Journal of Histochemistry and Cytochemistry, Vol. 45, 1629-1642, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Enzyme-based Antigen Localization and Quantitation in Cell and Tissue Samples (Midwestern Assay)

Kevin A. Rotha,c, Jennifer W. Brennera, Lee A. Selznicka, Murat Gokdena, and Robin G. Lorenza,b
a Department of Pathology, Washington University School of Medicine, St. Louis, Missouri
b Department of Medicine, Washington University School of Medicine, St. Louis, Missouri
c Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri

Correspondence to: Kevin A. Roth, Dept. of Pathology (Box 8118), Washington Univ. School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110.


  Summary
Top
Summary
Introduction
Methods and Results
Discussion
Literature Cited

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


  Introduction
Top
Summary
Introduction
Methods and Results
Discussion
Literature Cited

Enzyme-linked antibodies and insoluble enzyme substrates have been used for over 30 years to localize antigens in cells and tissue sections (Nakane and Pierce 1967 ). Unlike fluorescently-linked antibodies, enzyme-conjugated probes produce a signal amplification and do not require a fluorescence microscope for antigen localization. Quantitative procedures have demonstrated a clear relationship between the amount of deposited chromogen and the amount of antigen in the sample (Gin et al. 1988; Nabors et al. 1988 ; Hoyer and Kirkeby 1996 ; Watanabe et al. 1996 ). Most of these procedures involve the use of image analysis or microphotometric techniques (Witorsch 1982 ; Nibbering et al. 1985 , Nibbering et al. 1986 ; Nibbering and van Furth 1987 ; Mize et al. 1988 ; Masliah et al. 1990 ; Fritz et al. 1995 ; Hollingsworth et al. 1996 ). Despite their effectiveness, routine antigen quantitation by image analysis and/or microphotometry has not become widespread because of the specialized and expensive equipment, extensive training, and slow throughput typically associated with their use.

Enzyme-linked antibodies are also used to detect and quantitate antigens coated to solid surfaces (Eng-vall et al. 1971 ). Enzyme-linked immunosorbent assays (ELISAs) use enzyme substrates to produce soluble reaction products that are quantitated by spectrophotometric methods. Modified ELISAs are also used to quantitate cell surface antigens (Morris et al. 1982 ). In the modified ELISAs, whole cells are plated on the solid surface and cell surface antigens are quantitated on the basis of the generation of soluble enzyme reaction product. This cellular ELISA (CELISA) demonstrates that quantitative data can be generated from whole cells and enzyme-linked antibodies.

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.


  Methods and Results
Top
Summary
Introduction
Methods and Results
Discussion
Literature Cited

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 (Allen et al. 1973 ; Flaris et al. 1995 ). Sections were deparaffinized, boiled for 2 min in H2O to destroy endogenous AP activity, and tissue on each slide was encircled with a PAP pen (Research Products International; Mount Prospect, IL). Slides were then placed in PBS-blocking buffer (PBS-BB; 0.1 M, pH 7.2 containing 1% bovine serum albumin, 0.2% nonfat powdered milk, and 0.3% Triton X-100) for 10 min to inhibit nonspecific probe binding and incubated for 1 hr at room temperature (RT) in either PBS-BB or 10 µg/ml WGA-AP diluted in PBS-BB. All conditions were performed in duplicate on serial sections. After three 5-min washes in PBS, 200 µl of p-nitrophenyl phosphate (pNPP) solution (Sigma; St Louis, MO) was added to each slide. The pNPP solution and all other chromogen solutions used were made according to the manufacturer's instructions. The volume of chromogen solution added to each section was arbitrarily chosen but must be identical for all samples within the assay, must be sufficiently large to cover the sample, and must permit easy removal of an aliquot for spectrophotometric quantitation (see below). Slides were rotated at 40 cycles/min on a horizontal shaker to ensure continual mixing of the reaction product. After 30 min, 100 µl of pNPP solution was removed from each slide and placed in microplate wells containing 25 µl of 3 N NaOH to stabilize the pNPP reaction product. The amount of pNPP reaction product generated by each sample was quantitated spectrophotometrically with an automated microplate reader (EL311 SX; Bio-Tek Instruments, Winooski, VT) at 405{lambda}. 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 (DiFiglia et al. 1982 ).

