density of the enzyme sites exceeds the capacity to precipitate colorant from the chromogen. In other words the stain color is as dark as can be realized.
  • Cutoff occurs when the concentration of the primary antibody and enzyme gain of the secondary stain kit are too low, resulting in insufficient colorant precipitation to be seen. The two factors cause the darkness of the secondary line to shift to saturation (100%) or cutoff (0%) . Based on Figure 2, this movement is seen as the number of targets that are visible.
  • the common enzyme gains are 1, 2, 4, 5, 8, 10, 15, and 20.
  • the primary target array is present an increase in secondary enzyme gain shifts the stain density towards the low primary concentration dot. The same is true if the primary antibody concentration is increased.
  • the antigen retrieval process will cause both primary and secondary targets to be degraded to some level, which reverses the shift towards cutoff. If at the end of the IHC staining there are three or more dots that have disappeared the slide would be considered to have had excessive antigen retrieval duration, temperature, or both and too much antigen presence has been lost on the tissue making diagnostic interpretation marginal. This decision is independent of the efficacy of the primary antibody as the secondary staining is already been shown to be compromised. None on the antibody step can overcome this damage level.
  • AR damage that shifts the secondary array towards the 100%position by three or more dots is considered to be excessive and the slide should be redone using a higher enzyme gain secondary stain kit or a higher concentration of antibody.
  • the primary antigen target color density is thus the collective sum of the antibody concentration times the enzyme gain of the secondary stain kit. While the secondary target density is only that of the enzyme gain times the secondary target protein concentration.
  • the co-resident targets provide IHC process feedback as is illustrated in Figure 3.
  • the antigen retrieval process seeks to unmask the antigen sites by reversing the Schiff base bond between the formaldehyde and proteins.
  • the speed at which the antigens become exposed is largely dependent upon the temperature of the reaction. As the temperature is increased, the opportunity occurs for nucleated boiling. The nucleated boiling causes physical damage to both the tissue and protein deposits.
  • the antigen retrieval activity is uniform through the slide, but in practice that does not occur, resulting is areas having more or less antigen retrieval activity dependent on the method and environment used. Assuming uniform antigen retrieval activity the following can be used to indicate that the slide will be usable for diagnostic determination:
  • the secondary array may not be able to reflect the failure.
  • the two AR targets however, will signal the excessive failure conditions.
  • ⁇ Low AR is seen as the 2D/3D under fixed and 2D over fixed targets are both black.
  • the secondary arrays will appear as perfect with no AR shifting left of the targets.
  • ⁇ Low AR activity can occur from the following situations in the IHC stainer:
  • AR buffer has a neutral pH 7, rather than 6 or 9
  • ⁇ High AR activity can occur from the following situations in the IHC stainer:
  • the secondary array should be always increasing vs. site density. If not then the chromogen precipitation has exhausted the secondary reagent kit capacity. The solution is to increase the primary antibody dilution (same as reducing the antibody concentration) .
  • the chromogen reagent has deteriorated since being activated (often occurs with DAB) .
  • the solution is to use a new DAB mixture.
  • FIG. 3 illustrates the effect to the image as the illumination level is too dark (-5%from optimal) , optimal (+0) , and too bright as in (+10 or +15%) .
  • the illumination level is too dark (-5%from optimal) , optimal (+0) , and too bright as in (+10 or +15%) .
  • the light level is below optimal there is compression of stain density. In terms of cancer stages this could shift the diagnosis one stage higher than it should be.
  • the light level is above optimal there is bleaching of the image. In terms of cancer stages this could shift the diagnosis one stage lower than it should be.
  • the antigen color density and numeric ruler is developed from the primary and secondary targets and can be superimposed upon the WSI image.
  • the numerical scale is the independent term while the color density is the dependent term.
  • the numeric scale remains fixed as the user shifts the illumination level up or down.
  • the color density scale on the other hand shifts as the illumination level changes.
  • the advantage is that the user has the choice to shift the apparent illumination up/down to best ‘see’ features on the tissue image while never losing the numeric relationship to color density. This will also be functional as the magnification is changed.
  • Type A is based on the assumption that the primary antibody is always applied with less than 10%excess antibody vs. tissue antigen sites.
  • Type B uses the primary antigen gradient density array.
  • Type A Secondary Only Based Antigen Ruler
  • the passed in information that is imbedded in the 2-D bar code includes the (a) primary antibody data: host species for the antibody and dilution in -dBd and (b) secondary enzyme gain.
  • the secondary gradient density target array is composed of known concentrations of proteins following an -3dBd decrement between targets.
  • the maximum concentration is chosen by the least dilution that is used for the primary antibody. Most users take the concentration specification provided by the antibody reagent manufacturer and dilute to a constant intermediate concentration of 1ug/ml. From that all other dilutions are made as needed to accommodate the different tissue types.
  • the second set of primary antibody dilutions range between 1: 1 and 1,000: 1.
  • the secondary array must be composed of a wider range of dilutions.
  • SdBd the lowest dilution of the secondary array starts at 1,000: 1 or -60dBd, which is represented by SdBd.
  • the maximum of the 8-dot series then becomes -0dBd or 1: 1.
  • the action of the antigen retrieval degrades the secondary proteins which is represented by ARdBd.
  • Each dot, one of eight, in the secondary array represents an -3dBd increment.
  • the antigen retrieval loss for the loss of two targets would be +6dBd.
