Patent Application
20120077211
The present invention relates to the field of detection of biological targets in general, and nucleic acid and protein targets in particular, where oxidation of a chromogenic electron donor is utilized to generate a detectable signal.
Approximately 75% of the $1.4B histology market resides in the United States. The secondary staining segment (tissue analysis) is currently $600M and is expected to reach $1B by 2011, with 12% to 15% growth per annum. Key products within the histology area include immunohistochemistry (IHC) antibodies and detection reagents, H & E stains (for primary staining), special stains (for infectious disease), chromogenic in situ hybridization (CISH) reagents (e.g., DNA/RNA probes), automation systems (for tissue prep/staining) and imaging systems. Key customer drivers include quality and availability of stains and reagents, automation capabilities, breadth of menu (including antibodies, probes, and detection systems) and pricing.
Successful IHC, CISH, ELISA, and like assays depend on sensitive detection reagents with minimal background signals. Detection systems (e.g., kits) based on oxidation of a chromogenic electron donor such as 3,3′-diaminobenzidine (referred to herein as DAB) can be associated with weak signals, or no signals at all, as well as the presence of a significant amount of DAB background, i.e., the DAB chromogen reagent may undergo unwanted premature oxidation, turn dark brown and, on occasion, form precipitates. Accordingly, some CISH reagents, for example, which result from mixing DAB, hydrogen peroxide and an aqueous buffer, require use within one hour of being mixed.
In order to produce a chromogenic electron donor-based detection system that would be easy to manufacture, contain stable components, improve the resultant signal intensity and simplify the immunohistochemistry staining protocol to minimize user error(s), development efforts were focused on developing a two-component system including a stabilized formulation of DAB, as an exemplary chromogenic electron donor, and a stabilized formulation of hydrogen peroxide, as an exemplary peroxide.
Hydrogen peroxide decomposes to water and oxygen and requires stabilization when stored for prolonged periods of time. Patented compositions and methods for stabilization of hydrogen peroxide include those described in U.S. Pat. Nos. 3,811,833; 3,933,982; 4,070,442; 4,132,762; 4,133,869; 4,304,762; 4,770,808; 4,915,781; 4,981,662; 5,155,025; 5,804,404; and 6,677,466, the disclosures of which are hereby incorporated herein by reference. Classic hydrogen peroxide stabilizing agents described in the literature include: phosphoric acid; tin oxides, such as sodium stannate; dipicolinic acid; sodium pyrophosphate or organic phosphonic acids or their salts; acetone; 8-hydroxyquinoline; sulfolenes; sulfolanes; sulfoxides; sulfones; dialkylaminothiomethyl groups; thioalkylsulfonic acids; aliphatic amines; benzotriazole; nitro-substituted organic compounds, such as nitrobenzene sulfonic acids; thiosulfate; organic compounds, such as organic chelating agents or organic acids; ethylenediamine tetraacetic acid (EDTA); and amino tri-(lower alkylidene phosphonic acid). Most of the prior art compounds and compositions show some stabilization of hydrogen peroxide under acidic conditions, but have poor stabilizing effect under alkaline conditions.
In contrast to the vast amount of stabilizing agents described for hydrogen peroxide, there is little literature describing compositions or methods for stabilizing a chromogenic electron donor such as DAB. Temporary stabilization of DAB has typically been achieved by formulating under acidic conditions, where all four aromatic amino groups of DAB are protonated. However, despite the acidification, DAB continues to oxidize upon storage. Accordingly, a truly stabilized formulation of DAB has heretofore not been realized.
The present invention provides a novel, stabilized formulation of DAB, which formulation includes a chelating agent, an antioxidant, and an organic polyol the combination of which reduces unwanted oxidation and/or precipitation of DAB in aqueous solution.
The present invention further provides a novel, stabilized formulation of hydrogen peroxide, which formulation includes a buffer, a chelating agent, and a nitrogen-containing organic compound the combination of which reduces the rate of hydrogen peroxide decomposition in aqueous solution. Upon combination of the aforementioned stabilized formulations, the present invention also provides a horse radish peroxidase (HRP) reaction buffer wherein premature oxidation and/or unwanted precipitation of DAB in the absence of added HRP is reduced. Furthermore, combination of the aforementioned stabilized formulations essentially eliminates any requirement for immediate use of the HRP reaction buffer, thereby lending the stabilized formulations themselves and combinations thereof to use in automation.
Additional aspects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples that follow, while indicating preferred embodiments of the present invention, are given by way of illustration only. It is expected that various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art
Before describing the present invention in detail, it is to be understood that this invention is not limited to specific compositions or process steps per se, as such may vary. Further, it should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.