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 (Roth and Krause 1990 ); 1:20,000 in PBS-BB] containing 1 µM synthetic substance P (Sigma), an unrelated peptide (1 µM neuropeptide Y; Sigma), or no peptide. Sections were washed in PBS and incubated for 1 hr with biotin-labeled donkey anti-rabbit antibodies (1:1000 in PBS-BB; Jackson ImmunoResearch Laboratories, West Grove PA) followed by streptavidin-HRP [1:500 in Tris-blocking buffer (TBB); 20 mM Tris buffer, pH 7.6, 0.9% NaCl, 0.5% NEN blocking reagent; NEN Life Science Products, Boston, MA] for 30 min. Tyramide signal amplification (TSA; NEN Life Science Products) was used to increase signal intensity (Bobrow et al. 1989 ; Shindler and Roth 1996 ). TSA was performed for 5 min with biotinyl-tyramide (1:100) according to the manufacturer's protocol. Sections were then washed, incubated in streptavidin-AP (1:1000 in TBB; Jackson ImmunoResearch Laboratories) for 30 min, washed, and incubated with 250 µl of pNPP solution for 15 min. One hundred µl of pNPP solution was removed for spectrophotometric measurement. The remaining pNPP solution was washed off and slides were reacted for 15 min in BCIP/NBT to produce a localizable product.

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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Figure 1. Inhibition of substance P-like immunoreactivity by co-incubation of primary antiserum with excess substance P. Intense substance P-like immunoreactive nerve terminals can be seen adjacent to primary motor neurons in the anterior spinal cord (A), and this reactivity is abolished by co-incubation with 1 µM substance P (B). Solution-phase substance P-like reactivity decreased from 1.6 to 0.5 absorbance units in the presence of excess substance P. Bar = 25 µm.

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 (Roth et al. 1996 ). Serial fivefold dilutions of goat anti-BrdU antibody (Cohn and Lieberman 1984 ; a generous gift from Dr. Steven Cohn, Washington University) from 1:2000 to 1:250,000 were performed and antibody binding was detected with either donkey anti-goat HRP (1:1000, Jackson ImmunoResearch Laboratories) or donkey anti-goat HRP (1:1000) and TSA with biotinyl-tyramide (1:100) and streptavidin-HRP (1:1000). Sections were incubated with 250 µl of OPD for 5 min and 100 µl of OPD solution from each slide was then removed and placed in microplate wells containing 25 µl of 3 N HCl for quantitation of OD at 490{lambda}. 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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Figure 2. Simultaneous quantitation (A) and localization of BrdU immunoreactivity with (B) and without (C) TSA. Quantitation of BrdU immunoreactivity was significantly improved by TSA (A). At an antiserum dilution of 1:2000, localizable OPD signal was visualized with (B) but not without (C) TSA. Bar = 25 µm.

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 (Hanker et al. 1977 ), which consists of a mixture of PPD and pyrocatechol (PC) (PPD/PC; Fluka). Sections of mouse brain were incubated with WGA-HRP as described earlier and reacted for 5 min with OPD, PPD, or PPD/PC. The OD of the solution-phase reaction product was quantitated spectrophotometrically and the amount of deposited localizable reaction product was assessed with a Zeiss Axioskop and MC100 camera attachment (Figure 3). As above, OPD was capable of generating both soluble and insoluble products. However, it was relatively ineffective in generating localizable product compared to PPD and PPD/PC. PPD and PPD/PC produced roughly half of the soluble signal of OPD. PPD/PC produced approximately three times as much localizable signal as PPD alone and represents the best HRP chromogen substrate identified to date for simultaneous soluble and insoluble product generation. However, the applicability of PPD/PC to the Midwestern assay will require additional experiments to determine the relationships between the generation of soluble and insoluble products, enzyme reaction time, and antigen concentration.



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Figure 3. The relative ability of OPD, PPD, and PPD/PC to simultaneously produce soluble, quantifiable signal and deposited, localizable signal was determined using WGA-HRP and serial mouse brain sections. Soluble signal was quantitated spectrophotometric ally and localizable signal by photomicrographic exposure times in standard x40 fields. OPD was a sensitive soluble chromogen (A) but produced little deposited reaction product (B). PPD (not shown) and PPD/PC (C) were capable of producing both soluble and insoluble reaction products. Bar = 25 µm.

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 (Nakane 1968 ; Mason and Sammons 1978 ; Sternberger and Joseph 1979 ; Malik and Daymon 1982 ; Hermiston et al. 1992 ). To detect two antibodies raised in different species, an AP-conjugated secondary antibody to the first primary antibody can be used in conjunction with an HRP-conjugated secondary antibody to the second primary antibody. HRP quantitation and localization can then be performed sequentially with AP quantitation and localization. Alternatively, if both antibodies are detected with the same enzyme, after quantitation and localization of the first enzyme-linked antibody, the enzyme activity of the probe can be destroyed physically (e.g., boiling) or chemically (e.g., acid treatment) (Shi et al. 1997 ). The second primary antibody can then be applied and detected with a second enzyme-linked antibody. To detect two antibodies raised in a single species, the recently described dilutional neglect technique (Shindler and Roth 1996 ) or the enhanced polymer one-step staining system could be used (van der Loos et al. 1996 ).

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 (Biernat et al. 1992 ; Braak et al. 1994 ) and may exhibit neuron loss and reactive astrocytosis.