  • the secondary array is (–S+ AR) dBd for the 2D targets or [+6 to -54dBd] .
  • the antibody concentration and the secondary enzyme gain must now be factored in.
  • the antibody concentration would be AdBd, while the enzyme gain is EdBd.
  • the secondary array would be (-S+ AR –E) dBd, while the tissue would be (+AR –E + A) dBd.
  • the next factor that must be applied is the 100%2D to 3D differential.
  • the stain difference between the 3D objects in the 100%2D/3D target and the 100%2D represents the secondary stain chromogen precipitation constant, which is used to assign the color density to the numerical scale and is assigned to DdBd.
  • the difference in color density is applied to each of the 2D targets in the array.
  • the 2D array presents in stain color density as (+AR –E +A +D) dBd.
  • the 2D secondary array would then become: -14, -17, -20, -23, -26, -29, blank, blank dBd.
  • the two dots towards 0%having been damaged enough by the antigen retrieval process that they are unrecoverable by the staining and thus, blank.
  • the primary targets contain the same number of proteins per micron as the secondary the primary dilution from the 500ug/ml antibody master is then applied to the secondary array data to adjust the secondary color density to numeric antigen density.
  • Monitoring the secondary targets choose the target that has a middle color density. The middle color density being defined as the 50%point between maximum black and maximum white. The point then equates to 1.5dBd out of the 3dBd range. That point then functions as the anchor upon which the antigen density ruler is established. Using the last target range above the midpoint becomes -41.5dBd.
  • the secondary proteins are diluted to a 10ug/ml master dilution.
  • the standard target dot is 1mm in diameter. If the printed deposit is 1um thick, and the deposit concentration is 10ug/ml, 31.5x10 ⁇ 6 proteins will be deposited. A 1um diameter area would then have 31.5 proteins.
  • the secondary array uses the same number of proteins per deposit, but the ratio between Mouse or Rabbit and Donkey changes as the concentration of the Mouse or Rabbit is reduced.
  • the 100%target is entirely Mouse or Rabbit and is matched to the 0dBd point on the ruler.
  • the secondary will only stain on the tissue when a primary antibody binds to an antigen site on the tissue. It is not particularly dependent on the concentration of the applied antibody except that sufficient antibody concentration must be provided to bind to the available antigen sites.
  • the antigen density measurement on the tissue remains as a constant, but the numeric values must be corrected for antigen retrieval damage and secondary enzyme gain. The color density vs. numeric measurement must then be harmonized.
  • the enzyme gain is 10x and the antigen retrieval has caused the loss of two dots from the secondary array.
  • the enzyme gain is -20dBd while the antigen retrieval loss is +6dBd.
  • the result is -14dBd.
  • the dilutions then translate to:
  • Type B Primary Antigen Based Ruler
  • the passed in information that is imbedded in the 2-D bar code includes the (a) primary antibody data: host species for the antibody and dilution in dBdilution and (b) secondary enzyme gain.
  • the lot code data includes the information about which primary target combination is in use.
  • a primary target series If a primary target series is present it would be 3-dots wherein the most concentrated dot would be at the same 100%concentration as the secondary array, but the dots are spaced apart in -6dBd steps. In effect, the primary array and secondary array have the same dilution slope.
  • the primary targets become: -0, -6, -12dBd and are represented as PdBd. It is reasonable to expect that the antigen retrieval will damage will nearly identical to that of the secondary array.
  • the primary array is acted upon by the secondary stain and thus experiences the same enzyme gain function. Thus, the primary array would be (–A+ AR –E) dBd, where the primary target density is controlled by the primary antibody dilution. The only requirement is that P is always greater than A.
  • the primary array is -20, -26, -32 dBd.
  • the antigen retrieval loss does not act upon the primary targets enough to blank them out, based on the impact to the secondary array. While the secondary array is sufficient to produce the antigen density rulers it is important to verify that the primary dilution was correctly applied. Thus, the primary targets function in that capacity.
  • the aforementioned secondary protein target arrays are formed as two lines: one of Mouse IgG and the other Rabbit IgG mixed with a dummy IgG blood serum protein to form a five or more member gradient density series that progresses from max density to min density in a 20log (dilution) curve, wherein the dilutions may range between 1: 1 and 1,000: 1.
  • the Mouse &Rabbit target array reflects the 20log (dilution) curve of secondary stain kit chromogen precipitation.
  • the preferred solution for the method for forming the primary antigen density scale is predicated on successfully composing the target mixtures, depositing them onto the slide with a target protein concentration scale, and having a covalent bond between the adhesive and the target materials.
  • the primary stain may be selected from any IHC approved antibody that uses a Mouse or Rabbit host protein that is not also conjugated to a fluorescent marker or integrated with an enzyme site (such as HRP or AP) .
  • the secondary stain may be selected from but not limited to the secondary stains with enzyme gains of 1x through 25x, that are each uniquely independent between Mouse and Rabbit, which each use a different color chromogen.
  • the performance result in an absolute basis on one slide may not be identical to another slide done at another time. This comes from the fact that the secondary stain kits vary in performance lot to lot as does the primary conjugated primary antibody. However, the performance for any one slide with a target protein concentration scale the antigen scale will be valid and give close equivalence to another done using different stain reagents.
  • the primary antigen concentration scale is then applied to the co-resident tissue section to access the tissue section for detected cellular defects, such as cancer.
