ARTICLE |
Correspondence to: Kevin A. Roth, Dept. of Pathology (Box 8118), Washington Univ. School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110.
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Summary |
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Quantitation of antigen concentration in cell and tissue samples typically requires antigen extraction, which precludes antigen localization in the same sample. Similarly, antigen immunolocalization in fixed cells or tissue sections provides limited information about antigen concentration. We have developed a rapid and sensitive assay for simultaneous antigen localization and quantitation in cell and tissue samples that does not involve antigen extraction, radioactive materials, or image analysis. Fixed cells and/or tissue sections are used with antigen-specific enzyme-linked probes to generate soluble reaction products that are spectrophotometrically quantifiable and deposited reaction products that are microscopically localizable. The amount of soluble reaction product is dependent on several variables, including antigen concentration, probe specificity and sensitivity, sample size, and enzyme reaction time. These variables can be experimentally controlled so that soluble reaction product is proportional to antigen concentration in the sample. This assay was used in multiple applications including detection of Ki-67 nuclear antigen immunoreactivity in human brain tumors, in which it showed a clear relationship with visually determined Ki-67 cell labeling indexes. This assay, termed the Midwestern assay, should be applicable to a wide variety of antigens in both clinical and research samples. (J Histochem Cytochem 45:1629-1641, 1997)
Key Words: enzyme-linked, immunosorbent assay (ELISA), chromogens, tyramide signal amplification, (TSA), anti-nuclear antibodies (ANA), progesterone receptors, Ki-67 nuclear antigen
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Introduction |
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Enzyme-linked antibodies and insoluble enzyme substrates have been used for over 30 years to localize antigens in cells and tissue sections (
Enzyme-linked antibodies are also used to detect and quantitate antigens coated to solid surfaces (
The principles and procedures of enzyme immunohistochemistry have been combined with those of CELISA to create an assay that is capable of simultaneously localizing and quantitating target substances in biological samples. The development of this assay, termed the "Midwestern assay," is based on the observation that an enzyme-linked probe can produce both soluble and insoluble reaction products. Unlike insoluble reaction products, generation of soluble reaction products can occur without significant destruction of enzyme activity, thus permitting sequential soluble and insoluble chromogenic reactions from a single enzyme-linked probe. Alternatively, some enzyme substrates or combinations of substrates simultaneously produce both soluble and insoluble reaction products. Multilabel immunohistochemical techniques have been adapted to perform quantitation and localization of multiple substances in single samples. Multilabeling of single sections, or separate immunoassays on serial sections, permits a direct comparison between probes that allows standardization of Midwestern assay results to internal controls such as DNA content, protein antigens, or cell surface carbohydrates. The Midwestern assay should prove applicable to a wide variety of targets and biological samples.
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Methods and Results |
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Sequential Production of Soluble and Insoluble Reaction Products Using Single Enzyme-linked Probes
To determine if an enzyme-linked probe could sequentially produce a soluble reaction product for quantitation and an insoluble reaction product for localization, alkaline phosphatase (AP)-conjugated wheat germ agglutinin (WGA; EY Laboratories, San Mateo, CA) lectin was used to probe paraffin-embedded sagittal sections from a 4% paraformaldehyde-fixed embryonic Day 14 mouse. WGA binds to N-acetylglucosamine residues present on the surface of most cell types including neurons (. The remaining pNPP solution on each slide was rinsed off with PBS and the slides were incubated with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) solution (Sigma) for 5 min to generate deposited reaction product for antigen localization. Sections were then washed in PBS and mounted in PBS:glycerol (1:1).
The amount of solution-phase reaction product generated by duplicate sections incubated in PBS-BB was 0.032 and 0.028 absorbance units compared to 1.313 and 1.340 absorbance units for duplicate sections incubated in 10 µg/ml WGA-AP. The insoluble BCIP/NBT reaction product was easily localized in the sections incubated with WGA-AP but was absent in sections incubated in PBS-BB alone (data not shown). These results indicate that an enzyme-linked probe can sequentially generate solution-phase quantifiable reaction products and solid-phase localizable products.