As used herein, the term “Next Generation Detection Reagents” refers to the final paired formulations (developed herein) of the DAB chromogen, i.e., 200 mM DAB, 20 mM HCl, 10 mM DTPA, 1 mM Sodium Sulfite, 65% Propylene Glycol, and the Hydrogen Peroxide Buffer, i.e., 200 mM Sodium Acetate, pH 5.5, 50 mM Imidazole, 1 mM DTPA, and 0.03% Hydrogen Peroxide. “Next Generation DAB chromogen” refers to the final formulation of the DAB chromogen, i.e., 200 mM DAB, 20 mM HCl, 10 mM DTPA, 1 mM Sodium Sulfite, and 65% Propylene Glycol. “Next Generation Hydrogen Peroxide Buffer” refers to the final formulation of the Hydrogen Peroxide Buffer, i.e., 200 mM Sodium Acetate, pH 5.5, 50 mM Imidazole, 1 mM DTPA, and 0.03% Hydrogen Peroxide. “Next Generation” products as described herein are those that are based on “Next Generation Detection Reagents.”
The present invention provides compositions, assays, methods, and kits for use in applications that utilize oxidation of a chromogenic electron donor such as diaminobenzidine (DAB) to generate a signal. Applications include, but are not limited to, immunohistochemistry (IHC), chromogenic in situ hybridization (CISH), Western blots, Northern blots, Southern blots, ELISA assays, and microarray detection. The compositions, assays, methods, and kits of the present invention make use of a novel, stabilized formulation of DAB and a novel, stabilized formulation of hydrogen peroxide.
In one illustrative embodiment, the present invention provides a novel, stabilized formulation of DAB, which formulation includes a chelating agent, an antioxidant, and an organic polyol the combination of which reduces unwanted oxidation and/or precipitation of DAB in aqueous solution. In one illustrative aspect, chelating agents such as DTPA or EDTA, for example, may be used in the formulation. In another illustrative aspect, antioxidants such as sodium sulfite or sodium metabisulfite, for example, may be used in the formulation. In yet another illustrative aspect, organic polyols such as, for example, propylene glycol or a sugar (e.g., ribose) may be used in the formulation.
In another illustrative embodiment, the present invention provides a novel, stabilized formulation of hydrogen peroxide, which formulation includes a buffer, a chelating agent, and a nitrogen-containing organic compound the combination of which reduces the rate of hydrogen peroxide decomposition in aqueous solution. In one illustrative aspect, a buffer such as sodium acetate may be used in the formulation. In another illustrative aspect, chelating agents such as DTPA or EDTA, for example, may be used in the formulation. In yet another illustrative aspect, nitrogen-containing compounds such as imidazole, for example, may be used in the formulation.
Upon combination of the aforementioned stabilized formulations in another illustrative embodiment, the present invention also provides a horse radish peroxidase (HRP) reaction buffer wherein premature oxidation and/or unwanted precipitation of DAB in the absence of added HRP is reduced. In one illustrative aspect, combination of the aforementioned stabilized formulations essentially eliminates any requirement for immediate use of the resulting HRP reaction buffer.
In another illustrative embodiment, the present invention provides assays and methods for use in applications that utilize oxidation of a chromogenic electron donor such as diaminobenzidine (DAB) to generate a signal (see, paragraph [0026] for several exemplary applications). Assays and methods provided by the present invention make use of a novel, stabilized formulation of DAB and a novel, stabilized formulation of hydrogen peroxide. In one illustrative aspect, a novel, stabilized formulation of DAB (for use in assays and methods of the present invention) includes a chelating agent, e.g., DTPA, an antioxidant, e.g., sodium sulfite, and an organic polyol, e.g., propylene glycol, the combination of which reduces unwanted oxidation and/or precipitation of DAB in aqueous solution. In another illustrative aspect, a novel, stabilized formulation of hydrogen peroxide (for use in assays and methods of the present invention) includes a buffer, e.g., sodium acetate, a chelating agent, e.g., DTPA, and a nitrogen-containing organic compound, e.g., imidazole, the combination of which reduces the rate of hydrogen peroxide decomposition in aqueous solution.
In another illustrative embodiment, the present invention provides kits for use in detection applications that utilize oxidation of a chromogenic electron donor such as diaminobenzidine (DAB) to generate a signal (see, paragraph [0026] for several exemplary applications). Kits provided by the present invention for use in detection applications make use of a novel, stabilized formulation of DAB and a novel, stabilized formulation of hydrogen peroxide. In one illustrative aspect, a novel, stabilized formulation of DAB (for use in kits of the present invention) includes a chelating agent, e.g., DTPA, an antioxidant, e.g., sodium sulfite, and an organic polyol, e.g., propylene glycol, the combination of which reduces unwanted oxidation and/or precipitation of DAB in aqueous solution. In another illustrative aspect, a novel, stabilized formulation of hydrogen peroxide (for use in kits of the present invention) includes a buffer, e.g., sodium acetate, a chelating agent, e.g., DTPA, and a nitrogen-containing organic compound, e.g., imidazole, the combination of which reduces the rate of hydrogen peroxide decomposition in aqueous solution.