  • the slide with a target protein concentration scale is descripted below:
  • a slide comprising: a detection zone and a control zone, wherein
  • the detection zone is a space for a tissue section or loose cells to be applied for processing through immunohistochemistry (IHC) and subsequently examination; and
  • control zone comprises one or more sets of primary and/or secondary target arrays, wherein
  • the secondary target array includes one or more (eg. 1-50, 5-45, 10-40, 15-35 or 20-30, particularly, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) secondary target loading dots (the dot can be of any regular or irregular shape, such as circle, ellipse, square, diamond, etc. ) , each secondary target loading dot is a mixture of host protein (eg. IgG) and dummy protein (eg. IgG) fixed to the slide in a certain proportion.
  • host protein eg. IgG
  • dummy protein eg. IgG
  • dummy protein means the protein that is unreactive to the secondary antibody and used to mix with host protein to get gradient dilutions.
  • Preferred dummy protein is donkey protein (IgG) or horse protein (IgG) .
  • Term “host protein” means the protein (especially IgG) which has the same origin as the primary antibody, such as mouse, rat, rabbit, donkey, horse, and goat protein (IgG) .
  • Figure 1 One example of the slide is shown in Figure 1 with a detailed identification of the targets in Figure 2.
  • Figure 1 shows a representation of the portion of a slide containing the primary and secondary stain capturing targets after IHC staining with identification of the different targets.
  • Figure 2 shows the effects of antigen retrieval processing upon one of the secondary protein arrays after IHC staining.
  • Figure 3 shows a slide with a secondary protein concentration scale which has a co-resident tissue section and is subjected to IHC staining.

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Abstract

Provided in the disclosure relates to a method for IHC antigen imaging scale extrapolation. Provided in the disclosure particularly relates to a method by which an antigen concentration scale is developed from a known gradient density target series of secondary mammal IgG blood serums and optionally antigen concentrations. The primary application of the aforementioned method is to support image analysis on the slide with a target protein concentration scale. The method is used to form a primary antigen concentration scale from a secondary protein concentration scale. The primary antigen concentration scale is then applied to the co-resident tissue section to access the tissue section for detected cellular defects, such as cancer.

Description

METHOD FOR IHC ANTIGEN IMAGING SCALE EXTRAPOLATION
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priorities of US provisional Application number US62/520187 filed on June 15, 2017, and US provisional Application number 62/520319 filed on June 15, 2017, the disclosures of each of which are incorporated herein by reference in its entirety.
FIELD
The present invention relates to a method for IHC (immunohistochemical) antigen imaging scale extrapolation. The present invention particularly relates to a method by which an antigen concentration scale is developed from a known gradient density target series of secondary mammal IgG blood serums and optionally antigen concentrations. The primary application of the aforementioned method is to support image analysis on the slide with a target protein concentration scale. The method is used to form a primary antigen concentration scale from a secondary protein concentration scale. The primary antigen concentration scale is then applied to the co-resident tissue section to access the tissue section for detected cellular defects, such as cancer.
BACKGROUND
Immunoassays are used when an unknown concentration of an analyte within a sample needs to be quantified. To obtain the most accurate determination of the unknown concentration, an immunoassay must be developed based not only on the usual assay development criteria (standard deviation or optimal signal window) but also on how well the immunoassay can predict the value of an unknown sample. First, one needs to establish the assay critical success factors. Then the immunoassay needs to be developed, which establishes proof of concept. During the optimization phase, the quantifiable range of the immunoassay method is determined by calculating a precision profile in the matrix in which the experimental samples will be measured. A spiked recovery is then performed by spiking the analyte into the matrix and determining the percent recovery of the analyte in the matrix. If the precision profile is within the desired working range, then assaying spiked recovery  samples over several days completes the validation of the immunoassay. If the precision profile limits are not within the desired working range, further optimization of the immunoassay is required prior to validation.
The primary application of the method disclosed in the instant application is to support image analysis on the slide with a target protein concentration scale. The present invention discloses a method which is used to form a primary antigen concentration scale from a secondary protein concentration scale. The primary antigen concentration scale is then applied to the co-resident tissue section to access the tissue section for detected cellular defects, such as cancer.
SUMMARY
Generally, in one aspect of the present invention provides a method for IHC antigen imaging scale extrapolation.
In another aspect of the present invention, the invention discloses a method by which an antigen concentration scale is developed from a known gradient density target series of antigen concentrations and secondary mammal IgG blood serums.
In yet another aspect of the present invention, the primary application of the aforementioned method is to support image analysis on the slide with a target protein concentration scale.
In still another aspect of the present invention, the method is used to form a primary antigen concentration scale from a secondary protein concentration scale.
In yet another aspect of the present invention, the primary antigen concentration scale is then applied to the co-resident tissue section to access the tissue section for detected cellular defects, such as cancer.
Other aspects of the present invention are disclosed in the following description.
DETAILED DESCRIPTION
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. It is to be understood that this invention is not limited to the specific devices, methods, conditions or  parameters described and/or shown herein and that the terminology used herein is for the example only and is not intended to be limiting of the claimed invention. Also, as used in the specification including the appended claims, the singular forms ‘a’ , ‘an’ , and ‘the’ include the plural, and references to a particular numerical value includes at least that particular value unless the content clearly directs otherwise. Ranges may be expressed herein as from ‘about’ or ‘approximately’ another particular value. When such a range is expressed another embodiment. Also, it will be understood that unless otherwise indicated, dimensions and material characteristics stated herein are by way of example rather than limitation, and are for better understanding of sample embodiment of suitable utility, and variations outside of the stated values may also be within the scope of the invention depending upon the particular application.