The efficiency of sequential solution-phase and solid-phase enzyme reactions depends on the amount of enzyme activity present after the initial reaction. To determine the residual enzyme activity available for chromogen deposition after generation of the solution-phase reaction product, sections of paraformaldehyde-fixed mouse brain were incubated with WGA-AP probe and reacted with pNPP as described above, followed by a second identical pNPP reaction. The amount of pNPP reaction product generated in the second reaction was 84 ± 2% (mean ± SEM, n = 8) of that in the first reaction. Subsequent BCIP/NBT deposition was minimally affected by prior pNPP reactions (data not shown). Similar experiments performed with horseradish peroxidase (HRP)-conjugated WGA (Sigma) and the soluble HRP substrates o-phenylene diamine dihydrochloride (OPD; Sigma) and tetramethylbenzidine dihydrochloride (TMB; Sigma) indicated approximately a 30% loss of enzyme activity with each of these two soluble substrates (data not shown). This diminution of enzyme activity had a minimal effect on the subsequent deposition of 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) chromogen (data not shown). In contrast to the small loss of enzyme activity associated with soluble chromogen production, there is a dramatic decrease in enzyme activity associated with the generation of insoluble chromogens. For example, the deposition of DAB before the generation of solution-phase reaction product can result in complete abolition of HRP enzyme activity (data not shown).
In the experiments described above, sections were incubated in soluble chromogen solution for 30 min, as is typical of most ELISA protocols. However, the optimal solution-phase reaction time for any given immunoassay requires experimental determination and depends on several variables including probe sensitivity, antigen concentration, tissue size, and enzyme-substrate kinetics. In most instances, sufficient soluble chromogen was generated for accurate spectrophotometric measurements within reaction times of 15 min.
To determine if the amount of solution-phase product was proportional to the amount of sample antigen, one, two, or four serial sections of the same fixed mouse brain were placed adjacent to one another on slides and incubated with WGA-AP (1:200) or PBS-BB and reacted sequentially with pNPP and BCIP/NBT. The mean net OD values for one, two, and four brain sections were 0.79, 1.32, and 2.22, respectively, indicating a clear relationship between tissue antigen and solution-phase quantitation. However, because this relationship was not absolutely linear in this WGA-AP binding assay, care must be taken in extrapolating results across samples containing widely varying tissue amounts.
In the above experiments, antigen quantitation and localization were performed with an enzyme-conjugated lectin probe. To determine if the Midwestern assay worked with a nonlectin probe and with a probe that was not directly enzyme-conjugated, rabbit anti-substance P antibodies were used to quantitate and localize substance P immunoreactivity in formalin-fixed sections of human spinal cord. The spinal cord contains high concentrations of substance P in a well-defined neuroanatomic distribution (
Serial spinal cord sections were deparaffinized and incubated in 0.3% H2O2 in 0.2 N HCl for 30 min to destroy endogenous peroxidase and alkaline phosphatase activity. Sections were washed in H2O, PBS, and PBS-BB before overnight incubation in PBS-BB or rabbit anti-substance P antibodies [Code R5 (
Localizable substance P-like immunoreactivity in the spinal cord sections was dramatically reduced by addition of synthetic substance P to the primary antiserum solution (Figure 1A and Figure 1B). Similarly, approximately two thirds of the soluble substance P signal was blocked by antibody incubation with excess substance P (1.6 and 0.5 absorbance units without and with synthetic substance P, respectively). The unrelated peptide, neuropeptide Y, failed to decrease either the localizable or quantifiable substance P-like immunoreactivity (data not shown). Similar studies with other antibodies also demonstrated a competitive relationship between antibody binding to antigen in solution and binding to antigen in cells for both quantitation and localization (data not shown). The percentage of total soluble signal that was specific varied, depending on probe specificity and sensitivity, antigen concentration, sample size, and the use of signal amplification techniques, but was greater than 90% in some experiments (data not shown). Together, these data demonstrate the applicability of the Midwestern assay to nonenzymatically conjugated probes.