In order to produce a chromogenic electron donor-based detection system that would be easy to manufacture, contain stable components, improve the resultant signal intensity and simplify the immunohistochemistry staining protocol to minimize user error(s), it was first necessary to understand any limitations associated with a typical DAB-based detection kit.
Detection kits for anatomical pathology, for example those available from Invitrogen, have typically consisted of three components: Reagent B1 or I1, i.e., the buffer/substrate buffer component; Reagent B2 or I2, i.e., the DAB chromogen; and Reagent B3 or I3, i.e., hydrogen peroxide. Color development is performed by mixing each of the three components with 1 mL of water, which is supplied by the user. For in situ hybridization kits, one drop of each of the three reagents is added to 1 mL of water. However, for immunohistochemistry kits such as the Invitrogen SuperPicture™ kit and the Histostain® kit, two drops of the DAB chromogen are added with one drop of the buffer and hydrogen peroxide to 1 mL of water. The DAB chromogen is formulated in 85% methanol. Because of the inherent surface tension characteristics of methanol, the size of a DAB chromogen-containing drop can vary over a wide range of volumes.
The primary limitation of the SuperPicture™ kit was identified to be the Buffer/Substrate Buffer (Reagent B1 or I1) having insufficient buffering capacity. Addition of one drop of the DAB chromogen (Reagent B2 or I2) to one drop of Buffer/Substrate Buffer (Reagent B1 or I1), as is carried out with most Invitrogen detection kits, decreases the pH from ˜7 to pH 6.5-7.1. Addition of two drops of the DAB chromogen (Reagent B2 or I2) to one drop of Buffer/Substrate Buffer (Reagent B1 or I1), as is carried out with the SuperPicture™ kit, decreases the pH to 3.8 to 5.1. Therefore, as shown in Table 1, depending upon the actual mixture of components, the resulting pH may fall well outside of the buffering range possible for a Tris buffer, which buffering range is pH 7.5 to pH 9.0. Without knowing the optimal pH for a particular application, a recommendation could not be made as to an optimal final reaction pH.
Further, as shown in Table 1, the data also indicate that the pH drop is more pronounced with a new lot of Tris, whereas the drop in pH is similar for new and old lots of DAB. Therefore, although there appears to be less buffering capacity in a new lot of Tris as compared to an older lot, instructions for use of the SuperPicture™ kit do not call for adjusting the pH of the final solution. It is assumed that the final pH of the solution is controlled by mixing the appropriate amounts of Tris-HCl and Tris-Base.
The composition of Buffer/Substrate Buffer Reagent B1 is:
The composition of DAB chromogen Reagent B2 is:
DAB contains four amino groups all of which are protonated in the DAB-HCl used to make DAB chromogen Reagent B2.
DAB chromogen Reagent B2 contains a total of 205 mM in acid (4×50 mM+5 mM). Under the assumption that one drop is 50 μL, the final concentrations in the working detection reagent when one drop of DAB chromogen Reagent B2 is added are approximately 2.5 mM DAB and approximately 10.25 mM acid. Two drops, as in the SuperPicture™ DAB kit, results in 5 mM DAB and 20.5 mM in acid.
The final concentration of Tris in the Buffer/Substrate Buffer Reagent B1 is approximately 1M. Tris has a pKa of 8.06 at 23° C., and a workable buffered pH range between 7.5 and 9.0. Again under the assumption that one drop is 50 μL, the final concentration in the working detection reagent is 50 mM Tris.
As shown in Table 1, the addition of one or two drops of the DAB chromogen Reagent B2 to one drop of Buffer/Substrate Buffer Reagent B1 lowers the pH below the pH range over which Tris functions as a buffer. Therefore, small variations in acid introduced with the DAB solution would be expected to greatly affect the final pH of the mixture.
To ensure that additional acid was not being introduced with DAB chromogen Reagent B2, a pH titration curve was determined for a solution of DAB chromogen Reagent B2. Addition of 100 μL of DAB chromogen Reagent B2 should result in 5×10−6 moles of DAB and 2.05×10−5 equivalents of H+ ion. The titration data suggested that the solution may contain a small amount of additional acid. DAB chromogen Reagent B2 is formulated in methanol; thus, evaporation of some methanol, which would be expected to result in an increase in concentration of both the DAB and the acid, could explain the higher apparent concentration of acid in the stock.
The effect of Buffer/Substrate Buffer Reagent B1 on the pH titration curve for a solution of DAB chromogen Reagent B2 was also examined. It was observed that the drop in pH upon addition of the DAB solution was less (pH 4.18 versus 3.12) in the presence of Buffer/Substrate Buffer Reagent B1. Without being bound to theory, it is believed that there is an initial titration of the protons introduced with the DAB Chromogen Reagent B2 followed by Tris buffering the pH against further change with subsequent addition of base.