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 (van der Loos et al. 1996 ) and should be applicable to the Midwestern assay.



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Figure 4. Immunohistochemical detection of PHF/tau and GFAP immunoreactivity in Alzheimer's disease brain using sequential AP reactions (A-C). PHF/tau-immunoreactive neurons appear blue (A) and GFAP immunoreactive astrocytes brown (B) when immunodetection protocols are performed separately. Sequential immunodetection of PHF/tau and GFAP on single slides results in distinct localizable immunoreactivities. Blue PHF/tau-immunoreactive neurons and brown GFAP immunoreactive astrocytes are seen scattered throughout the cortex (C). Immunohistochemical detection of progesterone receptor and cytokeratin immunoreactivities in breast carcinomas using sequential HRP and AP reactions (D-F). Low magnification shows blue progesterone receptor-immunoreactive nuclei and brown cytokeratin-immunoreactive cytoplasm in a case of an invasive breast carcinoma (D). Higher magnification shows the discrete subcellular distribution of the two immunoreactivities (E). A control case that lacks progesterone receptors shows only brown cytokeratin immunoreactivity (F). Bars = 25 µm.

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 (Clark et al. 1983 ; Fritz et al. 1995 ). Several studies have shown that semiquantitative visual determination and quantitative image analysis assessment of estrogen receptor and progesterone receptor immunoreactivity on fixed tissue sections is highly correlated with biochemical measurements of receptor in frozen samples (Gin et al. 1988; Scheres et al. 1988 ). Case 1 had previously been shown to lack progesterone receptors by both immunohistochemical and biochemical analysis. In contrast, Case 2 had been determined to have abundant progesterone receptor immunoreactivity and a high concentration of receptor activity in a clinical assay (data not shown). Sections from both cases were deparaffinized, boiled for 15 min in 0.01 M citrate buffer, pH 6.0, incubated for 10 min in 3.0% H2O2 in MeOH, washed in H2O, and incubated for 30 min in PBS-BB. Sections were incubated overnight at 4C in PBS-BB or mouse anti-progesterone antibody (diluted 1:100 in PBS-BB; Novocastra, Newcastle upon Tyne, UK) followed by RT incubations in HRP-conjugated donkey anti-mouse antibodies (1 hr, 1:1000 in PBS-BB), biotinyl-tyramide plus reagent (5 min, 1:2000 in amplification buffer; NEN Life Science Products), and streptavidin-AP (30 min, 1:2000 in PBS-BB). A total of 250 µl of pNPP was added to each section and after 15 min 100 µl of the pNPP solution was removed for quantitation. Sections were rinsed and then incubated in BCIP/NBT solution for 10 min. The sections were subsequently boiled for 1 min to destroy residual enzyme activity from the first immunodetection protocol. Sections were then sequentially incubated in PBS-BB (30 min), rabbit anti-cyto-keratin serum (DAKO; 1:2000 in PBS-BB, overnight at 4C), TSA Plus reagent (a nonbiotinylated antigen-conjugated tyramide, 5 min, 1:2000 in amplification buffer; NEN Life Science Products), and anti-TSA Plus-HRP (1 hr, 1:20 in PBS-BB; NEN Life Science Products). Then 250 µl of TMB solution was added to each section and after 3 min 100 µl was removed for quantitation. Sections were rinsed and incubated in DAB/metal solution for 3 min.

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 (Homburger and Larsen 1996 ). Serial dilutions of serum from two ANA-positive patients (determined by clinical immunofluorescence testing) and positive and negative control sera (Kallestad Laboratories; Chaska, MN) were tested in the Midwestern assay to localize and quantitate ANA reactivity.

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{lambda}. 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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Figure 5. Immunohistochemical detection of ANA reactivity shows diffuse homogeneous labeling in the positive control serum (A), speckled in Sample 1 (B), rim pattern in Sample 2 (C), and no detectable staining in the negative control serum (D). Bar = 25 µm.

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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Table 1. Detection of anti-nuclear antibodies in human serum samples

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 (Gerdes et al. 1984 ). Antibodies against Ki-67 antigen have been used to identify proliferating cells in various neoplasms, and Ki-67 immunoreactivity in certain types of neoplasms correlates with a poor prognosis (Karamitopoulou et al. 1994 ). To determine if the Midwestern assay would be a useful technique for detecting Ki-67 reactivity in formalin-fixed, paraffin-embedded sections of human neoplasms, the visually determined Ki-67 cell labeling index was compared with the Ki-67 Midwestern assay results in 20 human brain biopsies. The cases consisted of two non-neoplastic samples, two medulloblastomas, four meningiomas, 11 glial neoplasms of various histological types and grades (including two glioblastoma multiformes), and a metastatic carcinoma. Sections were deparaffinized, boiled in 0.01 M citrate buffer, pH 6.0, incubated in 0.3% H2O2 in MeOH, and placed in PBS-BB for 30 min. Sections were then incubated overnight at 4C with either mouse anti-Ki-67 antibodies [MIB-1 (Immunotech, Westbrook ME), 1:1000], mouse anti-DNA antibodies (Pierce; anti-DNA, 121-3; 1:1000), or PBS-BB, followed by TSA, OPD incubation, and DAB/metal deposition as described above. Sections were lightly counterstained with hematoxylin, dehydrated, and mounted in Permount (Fisher Scientific; Fair Lawn, NJ).