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Simultaneous Production of Soluble and Insoluble Reaction Products Using Single Enzyme-linked Probes
In the experiments described above, enzyme-linked probes were used to sequentially generate soluble and insoluble reaction products. However, if soluble and insoluble products could be generated simultaneously, it would simplify and shorten the Midwestern assay. Therefore, the ability of several HRP and AP chromogenic substrates to produce both soluble and insoluble reaction products was examined. In these experiments, fixed sections of mouse brain were probed with either WGA-AP or WGA-HRP. The HRP substrates DAB, DAB/metal (Pierce; Rockford IL), 3-amino-9-ethylcarbazole (AEC, Sigma), o-Dianisidine (Sigma), and True Blue (Kirkegaard & Perry Laboratories; Gaithersburg MD) were found to generate effective insoluble reaction products for antigen localization but produced insufficient soluble reaction products for quantitation. 3,3', 5,5'-tetra-methylbenzidine (TMB; Sigma) and 5-amino salicylic acid (Sigma) generated soluble HRP reaction products but produced insufficient insoluble product for localization. 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) and OPD demonstrated some ability to generate both soluble and insoluble HRP reaction products.
OPD was used to simultaneously quantitate and localize goat anti-BrdU antibody binding to sections of Histochoice (Amresco; Solon OH)-fixed tissue from BrdU-treated mice. BrdU injections and tissue fixations were done as previously described (. Sections were washed, mounted in PBS:glycerol, and visualized with a microscope. Results are illustrated in Figure 2, and demonstrate the ability of OPD to simultaneously produce soluble and insoluble reaction products. However, the amount of deposited OPD chromogen was much less than that typically observed using DAB chromogen.
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To optimize simultaneous soluble and insoluble HRP chromogen generation, we compared OPD, the chemically related compound p-phenylene diamine dihydrochloride (PPD; Fluka, Buchs, Switzerland), and the Hanker-Yates reagent (
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Similar studies were performed with WGA-AP and AP substrates (data not shown). BCIP/NBT, New Fuchsin (Sigma), and Fast Blue naphthol (Sigma) produced insoluble and pNPP produced soluble reaction products. None of these substrates was effective in simultaneously producing soluble and insoluble products. Fast Red naphthol had a limited ability to generate both soluble and insoluble products, but the amount of soluble product generated was much less than that of pNPP. We found that a mixture of pNPP with BCIP/NBT was capable of producing both soluble and insoluble reaction products. However, sensitivity of both quantitation and localization was less than that when pNPP and BCIP/NBT reactions were performed sequentially (data not shown).
Multiprobe Quantitation and Localization
To be most useful, the Midwestern assay must be compatible with multiprobe detection. Several enzyme immunohistochemical methods have been published to accomplish multiprobe detection, and these are readily adapted to the Midwestern assay (
The feasibility of sequential AP reactions to detect multiple probes in the Midwestern assay was tested. Human brain sections from two cases of Alzheimer's disease were immunolabeled with antibodies against paired helical filament (PHF/tau) and glial fibrillary acidic protein (GFAP). Brains from patients with Alzheimer's disease contain increased amounts of PHF/tau (
Sections were deparaffinized, treated with 0.3% H2O2 in MeOH for 30 min, and incubated in PBS-BB. Two sections from each case were incubated with mouse anti-PHF/tau antibody [A8, (Polymedco; Cortlandt Manor, NY), 1:5000 in PBS-BB for 1 hr] and a third section was incubated with PBS-BB. Sections were then treated sequentially with biotin-conjugated donkey anti-mouse antibody (1:1000 for 1 hr), HRP-conjugated streptavidin (1:500 for 30 min), biotinyl-tyramide (1:100 for 5 min), and AP-conjugated streptavidin (1:1000 for 30 min). Three hundred µl of pNPP solution was placed on each section and after 6 min 100 µl was removed for spectrophotometric quantitation. Sections were rinsed and overlaid with BCIP/NBT for 10 min to generate a brownish-purple deposited product. Sections were boiled in water for 2 min to destroy the HRP and AP activities from the first detection procedure. This treatment converts the deposited BCIP/NBT chromogen to dark blue. One PHF/tau-reacted section from each case was coverslipped and examined under a microscope without further immunostaining.