This 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. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein for the purpose of description and should not be regarded as limiting. The use of “including” , “comprising” , “having” , “containing” , “involving” , and variations thereof as well as additional items.
Immunohistochemical (IHC) staining in general, used to assess the presence of specific antigen sites in a patient tissue section. Subjective interpretation is applied against the stain density on the tissue section to assign the diagnostic level of an abnormal or cancerous condition. In general, there is an assumption that the IHC processing always functions correctly and that the tissue section would be marked with visible chromogen markers identifying the abnormal or cancerous conditions if they are present. However, failure of the antigen retrieval or stain reagents leaves no signature identifying artefacts. Thus, there is considerable opportunity for not being able to render a valid diagnostic determination on the part of the Lab Tech or Pathologist. In other words, the physical morphology may not be enough to signal an abnormal condition, but without the antigen sites being marked, the slide offers nothing more than would be found on a Hematoxylin and Eosin (H&E) slide.
There are some passed in requirements that are necessary in order to complete the calculations. The parameters need to be known are the following:
1. Antibody A: Dilution ratio, ID, and host species (example would be 25, ER,  Mouse)
2. Antibody B: Dilution ratio and ID (example would be 20, Ki-67) . The reason that the host species is not needed as whatever the Antibody A uses Antibody B must be the opposite Mouse/Rabbit.
It is known that the primary antibody is composed of processed host blood serum obtained from the host animal (Mouse or Rabbit) that was inoculated by the desired antigen fraction. The host then produces blood serum proteins where the antigen sites now contain the antibody reactant to the antigen antagonist. When the antibody is subsequently brought in contact with a protein that contains target antigen the antigen and antibody bind together. The result is that the host species of the antibody (Mouse or Rabbit) is left free to react with the secondary stain kit.
The foundation upon which the IHC targets can be used to develop antigen density ruler against the co-resident tissue section follows the following sequence:
The slide’s adhesive binding site density exceeds the area displacement of a single protein by at least two orders of magnitude.
The primary antibody and secondary proteins have known atomic masses, kDa, which can be converted into weight as nanograms.
The primary and secondary targets have well defined and round deposition areas upon which the known dispensed volume of target material is applied. Since the protein deposits incorporate a cross-linking coupler they are not able to sink into the porosity of the slide coating more than a protein deep. Thus, knowing the protein’s atomic mass, the number of proteins of each protein type in the deposit, and the target’s area the target’s active surface protein density can be computed.
The applied concentration, dispensed volume, and surface area on slide exposed to the reagent of primary antibody are known. It can be reasonably assumed that during the exposure time of the reagent that most of the suspended antibodies will have fallen down and been captured by receptive antigen sites. Only those that fall directly over antigen sites will become captured and the balance washed away by a buffer wash step. Thus, the deposited antibody concentration can be established if the concentration is greater than 25%above cutoff and less than 25%from saturation.
a. Cutoff is defined as insufficient target site density to capture the applied the  protein concentration.
b. Saturation is defined as the inability to capture all of the applied protein concentration.
Knowing the primary dilution ration, the correct primary target density target can be chosen and the primary concentration can be validated.
Each secondary and primary target is a mix blend of [ (Mouse or Rabbit) + (Donkey + crosslinker + fungal inhibitor) ] or [ (KLH with antigen A or KLH with antigen B) + (unconjugated KLH + crosslinker + fungal inhibitor) ] . Each dot has the same volume of total proteins, but the mix ratio must be adjusted slightly as the atomic masses may be different between the proteins composing a specific target. For example,
A. Mouse IgG                      = 155kDa
B. Rabbit IgG                     = 150kDa
C. Donkey IgG                     = 160kDa
D. KLH subunits conjugated with   = 350 &390kDa
   antigen peptide strand, where the
   subunits are KLH1 and KLH2
The 2D secondary target series ranges between 10 to 100%following a 20log (dilution) profile, wherein the dilution ranges between 1: 1 and 1,000: 1. A single 2D/3D target is used to measure the stain density delta between a 2D base and the 3D particles. The delta can be applied to the balance of the 2D array to produce a color density scale that is a good match to the 3D behavior seen in or on the tissue section.
The secondary 100%2D/3D and 2D targets verify that the two deposits are matching in regards to the 2D stain density. This is a verification that the 3D particle component did not consume enough of the 100%protein material to cause shifting of the 2D component.
The secondary stain incorporates an enzyme gain function between 1 and 20x that is a function of the construction of the stain reagent. Therefore, as the gain rises the lower concentration secondary target will shift into saturation whereas when the gain drops to one only the high concentration secondary targets will be visibly stained.
If the secondary target array is not covalently fixed there will be damage to the  deposit from the antigen retrieval process that an IHC slide experiences. While that provides a measure of AR impact it is not useful in generating an antigen density scale that can be applied to the tissue as it will always be unknown what the AR impact to the tissue will be. Therefore, the antigen density scale can only reflect what is left on or within the tissue section. That is why two antigen retrieval targets are provided for the QC usage of evaluating the AR process.