A similar pH titration was measured when 1 M Tris, pH 8 was used in place of Buffer/Substrate Buffer Reagent B1. Replacement of Buffer/Substrate Buffer B1 by 1 M Tris, pH 8 resulted in effective buffering when the DAB chromogen Reagent B2 solution was added.
A similar pH titration was measured when 1 M Tris, pH 7.5 was used in place of Buffer/Substrate Buffer Reagent B1. Replacement of Buffer/Substrate Buffer Reagent B1 by 1 M Tris, pH 7.5 was somewhat less effective than 1 M Tris, pH 8 in buffering when the DAB chromogen Reagent B2 solution was added. Although both Buffer/Substrate Buffer Reagent B1 and 1 M Tris, pH 7.5 solutions are effective at buffering the pH around the pKa of Tris, there is a substantial difference in pH in the absence of added base. Specifically, when Buffer/Substrate Buffer Reagent B1 is the buffer, the resulting pH is over 0.6 pH units lower than when 1 M Tris, pH 7.5 is used as the buffer.
All of the above experiments were performed by addition of equal amounts of the Buffer/Substrate Buffer Reagent B1 to DAB Chromogen Reagent B2, as is the case with many Invitrogen detection kits. However, the SuperPicture™ detection kits introduce more DAB and acid by adding twice the amount of Chromogen Reagent B2 compared to Buffer/Substrate Reagent B1. Therefore, the effect of increasing volumes of DAB Chromogen Reagent B2 on the pH of a solution containing 100 μL of Buffer/Substrate Buffer Reagent B1 was investigated. The initial pH of the diluted Buffer/Substrate Buffer Reagent B1 was found to be 7.08. When an equivalent volume of DAB Chromogen Reagent B2 is added (i.e., one drop of each), the pH decreased to 4.0. When twice the volume of Chromogen Reagent B2 is added (i.e., one drop of Buffer/Substrate Buffer Reagent B1, and two drops of DAB Chromogen Reagent B2), the pH decreased to around 3.1.
In the above experiments, the volumes of Buffer/Substrate Buffer Reagent B1 and DAB chromogen Reagent B2 were actually measured. However, it is likely that the use of drops, rather than actually measured volumes, may result in significant differences in the volume of each reagent actually added, thereby resulting in large variation in the reaction pH. Aging of DAB chromogen Reagent B2, which contains 85% methanol, can be expected to lead to increased concentrations of DAB and acid through evaporative loss of methanol, while also contributing to fluctuations in the amount of acid added to the final reaction mixture.
The pH optimum for Horseradish Peroxidase (HRP) reported in the literature varies. The pH optimum for oxidation of DAB by HRP has been reported to be 4.3, with a sharp drop off in activity above and below this pH (see, Herzog, V. and Fahimi, H D. (1973). A New Sensitive Colorimetric Assay for Peroxidase Using 3,3′-Diaminobenzidine as Hydrogen Donor. Analytical Biochemistry 55: 554-562). This extreme dependence of DAB oxidation on pH is consistent with the problems described herein observed upon use of newer lots of Tris.
In light of the data obtained in support of the present invention, it was deemed advisable to determine the optimal pH for the SuperPicture™ kit and then select a buffer that has a pKa in this range to be formulated at a concentration sufficient to control the final pH. Further, it was deemed advisable to reformulate the DAB chromogen Reagent B2 in order to i) remove the methanol from the Reagent and (based on results described below) ii) include a chelating agent to reduce metal-oxygen induced oxidation, and likely concomitant color change and/or precipitation, of the DAB.
Work was also initiated to identify buffer formulations that would result in stronger CISH signals. The incorporation of nitrogenous ligands has been reported to interact with HRP thereby increasing its activity and extending the pH optimum for the reaction (see, Kuo, Che-Fu and Fridovich, Irwin. (1988). Stimulation of the Activity of Horseradish Peroxidase by Nitrogenous Compounds. Journal of Biological Chemistry. 263, No. 8: 3811-3817; Fridovich, Irwin. (1963). The Stimulation of Horseradish Peroxidase by Nitrogenous Ligands. Journal of Biological Chemistry. 283, No. 12: 3821-3927; and Claiborne, Al and Fridovich, Irwin. (1979). Chemical and Enzymatic Intermediates in the Peroxidation of o-Diansidine by Horseradish Peroxidase. 2. Evidence for a Substrate Radical-Enzyme Complex and Its Reaction with Nucleophiles. Biochemistry. 18, No. 11: 2329-2335). Accordingly, a series of buffer compositions covering the pH range from pH 3.0 to 7.5, with and without the addition of imidazole, were examined. CISH was performed on non-amplified breast cancer tissue using the digoxigenin-labeled HER2 probe. Development of HRP was performed with DAB as the substrate using the reaction mixtures outlined in Table 2. The final pH of the buffers following addition of the DAB was measured as was the color of the solution (Table 2).