The second PHF/tau-immunostained section and an additional control section were sequentially incubated with rabbit anti-GFAP serum (DAKO; Carpinteria, CA) (1:2000 overnight at 4C) and AP-conjugated goat anti-rabbit serum (Jackson ImmunoResearch Laboratories; 1:500 for 1 hr). The immunodetection of GFAP in sections with or without prior PHF/tau detection allowed assessment of the effect of the first immunodetection procedure on the second. pNPP and BCIP/NBT reactions were then performed as described above, except that boiling was not done after the BCIP/NBT reaction, thus leaving the deposited BCIP/NBT product from the first AP reaction blue and that from the second brownish-purple. Both PHF/tau and GFAP immunoreactivities were readily detected in Alzheimer's brain sections. Blue PHF/tau-immunoreactive neurons (Figure 4A) and brownish-purple GFAP immunoreactive astrocytes (Figure 4B) resulted when these reactions were performed individually. When the two reactions were performed sequentially on the same section, the two reactivities could be distinguished (Figure 4C). The net OD values for PHF/tau and GFAP reactivity were 1.35 and 0.23 in Case 1 and 1.64 and 0.85 in Case 2, respectively. Previous PHF/tau immunodetection decreased subsequent GFAP immunodetection by approximately 25%. In this example, PHF/tau was found in neurons and GFAP was localized in astrocytes, making simultaneous detection of both chromogens in single cells unnecessary. Chromogens capable of producing "mixed" colors when co-localization of reactivity occurs have recently been described (
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To demonstrate the feasibility of multiprobe AP and HRP immunodetection in the Midwestern assay, sequential AP and HRP reactions to detect progesterone receptor and cytokeratin immunoreactivity in two cases of surgically resected, formalin-fixed human breast cancer were performed. The presence of estrogen and progesterone receptors in human breast carcinomas correlates with both response to hormonal therapy and survival (
Microscopic examination of the dual immunostained sections showed dark brown (DAB/metal) cytokeratin immunoreactivity in the invasive ductal carcinomas in both cases (Figure 4D-F). In contrast, only the neoplastic cells in Case 2 exhibited dark blue (BCIP/NBT) progesterone receptor immunoreactivity (Figure 4D and Figure 4E). No DAB/metal or BCIP/NBT deposits were observed in sections in the absence of primary antibodies. The solution-phase quantitation of cytokeratin immunoreactivity was comparable between the two cases (0.421 vs 0.504 absorbance units in Case 1 and 2, respectively). In contrast, progesterone receptor immunoreactivity was essentially undetectable in Case 1 (0.003 absorbance units) but was easily quantitated in Case 2 (0.685 absorbance units), consistent with previous biochemical analyses. This example illustrates the ability of the Midwestern assay to quantitate and localize multiple probes using sequential AP and HRP reactions.
Applications of the Midwestern Assay
Anti-nuclear Antibody Testing.