Because of the considerable size difference between the secondary and primary target proteins the protein concentration density will be established by the primary proteins. If we accept the premise that the KLH subunits KLH1 &KLH2 have a 50: 50 distribution then their average value, 370kDa, can be used to set the primary target dilution.
With an average primary antibody atomic mass of 150kDa, the weight of a single antibody is 150kDa (1.6605x10^-12) which equates to a weight of 249x10^-12ηg. If we elect to have a single area of the slide as the only part exposed then we can develop the amount of applied primary reagent. Therefore, with a closed capillary gap within inside dimensions of 20.3mmsq x 0.14mm high the volume is 57.2μl. Ratio for a target zone of 1mm diameter, representing the area of one of the target dots, which yields 0.1μl of the applied primary antibody reagent.
The primary antibody reagent is diluted from its concentrate to a range between 1 to 100μg/ml. Therefore, for the applied primary dilution of 1-100μg/ml the target is exposed to 0.1 to 1μg of antibodies. Given the weight of the antibody is nominally 249x10-12ηg the 1mm target maximum protein exposure range will be 41.06 to 4106 antibodies.
To ensure 100%capture ability the primary target should have a safety factor of 100 to 1000x. Choosing the 1000x option then the primary target needs to contain 4x10^6 antigen sites. While the KHL subunits are bigger than the applied antibodies, the increase is not enough to change the number of captured antibodies beyond 1: 1. Each KLH subunit has an average atomic mass of 370kDa which equates to a weight of 614.4x10^-12ηg.
The volume of a protein molecule can be approximated very simply and reliably from the molecular weight of the protein and an average protein partial specific volume. (Partial specific volume = volume /molecular weight. ) The average of experimentally determined partial specific volumes for soluble, globular proteins is ～0.73 cm 3/g. This value varies from protein to protein, but the range is rather narrow. The equation reduces down to a  protein volume of ～ (1.212 x10^3 x MW) ηm 3. Thus, for the KLH subunit the individual volume is 448.44ηm 3. If the protein is modeled as a sphere then the diameter of the sphere become 0.132 *MW^1/3 in ηm. For the KLH subunit this is 9.436ηm.
For the target diameter of 1mm a monolayer of the KLH subunits requires 11.237*10^27 proteins. For the active target density of 4x10^6 proteins the minimum dilution ratio becomes 1: 2.8x10^21. In practical terms, any dilution approaching 1: 1000 is workable as the evaluation of the primary antibody is dominated by its active protein concentration. Thus, the target density is only limited by its low concentration floor value.
The secondary target arrays are stepped dilution increments between 1: 1 and 1,000: 1. A linear slope for the dilution occurs as -20log (dilution) , hereafter call dBd. For the dilution range listed between 1: 1 and 1,000: 1, the semi-log range is 0 to -60dBd. Choosing -3dBd dilution steps the secondary target dilutions become 0, -3, -6, -9, -12, -15, -18, -21 dBd.
The staining can experience saturation or cutoff as a function of the concentration of the primary antibody and the enzyme gain of the secondary stain kit. Saturation is when the density of the enzyme sites exceeds the capacity to precipitate colorant from the chromogen. In other words the stain color is as dark as can be realized. Cutoff occurs when the concentration of the primary antibody and enzyme gain of the secondary stain kit are too low, resulting in insufficient colorant precipitation to be seen. The two factors cause the darkness of the secondary line to shift to saturation (100%) or cutoff (0%) . Based on Figure 2, this movement is seen as the number of targets that are visible. As the secondary enzyme gain increases the 100%dot density shifts towards the 0%position. The common enzyme gains are 1, 2, 4, 5, 8, 10, 15, and 20. These translate into shifting the secondary array towards the 0%position by:
· 20x all targets shift -26dBd
· 15x all targets shift -23.52
· 10x all targets shift -20
· 5x all targets shift -13.98
· 4x all targets shift -12.04
· 2x all targets shift -6.02
· 1x only 2D 100%dot near black
If the primary target array is present an increase in secondary enzyme gain shifts the  stain density towards the low primary concentration dot. The same is true if the primary antibody concentration is increased. The antigen retrieval process will cause both primary and secondary targets to be degraded to some level, which reverses the shift towards cutoff. If at the end of the IHC staining there are three or more dots that have disappeared the slide would be considered to have had excessive antigen retrieval duration, temperature, or both and too much antigen presence has been lost on the tissue making diagnostic interpretation marginal. This decision is independent of the efficacy of the primary antibody as the secondary staining is already been shown to be compromised. Nothing on the antibody step can overcome this damage level.
Typically, AR damage that shifts the secondary array towards the 100%position by three or more dots is considered to be excessive and the slide should be redone using a higher enzyme gain secondary stain kit or a higher concentration of antibody.
The primary antigen target color density is thus the collective sum of the antibody concentration times the enzyme gain of the secondary stain kit. While the secondary target density is only that of the enzyme gain times the secondary target protein concentration.
Depending on the digital imaging system, changes in the illumination intensity will shift the dynamic range of the image into compression (getting darker) or saturation (getting lighter) . These changes will shift the antigen color scale while the antigen density numeric scale will not. Thus, the numeric scale is independent and the color scale dependent on the illumination intensity.