Many of the solutions containing DAB and hydrogen peroxide turned various shades of brown (Table 2). The brown coloration was most intense in the solutions containing sodium acetate (Solutions 5, 6, 9 and 10 in Table 2). The intensity of the brown coloration increased with time. The brown coloration seen in many of the solutions was more intense at the interphase between the liquid and the atmosphere, thereby suggesting that the mechanism of color formation involved oxidation in the presence of molecular oxygen. With overnight incubation, precipitation was noted even when no HRP was present.
The resulting CISH intensity scores obtained when each of the buffers were used for HRP color development are summarized in Table 3. The use of acetate-containing buffers resulted in very high backgrounds. Incorporation of imidazole in the HRP reaction increased the signal intensity. Optimal HER2 CISH signals were obtained in buffers 11, 12, 15, 16, 20 and 22 (Table 3).
Oxidation frequently is accelerated by the presence of metals. Therefore, it seemed reasonable that the addition of chelating agents to the DAB solution might reduce the formation of the brown product. Incubation of DAB and hydrogen peroxide in acetate-containing buffers for prolonged periods was observed to result in the formation of a brown precipitate Addition of 0.8 mM DTPA was found to prevent formation of brown color as well as the development of a precipitate. These results suggest that acetate-containing buffers may contain a sufficient amount of trace metals such that, in combination with oxygen, oxidation of DAB is promoted. Addition of HRP to solutions containing 165 mM Acetate, pH 4, 2.0 mM DAB, 0.8 mM DTPA and 0.025% hydrogen peroxide resulted in the formation of a large precipitate, indicating that addition of the DTPA did not inhibit the enzymatic reaction.
The effect of three different buffers (MES, HEPES and Tris), the presence of imidazole, and the presence of DTPA in the final HRP color development step were examined for HER2 CISH detection. The resulting data are summarized in Table 4. Buffers containing MES or HEPES resulted in very strong CISH HER2 signals. The presence of higher concentrations of Tris showed stronger, more consistent signals than the Tris concentration typically used in Invitrogen HER2 CISH kits. Incorporation of imidazole resulted in more consistent and darker CISH signals. The presence of DTPA in the buffer did not negatively impact the signal intensity or increase background.
A broader range of DTPA and imidazole concentrations were evaluated in Tris and HEPES buffers. The resulting data are summarized in Table 5. The addition of DTPA reduced the amount of precipitate that formed in the final reaction mixture (containing substrate, buffer and hydrogen peroxide) without having a negative impact on the intensity of the final CISH signal. The presence of imidazole increased the signal intensity in all buffer conditions. The presence of DTPA up to 9 mM concentration did not negatively impact signal intensity. Therefore, the addition of DTPA results in a more stable final reaction mixture (containing buffer, hydrogen peroxide and DAB) without having a negative impact on CISH signal intensity.
Two problems were sought to be overcome with a typical DAB formulation as presented by an Invitrogen DAB formulation. First, storage of such a DAB formulation results in sporadic oxidation and precipitation of DAB; DAB-containing vials which show intense color and those that show some precipitation result in increased backgrounds when used in CISH. Second, formulation of a reaction mixture consisting of Buffer/Substrate Buffer (Reagent B1 or I1), DAB chromogen (Reagent B2 or I2), and hydrogen peroxide (Reagent B3 or I3) results in varying rates of DAB oxidation and precipitation following mixing of the three components. Typically, the presence of intense coloration and/or precipitation resulting from oxidation also contributes to increased background when used in CISH. Because this oxidation occurs as a function of time, typical Invitrogen kits have recommended using the reaction mixture essentially immediately after mixing the three aforementioned components (i.e., Reagent B1 or I1+Reagent B2 or I2+Reagent B3 or I3). Requiring such immediate use of the reaction mixture is clearly disadvantageous for automation. Formulation of a more stable reaction mixture would reasonably be expected to result in a more robust kit as well as increased compatibility with automation.
The absorbance spectrum of DAB formulated in water shows a strong absorbance in the UV region of the spectrum with a maximum absorption around 270 nm. Upon storage, there is an increase in brown coloration with a corresponding increase in absorbance in the 465 nm to 520 nm region of the spectrum. These peaks are likely due to the formation of DAB oxidation products. Upon addition of imidazole to 100 mM concentration and hydrogen peroxide to 0.03%, there is a splitting of the UV peak with resulting absorption maxima around 270 nm and 310 nm. In addition, the visible peak shifts from an absorption maximum of 520 nm to around 460 nm. Following incubation of the mixture in the presence of HRP there is a loss of both the visible and UV peaks due to precipitation of the DAB.
The absorbance spectra of a DAB reaction mixture containing DAB, Tris (pH 7.4) and hydrogen peroxide were measured upon incubation at room temperature. After 14 hours of incubation, conjugate containing HRP was added and the mixture was incubated for an additional 5 minutes. The absorbances at 280 nm and 307 nm that were observed are similar to those described when DAB is incubated in water. Also similar to what was found when DAB is incubated in water are absorbance peaks that appear in the range of 465 nm to 478 nm and increase upon incubation at room temperature.