Different patterns of anti-nuclear antibody (ANA) labeling obtained from human serum samples are described as homogeneous (diffuse), speckled, rim, centromere, or nucleolar, and are clinically associated with particular diseases (
HEp-2 human carcinoma cells (Kallestad Laboratories) served as substrate for ANA tests. Cell samples were blocked in PBS-BB and then reacted with either control or serially diluted patient serum samples in PBS-BB for 1 hr. Cell samples were then washed three times with PBS, incubated with HRP-conjugated anti-human IgG antibodies (Jackson ImmunoResearch Laboratories; 1:500 in PBS-BB for 1 hr), and then washed again three times with PBS. Next, 60 µl of OPD solution was added to each cell sample and reacted for a total of 15 min. The OPD solution was removed and combined with 15 µl of 3 N HCl and absorbance was measured at 490. Cell samples were washed with PBS to remove excess substrate solution, and a DAB/metal solution was added for 5 min. Cell samples were washed, coverslipped with PBS:glycerol (1:1), and observed with a light microscope.
The patterns of immunohistochemical staining produced by the separate serum samples were distinct. The positive control showed a diffuse homogeneous nuclear staining pattern suggestive of autoantibodies to native DNA, histones, or deoxyribonucleoprotein (Figure 5A). A pattern of small speckles was observed within the nuclei of cells reacted with serum Sample 1, suggestive of the presence of autoantibodies to nonhistone proteins (Figure 5B). The chromogen deposited on cells reacted with serum Sample 2 was predominantly at the rim of each cell nucleus, a pattern consistent with autoantibodies against DNA and histones (Figure 5C). The negative control showed no apparent chromogenic deposit (Figure 5D).
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In the quantitative analysis, the positive control produced the greatest amount of solution-phase chromogen (Table 1). Serum Sample 1 produced a lower amount of solution-phase chromogen than Sample 2, consistent with the relative titers of ANA known to exist in these patients' serum as determined by classical ANA fluorescent detection methods (titers of 1:1280 and >1:5120, respectively). Thus, both the pattern of ANA distribution within the cell as well as the quantitative amount of ANA was determined on each of the samples.
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Ki-67 Detection in Brain Tumors.
Ki-67 nuclear antigen is expressed in cells traversing the cell cycle but is absent from resting cells in G0 (
Microscopic examination showed many Ki-67-immunoreactive cells in both cases of glioblastoma multiforme (Figure 6A) and in other high-grade neoplasms. Only rare Ki-67 immunoreactive cells were detected in the non-neoplastic brain (Figure 6B). Visually determined Ki-67 cell labeling ranged from zero to 51 percent of cell nuclei (Figure 6D). The Midwestern assay quantitation of Ki-67 immunoreactivity, expressed as a ratio of Ki-67 to DNA immunoreactivity to control for differences in cellularity between samples, ranged from zero to 1.41 (Figure 6D). Linear regression analysis showed a significant correlation between the two determinations (r2 = 0.60, F = 26.1, p<0.005). Although there was a significant correlation between these two determinations, it is important to point out that the Midwestern assay quantitation of the Ki-67 to DNA ratio is not equivalent to the Ki-67 cell labeling index. In a visually determined cell labeling index, a cell is considered either positive or negative and a weakly positive cell is equivalent to a strongly positive cell. In the Midwestern assay, antigen immunoreactivity is determined for the entire sample, and therefore any given positive cell can contribute various amounts of reactivity to the entire sample. Because the Midwestern assay results in the formation of both soluble and localizable signals, additional studies can be readily performed to determine the relative advantages and disadvantages of these two determinations in different applications.
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Discussion |
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A variety of techniques exist for quantitation of substances in cells and tissues, including Western, Southern, and Northern blots, radioimmunoassays (RIAs), ELISAs, and dot-blot assays. Although these techniques can provide useful quantitative data, they require target extraction and/or tissue disruption, which precludes localization of the target substance in the sample. Only a few reports have been published on approaches other than image analysis to achieve both antigen quantitation and localization in single samples (
Simultaneous antigen localization and quantitation of autoantibodies in single human serum samples can be performed by using either a mixture of enzyme-conjugated anti-human antibodies and fluorophore-conjugated anti-human antibodies, or anti-human antibodies conjugated to both an enzyme and a fluorophore (
We have devised a simple method for simultaneously localizing and quantitating one or more target substances in biological samples using enzyme-linked probes. The assay utilizes fixed cells or tissue sections and probes such as primary antibodies or lectins that specifically bind to their targets and are either themselves enzyme-conjugated or are linked to enzyme-conjugated secondary reagents. The enzyme-linked probe is used to generate a first reaction product which is soluble in the bathing medium and is quantitatively related to the amount of target substance in the sample, and a second reaction product which deposits at the site of target-probe interaction and is localizable with a microscope.