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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Figure 6. Immunohistochemical detection of Ki-67 and DNA immunoreactivity in human brain specimens. Frequent, strong nuclear Ki-67 signal is observed in a case of glioblastoma multiforme (A) but is absent in non-neoplastic brain (B). Nuclear DNA immunoreactivity is observed in all cases (C). Linear regression analysis (D) of Ki-67 cell labeling (expressed as a percentage of Ki-67-positive nuclei/total cell nuclei as counted in four randomly selected x40-magnification microscopic fields) and Midwestern assay determination of the ratio of Ki-67 to DNA reactivity show a significant correlation between the two values (r2 = 0.60, F = 26.1, p < 0.005).


  Discussion
Top
Summary
Introduction
Methods and Results
Discussion
Literature Cited

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 (Reeves et al. 1996 ). van Leuven et al. 1978 described a technique in which an enzyme-linked probe was used for both immunolocalization of a target and its quantitation. However, quantitation required liberation of the enzyme conjugate from the target antigen-antibody complexes in the sample, precluding target localization in the same sample. Therefore, to perform both antigen localization and quantitation, duplicate samples were required. Similarly, Makler et al. 1981a , Makler et al. 1981b described a technique for localizing and quantitating cell surface antigens on red blood cells using enzyme-linked antibodies that required separate aliquots of the red blood cell sample/enzyme-linked antibody mixture for antigen localization and quantitation. Although simultaneous quantitation and localization were not achieved, both of these methods demonstrated the feasibility of generating quantitative data from fixed cell and tissue samples using enzyme-linked probes.

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 (Coates and Binder 1984 ). Similarly, hybridoma binding to cells cultured in 96-well plates can be simultaneously quantitated and localized using fluorescently conjugated secondary antibodies and a fluorescence microplate reader, and a fluorescence microscope, respectively (Su et al. 1997 ). However, these techniques preclude routine light microscopic examination and do not use enzyme amplification of the localizable and/or quantifiable signal, thus limiting sensitivity. In addition, none of these procedures uses multiprobe immunodetection on single samples. Therefore, variance in sample size, which will affect target quantitation, cannot be determined with these techniques.

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 (Watanabe et al. 1996 ), and similar studies with the Midwestern assay will be required if an absolute antigen quantitation is necessary. However, in many instances the relative quantitation of antigen immunoreactivity in identically processed biological samples by an objective measurement is sufficiently justified to make the Midwestern assay a useful addition to standard assay techniques.

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 (Zaitsu and Ohkura 1980 ; Murray and Ewen 1992 ; Huang et al. 1993 ) and could be used for target quantitation and localization with a fluorimeter and fluorescence microscope, respectively. Although in most of the examples described above the enzyme-linked probe directly generated the chromogenic reaction products, an enzyme-linked probe can be used to generate a reaction product that is neither a fluorogen or chromogen. For example, the deposition of biotin-conjugated tyramide in the TSA procedure provides a substrate with which enzyme linked- or fluorescently conjugated strep-tavidin can bind, providing both tremendous signal amplification and assay flexibility. The need for signal amplification in the Midwestern assay depends on a variety of factors including target concentration, probe specificity and sensitivity, target preservation and/or the need for antigen retrieval, enzyme activity, duration of the enzyme reaction, and substrate properties. Therefore, for each target and probe combination the Midwestern assay requires experimental optimization. The Midwestern assay does not require image analysis to quantitate target concentration in the sample. However, the assay is not intended as a replacement for image analysis and is perfectly compatible with it as a visualizable reaction product is produced.

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 (Hermiston et al. 1992 ). In addition, if both targets are localized in the same cellular structures, dual detection may be difficult. Recently, several combinations of HRP and AP chromogens have been reported to be capable of producing "mixed" signals, thus permitting true co-localization of chromogenic signals (van der Loos et al. 1996 ). Alternatively, the use of fluorogenic enzyme substrates and/or fluorescent conjugates that can bind to deposited enzyme reaction products (e.g., biotinyl-tyramide) would provide the means to perform multitarget quantitation and immunolocalization.

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.


  Acknowledgments

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.


  Literature Cited
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Summary
Introduction
Methods and Results
Discussion
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