For purposes of the present invention, the absorbance at 465 nm to 520 nm was used to monitor the appearance of the oxidation product(s) of DAB. The absorbance at 280 nm or 307 nm was used to monitor the loss of DAB due to precipitation.
The effect of a series of additives on the stability of DAB formulated in 100 mM Tris, pH 8; 100 mM HEPES, pH 7.4; 100 mM MES, pH 6.5; and 100 mM Imidazole were evaluated based on color changes and the appearance of precipitation upon incubation at 37° C. (Table 6 and Table 7). Loss of DAB through precipitation was monitored by the absorbance at 280 nm (Table 8) while DAB oxidation was monitored by the absorbance at 478 nm (Table 9). With the exception of ascorbic acid and EGTA, most of the additives resulted in reduced DAB precipitation upon incubation at 37° C. (Table 8). Depending on the buffer, the additives propylene glycol, acetonitrile, ribose and DTPA reduced the background oxidation rate of DAB when incubated at elevated temperature (Table 9). The effect of the additives on HRP activity was evaluated following addition of Anti-Mouse HRP Polymer conjugate. The addition of metals has been reported to increase the intensity of the DAB signal generated from the HRP reaction. However, the addition of metals (cobalt, copper and magnesium) resulted in significant precipitation of DAB prior to the addition of enzyme.
The effect of a series of additives on DAB stability when formulated in a wider range of buffers and pH conditions was evaluated spectrophotometrically. Under most of the conditions examined, there was little visible precipitation of DAB with corresponding little loss of UV absorbance (Table 10). The significant loss of absorbance at 280 nm resulting from DAB precipitation upon addition of HRP suggested that the enzyme remained active in all evaluated buffers. Oxidation of DAB was monitored by increased absorbance at 520 nm (Table 11). In general, the incorporation of chelating agents (i.e., DTPA, EDTA, EGTA, 1,10-phenanthroline or diethylene-triaminepentamethylenephosphonic acid) reduced DAB oxidation rates in many of the buffers. With some of the buffers, the additives ascorbic acid, glycerol, and ribose also had a protective effect.
When DAB is formulated in water, there is a 230-fold increase in absorbance at 520 nm upon incubation at 37° C. for 5 days (Table 12). Formulation in 85% methanol had the greatest effect in preventing DAB oxidation (Table 12). The protective effect of methanol shows a dose dependence (Table 12). D-ribose, ascorbate and the chelating agents DTPA, EDTA and EGTA had the greatest effect in reducing DAB oxidation. Upon storage, solutions containing ascorbate and D-ribose turned orange resulting in modification of the corresponding absorption spectra.
By plotting the increase in absorbance at 520 nm with time (
A study to examine the ability of DTPA, EDTA, 1,10-phenanthroline, D-ribose, polyethylene glycol, sodium metabisulfite and ascorbate concentration to decrease DAB oxidation was performed (Table 14). Optimum protection occurred at 2 mM DTPA, 20 mM D-ribose, 50 mM polyethylene glycol, 5 mM sodium metabisulfite and at all ascorbate concentrations.
Combinations of agents were evaluated at two concentrations (Table 15 and Table 16). Combinations containing DTPA, sodium metabisulfite and/or D-ribose showed the greatest protective effect. Formulations containing 10 mM sodium metabisulfite contained visible precipitation, while those containing D-ribose or ascorbate became dark orange or brown (Table 16). As a result of the color change, D-ribose and ascorbate were avoided. The absorbance at 520 nm of each of the buffers is summarized in
Additional formulations and stability studies revealed that sodium metabisulfite tended to form precipitates even when formulated at 1 mM concentration. Sodium sulfite was evaluated as a potential alternative to sodium metabisulfite (Table 17). Whereas concentrations of sodium metabisulfite above 1 mM resulted in precipitation, concentrations of sodium sulfite as high as 50 mM did not show significant precipitation (Table 17). Concentrations of sodium sulfite in the range of 1 mM to 50 mM displayed a protective effect against DAB oxidation similar to that seen with 1-10 mM sodium metabisulfite.
Additional DAB stabilizers were also tested. Polyethylene glycol (PEG), propylene glycol (PG), dimethylsulfoxide (DMSO), glycerol, and 1-methyl-2-pyrrolidone were added to a 50 mM DAB solution containing 10 mM DTPA and 1 mM sodium metabisulfite. The addition of propylene glycol and PEG had a positive effect on DAB stability, reducing the absorbance maximum more than the DAB solution without these additives. Addition of DMSO, glycerol or 1-methyl-2-pyrrolidone had a negative effect as absorbance values increased compared to the control solution. Higher concentrations of PG were also tested. DAB solutions containing 60% PG and 70% PG displayed significantly reduced absorbance maxima compared to DAB solutions without PG and a DAB/85% Methanol solution over the same time course.