Antigen quantitation in the Midwestern assay can be either relative or absolute. Absolute quantitation requires a comparison between the amount of soluble reaction product generated by an enzyme-linked probe and the amount of antigen determined in an identical sample, as measured by a separate quantitative technique such as an RIA or ELISA. Alternatively, in the Midwestern assay standard antibody-antigen competition curves can be generated by addition of known amounts of antigen to the primary antibody solution. Just as antigen quantitation in an ELISA or RIA will be affected by the efficiency of the antigen extraction conditions and aliquot size, antigen quantitation in the Midwestern assay will be affected by tissue fixation (antigen preservation) and sample size. Therefore, the Midwestern assay will be most applicable to target quantitation in identically processed samples of similar size and consistency. Experimental evidence for the validity of immunohistochemical quantitation of some antigens by image processing measurements has recently been provided (
Both AP and HRP enzyme conjugates have been successfully used in the Midwestern assay; other enzymes, such as ß-galactosidase, glucose oxidase, and chloramphenicol acetyl transferase, should prove applicable if appropriate soluble and depositable reaction products are identified. The enzyme reaction products that have typically been utilized are chromogens and can be quantitated spectrophotometrically in solution and visualized by light microscopic examination of the sample. Fluorogenic enzyme substrates for both AP and HRP have been described (
Because the Midwestern assay is based on enzyme immunohistochemistry, multilabel immunohistochemical techniques are readily adaptable for multiprobe quantitation and localization. Some caution must be exercised when sequential deposited chromogenic reactions are performed because chromogen deposition can interfere with subsequent immunohistochemical detection (
As with any quantitative assay, accurate Midwestern assay determinations require appropriate controls. In the examples presented earlier, several criteria were used to establish specificity, including antigen blocking controls, probe omission, antigen-positive and -negative controls, and internal standards to control for sample size. The Midwestern assay has several advantages over other quantitative immunohistochemical assays. First, it is a simple assay requiring only routine laboratory equipment. Second, because antigen quantitation is performed across the entire sample, heterogeneous antigen distribution does not bias quantitation (i.e., "randomly" selected or "representative" fields are not the basis for quantitation). Third, in contrast to semiquantitative visual determinations of immunopositive vs immunonegative signals which require a subjective assessment of "intermediate" or "weakly positive" structures, Midwestern quantitation is entirely objective. However, unlike image processing techniques in which quantitation can be performed on an individual cell basis, Midwestern quantitation integrates antigen reactivity across the entire specimen. The ability to trim paraffin-embedded sections by scraping away unwanted tissue with a razor blade before the Midwestern assay can reduce but not eliminate this problem.
The Midwestern assay has been successfully applied to a variety of cell and tissue samples, fixation conditions, and probes. In our initial experiments on human brain neoplasms, Midwestern assay quantitation of Ki-67 immunoreactivity was highly correlated with visually determined Ki-67 cell-labeling indexes. Because the assay is a modification of existing immunohistochemical techniques, it should prove widely applicable to both basic science and clinical laboratory settings and to a variety of targets and samples.
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Acknowledgments |
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Supported by funds from the Washington University School of Medicine, Department of Pathology.
We thank Dr Mark N. Bobrow (NEN Life Science Products) for the generous gifts of tyramide signal amplification reagents and for reviewing the manuscript. We also thank Drs Jack Ladenson, Anne Marie Yunker, and Kenneth Shindler for valuable discussions and reviewing of the manuscript.
Received for publication March 4, 1997; accepted June 9, 1997.
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