In QC mode the co-resident targets provide IHC process feedback as is illustrated in Figure 3. There are four lines of secondary arrays shown with the difference being the degree of antigen retrieval performed from within nominal, over, very over, and excessively over, 5, 10, 30, and 40%respectively. The antigen retrieval process seeks to unmask the antigen sites by reversing the Schiff base bond between the formaldehyde and proteins. The speed at which the antigens become exposed is largely dependent upon the temperature of the reaction. As the temperature is increased, the opportunity occurs for nucleated boiling. The nucleated boiling causes physical damage to both the tissue and protein deposits. Ideally, the antigen retrieval activity is uniform through the slide, but in practice that does not occur, resulting is areas having more or less antigen retrieval activity dependent on the method and environment used. Assuming uniform antigen retrieval activity the following can be used to indicate that the slide will be usable for diagnostic determination:
If the AR is minimal or excessive, the secondary array may not be able to reflect the failure. The two AR targets however, will signal the excessive failure conditions.
·Low AR is seen as the 2D/3D under fixed and 2D over fixed targets are both black. The secondary arrays will appear as perfect with no AR shifting left of the targets.
·Low AR activity can occur from the following situations in the IHC stainer:
AR Heater not working or set well below 80℃
AR buffer has a neutral pH 7, rather than 6 or 9
Exposure time too short
·High AR is seen as the 2D/3D under fixed is very bleached and the 2D over fixed target is less than 50%black. The secondary arrays will be largely bleached out as well.
·High AR activity can occur from the following situations in the IHC stainer:
Heater operating at temperature >95℃
Exposure time too long
·Chromogen precipitation error can arise under two situations:
If at the high concentration secondary targets the stain intensity dips rather than is at maximum darkness. The secondary array should be always increasing vs. site density. If not then the chromogen precipitation has exhausted the secondary reagent kit capacity. The solution is to increase the primary antibody dilution (same as reducing the antibody concentration) .
The chromogen reagent has deteriorated since being activated (often occurs with DAB) . The solution is to use a new DAB mixture.
Viewing a microscope slide through a conventional microscope is subjective in regards to the illumination level. In whole slide imaging (WSI) , the scanner uses a perfect white and black hole to establish the white balance and contrast. Such is not the case with manual microscopes. Figure 3 illustrates the effect to the image as the illumination level is too dark (-5%from optimal) , optimal (+0) , and too bright as in (+10 or +15%) . When the light level is below optimal there is compression of stain density. In terms of cancer stages this could shift the diagnosis one stage higher than it should be. When the light level is above optimal there is bleaching of the image. In terms of cancer stages this could shift the diagnosis  one stage lower than it should be. The antigen color density and numeric ruler is developed from the primary and secondary targets and can be superimposed upon the WSI image. The numerical scale is the independent term while the color density is the dependent term. When the antigen density color and numeric ruler is applied to the WSI the numeric scale remains fixed as the user shifts the illumination level up or down. The color density scale on the other hand shifts as the illumination level changes. The advantage is that the user has the choice to shift the apparent illumination up/down to best ‘see’ features on the tissue image while never losing the numeric relationship to color density. This will also be functional as the magnification is changed.
There are two forms by which the antigen density ruler can be developed. Type A is based on the assumption that the primary antibody is always applied with less than 10%excess antibody vs. tissue antigen sites. Type B uses the primary antigen gradient density array.
Type A: Secondary Only Based Antigen Ruler
This form uses only the secondary target array. The passed in information that is imbedded in the 2-D bar code includes the (a) primary antibody data: host species for the antibody and dilution in -dBd and (b) secondary enzyme gain.
The secondary gradient density target array is composed of known concentrations of proteins following an -3dBd decrement between targets. The maximum concentration is chosen by the least dilution that is used for the primary antibody. Most users take the concentration specification provided by the antibody reagent manufacturer and dilute to a constant intermediate concentration of 1ug/ml. From that all other dilutions are made as needed to accommodate the different tissue types. In general, the second set of primary antibody dilutions range between 1: 1 and 1,000: 1.
To accommodate the range of secondary enzyme gain the secondary array must be composed of a wider range of dilutions. Thus, with -3dBd steps the lowest dilution of the secondary array starts at 1,000: 1 or -60dBd, which is represented by SdBd. The maximum of the 8-dot series then becomes -0dBd or 1: 1. The action of the antigen retrieval degrades the secondary proteins which is represented by ARdBd. Each dot, one of eight, in the secondary array represents an -3dBd increment. The antigen retrieval loss for the loss of two targets (no longer visible) would be +6dBd. This means the secondary array is (–S+ AR) dBd for the 2D targets or [+6 to -54dBd] . The antibody concentration and the secondary enzyme gain  must now be factored in. The antibody concentration would be AdBd, while the enzyme gain is EdBd. Thus, the secondary array would be (-S+ AR –E) dBd, while the tissue would be (+AR –E + A) dBd. The next factor that must be applied is the 100%2D to 3D differential. The stain difference between the 3D objects in the 100%2D/3D target and the 100%2D represents the secondary stain chromogen precipitation constant, which is used to assign the color density to the numerical scale and is assigned to DdBd. The difference in color density is applied to each of the 2D targets in the array. Thus, the 2D array presents in stain color density as (+AR –E +A +D) dBd.