Several modifications of the final components were retested to ensure robustness. Sodium sulfite was again compared to sodium metabisulfite. The performance of two DAB solutions formulated with either sodium metabisulfite or sodium sulfite was compared using IHC. Replacement of sodium metabisulfite with sodium sulfite in the DAB formulation slightly improved signal intensity although both formulations outperformed the current Invitrogen-Zymed DAB solution. Based on the results obtained, DAB was formulated with 10 mM DTPA, 1 mM sodium sulfite and 65% propylene glycol.
Notwithstanding the observation that prototypical DAB formulations containing sodium sulfite, DTPA, and propylene glycol display greatly improved DAB stability, the concomitant staining intensity thereof was lower than that of DAB formulations from many competitors. A higher staining intensity was produced by increasing the concentration of DAB in the stock solution (Table 18). The prototypical DAB chromogen solution containing 5 mM DAB outperformed 3 of 4 competitor products when tested by IHC. Higher concentrations of DAB resulted in an increase in signal intensity, but also generated an increase in background staining.
Based on the data obtained, a final formulation of DAB including 200 mM DAB, 20 mM HCl, 10 mM DTPA, 1 mM Sodium Sulfite, and 65% Propylene Glycol was deemed optimal.
Another improvement sought to be developed for Invitrogen detection kits, which kits have heretofore consisted of three components (see paragraph [0033] above), is a two-component system, including a DAB chromogen component and a hydrogen peroxide component, in which the hydrogen peroxide component would be stably formulated in a reaction buffer.
An assay for HRP activity was developed based on Herzog and Fahimi (see, Herzog, V. and Fahimi, H D. (1973). A New Sensitive Colorimetric Assay for Peroxidase Using 3,3′-Diaminobenzidine as Hydrogen Donor. Analytical Biochemistry 55: 554-562) to indirectly assay for active hydrogen peroxide concentration (
With respect to pH, the greatest activity of HRP is found with acidic buffers (Table 20). With the exception of 200 mM MES, pH 6.5, 200 mM HEPES, pH 7.4, and 50 mM Imidazole, hydrogen peroxide appears stable when stored at 37° C. for 112 hours (Table 20). Based on the activity of HRP and hydrogen peroxide stability, focus was placed on citrate and acetate buffers in the pH range of 4.0 to 5.0. A reaction mixture containing 200 mM Sodium Citrate, pH 5.0, 1 mM DTPA, 50 mM imidazole, and 0.03% hydrogen peroxide was observed to lose only a small portion of its activity when stored at 37° C. for 9 days (
The effect of DAB concentration on HRP activity was evaluated in buffers containing 200 mM sodium acetate, pH 5.0, 1 mM DTPA, 50 mM imidazole and 0.015% hydrogen peroxide (Table 21,
Although the HRP rate increased with increasing imidazole concentration, the total amount of signal seen at 5 minutes remained constant (Table 22). The effect of imidazole concentration was investigated using three different buffering systems: 0 to 1 M imidazole, and in the absence of imidazole either 50 mM Tris at pH 8 or 100 mM phosphate at pH 6 and 7. As was seen with acetate buffers, the enzymatic HRP rate was affected more than the total amount of DAB signal. The HRP rate reached a maximum at around 100 mM to 200 mM imidazole. In contrast, the DAB signal reached a maximum value at 50 mM imidazole.
The effect of a few other additives in the reaction buffer was also investigated (Table 23). Although higher concentrations of dextran sulfate increased the overall catalytic rate of HRP, the total amount of signal did not increase.
The stability of the hydrogen peroxide-containing buffer was tested in CISH. Reaction buffer consisting of 200 mM sodium acetate, pH 5.0, 1 mM DTPA, 50 mM Imidazole and 0.03% hydrogen peroxide was stored at 37° C. for 27 days. When used as the detection buffer in CISH with 50 mM DAB, 10 mM DTPA, 65% propylene glycol and 10 mM sodium sulfite, the signal intensity was stronger than that seen using the control reagents (
Based on the data obtained, a final formulation of stable reaction buffer including 200 mM Sodium Acetate, pH 5.5, 1 mM DTPA, 50 mM Imidazole, and 0.03% Hydrogen Peroxide was deemed optimal.
A detailed description of the invention having been provided above, the following examples are given for the purpose of illustrating the invention and shall not be construed as being a limitation on the scope of the invention or claims.
Several competitive DAB-based HRP detection systems were analyzed. In initial screening of competitive reagents, the most intense signals were obtained with the PowerVision™ Plus and Vector ImmPACT™ reagents. The pH of the components from a number of the kits were measured (Table 24).
The effect of several competitive detection systems on HRP activity was assessed (Table 25). The PowerVision™ and ImmPACT™ DAB kits displayed significantly higher HRP catalytic activity than that found with standard Invitrogen reagents. With the PowerVision™ buffer system, 1.5 mM DAB was found to be less effective than the PowerVision™ chromogen. In contrast, 200 mM NaOAc, pH 5.0, 1 mM DTPA, 50 mM Imidazole, 0.03% H2O2 was found to be more active than the PowerVision™ buffer system with the PowerVision™ chromogen. The ImmPACT™ buffer system was found to be more active with 1.5 mM DAB than a 200 mM sodium acetate, pH 5.0, 1 mM DTPA, 50 mM Imidazole, 0.03% H2O2 formulation of the present invention.
Each of the components of the PowerVision™ detection system was assessed in the HRP assay (
The absorption spectrum of the PowerVision™ Plus chromogen reagent suggested that this chromogen reagent utilizes a modified DAB. Evaluation of the ImmPACT™ and ImmunoVision™ buffers revealed their superiority over the formulation in current Invitrogen CISH kits. Using imidazole in the buffer did not improve the enzymatic activity to the level found in the two competitive kits (
The final formulations of the paired DAB chromogen (i.e., 200 mM DAB, 20 mM HCl, 10 mM DTPA, 1 mM Sodium Sulfite, and 65% Propylene Glycol) and Hydrogen Peroxide Buffer (i.e., 200 mM Sodium Acetate, pH 5.5, 50 mM Imidazole, 1 mM DTPA, and 0.03% Hydrogen Peroxide) solutions, referred to herein as “Next Generation Detection Reagents,” were tested by IHC for reproducibility, robustness of the DAB source material, general stability of the components, and equivalency to current Invitrogen products and competitor offerings.
To evaluate the repeatability and reproducibility of the Next Generation Detection reagents, as provided by the present invention, multiple tissue types and protein targets were tested to assess for broad spectrum functionality (Table 26, Table 27, Table 28, and Table 30). Using IHC, three independently manufactured R&D lots of the final formulation of the Next Generation DAB chromogen and Hydrogen Peroxide Buffer were compared to assess lot-to-lot reproducibility (Table 26). With regard to signal intensity, each lot produced similar results (signal intensity score varied <0.5 units) when tested by IHC using two different primary antibodies. Day-to-day reproducibility (Table 28) and intra-run reproducibility (Table 27) were also assessed and exceeded the Design Input Specifications for this program. Specifically, using the Next Generation DAB chromogen and Hydrogen Peroxide Buffer, 90% of the samples (n=10) gave the same signal intensity when run on 3 separate days and 100% of the samples (n=12) gave the same signal intensity (≦0.43) when run in triplicate in the same assay.
Four sources of DAB from different manufacturers were tested to determine the robustness of the new DAB detection system (Table 29). Four identical formulations of Next Generation DAB chromogen solution were prepared with each DAB source and compared by IHC. Regardless of the DAB source material, the signal intensity was reproducible and consistent. Each of the four Next Generation DAB formulations outperformed the Invitrogen-Zymed DAB chromogen and buffer currently supplied in the Invitrogen SuperPicture™ Polymer Detection Kit (87-9663).
The final formulation of the Next Generation DAB Chromogen and Hydrogen Peroxide Buffer were first tested for long term stability by assessing performance of these formulations after storage at 37° C. for 9 days (comparable to 6 months to 1 year at 4° C.; see, Anderson, Geoffrey and Scott, Milda. (1991). Determination of Product Shelf Life and Activation Energy for Five Drugs of Abuse. Clin. Chem. 37, No. e: 398-402.). As shown in
IHC was performed to evaluate the effectiveness of the Next Generation Detection platform versus the Invitrogen SuperPicture™ detection kit and four competitor product lines (Table 30). The Next Generation detection platform consistently outperformed the Invitrogen SuperPicture™ Detection system as evidenced by an increase in mean signal intensity (3.5 vs. 3.18) when compared across 11 tissues using three different primary antibodies. On average, the Next Generation DAB kit outperformed 2 of 4 competitive DAB products and was equivalent to the other two competitive products tested (Table 30).
Each of the above-cited references are hereby incorporated herein by reference as if set forth fully herein.
NA#
#Not Available
#IHC was performed to detect expression of Estrogen Receptor and Ki67 according to the manufacturer's instructions.
#Staining intensity was scored following Invitrogen Quality Procedures, Document No. TM-041. Maximum staining intensity score = 4.0
#Staining intensity was scored following Invitrogen Quality Procedures, Document No. TM-041. Score range = 0.0-4.0
This application is a U.S. National Stage Application of PCT application no. PCT/US2009/068067, filed Dec. 15, 2009, which claims priority to U.S. application No. 61/122,692, filed Dec. 15, 2008, which disclosures are herein incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US09/68067 | 12/15/2009 | WO | 00 | 12/19/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61122692 | Dec 2008 | US |