If for example, the enzyme gain was 10x then E=-20dBd. The 2D secondary array would then become: -14, -17, -20, -23, -26, -29, blank, blank dBd. The two dots towards 0%having been damaged enough by the antigen retrieval process that they are unrecoverable by the staining and thus, blank. If for example the 2D/3D color density difference is 10x then D=+20dBd bringing the 3D secondary array to -34, -37, -40, -43, -46, -49, blank, blank dBd. It is assumed that the primary antibody reagents will find suitable antigen sites in the primary targets that 100%yield takes place. It is also assumed that while there are many more than two antigen peptide strands per KLH protein that only one antibody can effectively bind and become stained per KLH protein. Any additional antibodies finding a suitable antigen on the same KLH protein will be prevented from completion by the secondary stain because of overlapping occupancy. Therefore, the number of antigen sites per primary antigen carried protein that can become detected is one. Since the primary targets contain the same number of proteins per micron as the secondary the primary dilution from the 500ug/ml antibody master is then applied to the secondary array data to adjust the secondary color density to numeric antigen density. Monitoring the secondary targets, choose the target that has a middle color density. The middle color density being defined as the 50%point between maximum black and maximum white. The point then equates to 1.5dBd out of the 3dBd range. That point then functions as the anchor upon which the antigen density ruler is established. Using the last target range above the midpoint becomes -41.5dBd.
The secondary proteins are diluted to a 10ug/ml master dilution. Each array is a blend of Mouse or Rabbit mixed with Donkey IgG proteins. While the proteins all have different atomic masses the following will assume all are 150kDa and that the total number of proteins per target dot is constant the mix ratio is not. For now, only the reactive protein concentration is being considered. At 150kDa the individual protein molecular weight MW =  249.07x10^-12ng. The standard target dot is 1mm in diameter. If the printed deposit is 1um thick, and the deposit concentration is 10ug/ml, 31.5x10^6 proteins will be deposited. A 1um diameter area would then have 31.5 proteins. If we allow that one protein equates to 1 antigen site then the antigen density can be established. The secondary array uses the same number of proteins per deposit, but the ratio between Mouse or Rabbit and Donkey changes as the concentration of the Mouse or Rabbit is reduced. The 100%target is entirely Mouse or Rabbit and is matched to the 0dBd point on the ruler.
The secondary will only stain on the tissue when a primary antibody binds to an antigen site on the tissue. It is not particularly dependent on the concentration of the applied antibody except that sufficient antibody concentration must be provided to bind to the available antigen sites. Thus, the antigen density measurement on the tissue remains as a constant, but the numeric values must be corrected for antigen retrieval damage and secondary enzyme gain. The color density vs. numeric measurement must then be harmonized.
In the previous example the enzyme gain is 10x and the antigen retrieval has caused the loss of two dots from the secondary array. The enzyme gain is -20dBd while the antigen retrieval loss is +6dBd. The result is -14dBd. The dilutions then translate to:
Figure PCTCN2018091689-appb-000001
Type B: Primary Antigen Based Ruler
This form uses both the primary and secondary target arrays. The passed in information that is imbedded in the 2-D bar code includes the (a) primary antibody data: host species for the antibody and dilution in dBdilution and (b) secondary enzyme gain. The lot code data includes the information about which primary target combination is in use.
If a primary target series is present it would be 3-dots wherein the most concentrated dot would be at the same 100%concentration as the secondary array, but the dots are spaced apart in -6dBd steps. In effect, the primary array and secondary array have the same dilution slope. The primary targets become: -0, -6, -12dBd and are represented as PdBd. It is reasonable to expect that the antigen retrieval will damage will nearly identical to that of the secondary array. The primary array is acted upon by the secondary stain and thus experiences the same enzyme gain function. Thus, the primary array would be (–A+ AR –E) dBd, where the primary target density is controlled by the primary antibody dilution. The only requirement is that P is always greater than A. For 10x enzyme gain = -20dBd and +6dbd antigen retrieval loss the primary array is -20, -26, -32 dBd. The antigen retrieval loss does not act upon the primary targets enough to blank them out, based on the impact to the secondary array. While the secondary array is sufficient to produce the antigen density rulers it is important to verify that the primary dilution was correctly applied. Thus, the primary targets function in that capacity.
This application incorporates the reference of the definition of a slide with a target protein concentration scale disclosed in application number 62/520319 entitled “Process Record Slide for Immunohistochemical Staining” into its entirety. The aforementioned slide with a target protein concentration scale as also defined in the application number 62/520319 is composed of two secondary protein target arrays at a minimum and optionally one or more primary antigen target arrays.
In one embodiment of the present invention, the aforementioned secondary protein target arrays are formed as two lines: one of Mouse IgG and the other Rabbit IgG mixed with a dummy IgG blood serum protein to form a five or more member gradient density series that progresses from max density to min density in a 20log (dilution) curve, wherein the dilutions may range between 1: 1 and 1,000: 1.
In another embodiment, in the last process step those antigen sites identified become colored by chromogen precipitation. Thus, the Mouse &Rabbit target array reflects the 20log  (dilution) curve of secondary stain kit chromogen precipitation.
In another embodiment, the preferred solution for the method for forming the primary antigen density scale is predicated on successfully composing the target mixtures, depositing them onto the slide with a target protein concentration scale, and having a covalent bond between the adhesive and the target materials.
In another embodiment, deducing that the target arrays are successfully applied and the both the primary and secondary stain reagents perform reasonably then the curve fitting between the data sets can be easily done by computer algorithm. In another embodiment, the primary stain may be selected from any IHC approved antibody that uses a Mouse or Rabbit host protein that is not also conjugated to a fluorescent marker or integrated with an enzyme site (such as HRP or AP) . In another embodiment, the secondary stain may be selected from but not limited to the secondary stains with enzyme gains of 1x through 25x, that are each uniquely independent between Mouse and Rabbit, which each use a different color chromogen.
In another embodiment of the present invention, it is pertinent to note that the performance result in an absolute basis on one slide may not be identical to another slide done at another time. This comes from the fact that the secondary stain kits vary in performance lot to lot as does the primary conjugated primary antibody. However, the performance for any one slide with a target protein concentration scale the antigen scale will be valid and give close equivalence to another done using different stain reagents.
In another embodiment, the primary antigen concentration scale is then applied to the co-resident tissue section to access the tissue section for detected cellular defects, such as cancer.
In another embodiment, the slide with a target protein concentration scale is descripted below:
A slide, comprising: a detection zone and a control zone, wherein
the detection zone is a space for a tissue section or loose cells to be applied for processing through immunohistochemistry (IHC) and subsequently examination; and
the control zone comprises one or more sets of primary and/or secondary target arrays, wherein
the secondary target array includes one or more (eg. 1-50, 5-45, 10-40, 15-35 or 20-30, particularly, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) secondary target loading dots (the dot can be of any regular or irregular shape, such as circle, ellipse, square, diamond, etc. ) , each secondary target loading dot is a mixture of host protein (eg. IgG) and dummy protein (eg. IgG) fixed to the slide in a certain proportion.
Term “dummy protein” means the protein that is unreactive to the secondary antibody and used to mix with host protein to get gradient dilutions. Preferred dummy protein is donkey protein (IgG) or horse protein (IgG) .
Term “host protein” means the protein (especially IgG) which has the same origin as the primary antibody, such as mouse, rat, rabbit, donkey, horse, and goat protein (IgG) .
One example of the slide is shown in Figure 1 with a detailed identification of the targets in Figure 2.
Various embodiments are described herein as examples. It will be apparent to those skilled in the art that various modifications may be made and other embodiments can be used without departing from the broader scope of the invention (s) presented herein. These and other variations upon the exemplary embodiments are intended to be covered by the present invention (s) .
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a representation of the portion of a slide containing the primary and secondary stain capturing targets after IHC staining with identification of the different targets.
Figure 2 shows the effects of antigen retrieval processing upon one of the secondary protein arrays after IHC staining.
Figure 3 shows a slide with a secondary protein concentration scale which has a co-resident tissue section and is subjected to IHC staining.

Claims (10)

  1. A method for IHC imaging extrapolation by which an antigen concentration scale is developed from a known gradient density target series of antigen concentrations and secondary mammal IgG, comprising the following steps:
    (a) exposing a microscopic slide to a primary stain reagent composed of an antibody;
    (b) capturing the antibodies onto their matching antigen targets;
    (c) binding between the antibody in step (a) and the secondary target arrays with a secondary stain reagent kit for antigen identification;
    (d) coloring the identified antigen of step (c) by chromogen precipitation; and
    (e) calculating/comparing color intensity;
    wherein,
    the microscopic slide comprises a detection zone and a control zone, wherein
    the detection zone is a space for a tissue section or loose cells to be applied for processing through immunohistochemistry (IHC) and subsequently examination; and
    the control zone comprises two secondary mammal IgG arrays and optionally one or more sets of antigen arrays fixed to the slide in a certain proportion.
  2. The method as claimed in claim 1 wherein, the antibody is a Mouse IgG or Rabbit IgG type antibody.
  3. The method as claimed in claim 1 or 2, wherein, all antigen and secondary mammal IgG loading dots can be of any regular or irregular shape, such as circle, ellipse, square, diamond, etc.
  4. The method as claimed in any one of claims 1 to 3 wherein, the antibody is selected from IHC approved antibody that uses a Mouse or Rabbit host protein that is not also conjugated to a fluorescent marker or integrated with an enzyme site (such as HRP or AP) .
  5. The method as claimed in any one of claims 1 to 4 wherein, the secondary stain reagent may be selected from secondary stains with enzyme gains of 1x through 25x, that are each uniquely independent between Mouse and Rabbit, which each use a different color chromogen.
  6. The method as claimed in any one of claims 1 to 5 wherein, said method may be implemented on an adhesive coated microscope slide having arrays of targets, the minimum of which has gradient density arrays of Mouse and Rabbit IgG protein co-resident with a tissue section.
  7. The method as claimed in any one of claims 1 to 6 wherein, all array targets are members on a 20log (dillutions) curve and the dilutions may range from 1: 1 to 1,000: 1.
  8. The method as claimed in any one of claims 1 to 7 wherein, an expanded antigen scale is projected from three points on a 20log (dilutions) profile to an expanded scale using a larger number of points from a secondary protein series also with a 20log (dilutions) profile while compensating for reactivity variations in the stain reagents themselves.
  9. The method as claimed in any one of claims 1 to 8 wherein, each antigen scale can be applied to measure the same antigen density in the co-resident tissue section with objective measure.
  10. The method as claimed in any one of claims 1 to 9 wherein, the primary antigen concentration scale is then applied to the co-resident tissue section to access the tissue section for detected cellular defects, such as cancer.
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