ARTICLE |
Correspondence to: Richard D. Powell, Nanoprobes, Inc., 25 East Loop Road, Suite 124, Stony Brook, NY 11790-3350.
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Summary |
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Immunoprobes that combine a fluorescent label with a 1.4-nm gold cluster compound have been prepared by covalent conjugation with polyclonal antibody Fab' fragments. These new immunoconjugates allow the collection of two complementary sets of data, from fluorescence and electron microscopy, from a single labeling experiment. We find that incorporation of one or more fluorescein moieties into the coordinated ligands of the 1.4-nm Nanogold gold cluster label yields a stable, dual-function immunolabel in which fluorescence quenching is negligible. In a second synthetic strategy, Nanogold and fluorescein were separately covalently conjugated to Fab' fragments to yield a probe with very similar properties. With the use of Fab' fragments, the entire probe is smaller than a whole IgG molecule, and it exhibited excellent penetration properties. It was used to localize the pre-mRNA splicing factor SC35 in the HeLa cell nucleus by both fluorescence and electron microscopy. (J Histochem Cytochem 45:947-956, 1997)
Key Words: immunogold, immunofluorescence antibody, fluorescence microscopy, electron microscopy, immunoprobe, cluster, nucleus
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Introduction |
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Microscopic STUDIES of cells and biological processes utilize a wide variety of immunoconjugates and labeled probes (
Gold cluster complexes used as labels offer a number of advantages over colloidal gold. Reactive groups can be incorporated during synthesis and used for covalent, site-specific attachment to biomolecules. An example is the undecagold cluster; a reactive form of this compound, with a single peripheral maleimide group, has been used to label Fab' fragments at a hinge thiol site (
Recently, a larger gold cluster complex, with a gold core 1.4 nm in diameter, has been developed for use in the same manner. This compound, known as Nano-gold, can be visualized in the transmission electron microscope (
Synthetic modification of gold cluster coordination compounds affords unique opportunities to tailor their properties to specific applications or to introduce additional components with secondary functions. Here, we show how both a gold label and a fluorophore can be incorporated into a single probe in which fluorescence quenching is shown to be almost absent. These combined Nanogold-fluorescent probes enable simultaneous fluorescence and immunogold labeling in a single immunostaining procedure, providing a significant advantage for correlative microscopic studies.
We describe here the preparation and properties of two types of probes (Figure 1), which combine the Nanogold cluster and fluorescent groups into a single covalently linked conjugate. In the first, fluorescein moieties were incorporated into the ligand sphere of the Nanogold cluster; this label was site-specifically conjugated to Fab' fragments (Figure 1A). In the second type, Fab' fragments were first covalently conjugated with unmodified Nanogold (i.e., without attached fluorophore), then in a second reaction this conjugate was covalently labeled with fluorescein (5- and 6-carboxy)-N-hydroxysuccinimidyl ester (Figure 1B). These conjugates were evaluated as secondary probes against a monoclonal primary antibody to localize the SC35 pre-mRNA splicing factor within the nuclei of mammalian cells. Because the fluorescein is tethered to the gold cluster in the first type, yet can be relatively remote from it in the second, it was anticipated that the nature of any interaction between the gold particle and the fluorophore could be inferred by comparing the fluorescence and spectroscopic properties of the two types of probes.
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Materials and Methods |
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Antibodies, Biologicals, and Chemicals
Antibody Fab' fragments were prepared from F(ab')2 fragments purchased either from Jackson Immunoresearch (West Grove, PA; grades with minimal crossreactivity towards human, bovine and horse serum proteins) or Sigma (St Louis, MO). Sheep red blood cells and antibodies against them were purchased from Sigma. Triethylammonium bicarbonate 2.0 M stock was prepared by bubbling gaseous CO2 through 2.0 M triethylamine/water, followed by degasing briefly by water aspiration, then stored and diluted as necessary. Phosphine ligands were synthesized using previously described synthetic methods. GH25 and GCL90 gel filtration media were obtained from Amicon (Danvers, MA), degased using a water aspirator, and packed in glass columns (Omnifit; Cambridge UK). Pre-packed Superose-12 HR (30 cm x 1 cm) columns (Pharmacia; Uppsala, Sweden) were used for separation of labeled conjugates.
Measurements
UV/visible spectra were recorded using a Hewlett-Packard 8452A spectrophotometer, and fluorescence intensities were recorded using a Sequoia-Turner 450 fluorimeter fitted with a narrow-band 490-nm excitation and 515-nm sharp cutoff filters. HPLC was performed using a SSI model 222B or 222C titanium head pump and Pharmacia model UV-1 single-wavelength detector to monitor absorbance at 280 nm.
Preparation of a Combined Nanogold-Fluorescein Cluster Label
Fluorescein was incorporated into a combined label with Nanogold by utilizing substituents of the coordinated tris (aryl) phosphine ligands of the cluster as attachment sites for the fluorescein moieties. The ligands used for this project are shown in Figure 2. Preparations of (1) and (3) have been described previously (
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After formation, the product was purified by HPLC on a desalting column (GH25, Amicon) eluted with 0.6 M triethylammonium bicarbonate in 20% isopropanol/water. The fluorescein-conjugated gold cluster complex was eluted first, in the void volume, followed by smaller species such as uncoordinated phosphine ligands. Fractions containing the desired product were combined, re-evaporated to dryness, then re-chromatographed in the identical manner to ensure complete removal of unattached fluorescein. Fractions containing pure cluster, identified by UV/visible absorption spectra, were combined.
Fab' Labeling with Combined Nanogold-Fluorescein Cluster Label
Goat anti-mouse F(ab')2 fragments (1.0 mg, 10 nmol) were reduced with 40 mM mercaptoethylamine hydrochloride in 0.1 M sodium phosphate buffer, pH 6.0, at room temperature (RT) for 1 hr 10 min, with 5 mM EDTA to prevent reoxidation. The resulting Fab' fragments were separated from the reducing agent using a desalting column (GH25; Amicon).
The fluorescein/1.4-nm gold cluster was evaporated to dryness (rotary evaporator), then re-evaporated to dryness five times from methanol to remove the volatile triethylammonium bicarbonate, then converted to the maleimide as follows: 300 nmol was dissolved in DMSO (0.5 ml) and 0.1 M sodium phosphate, pH 7.50 (0.90 ml) and added to a solution of sulfo-succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC: Pierce, Rockford, IL; 50-fold excess) dissolved in 0.1 ml DMSO. The mixture was incubated at RT for 90 min with gentle agitation. Maleimido-cluster label was separated from excess sulfo-SMCC by HPLC on a desalting column (GH25; Amicon) eluted with 0.02 M sodium phosphate buffer, pH 6.50, with 150 mM sodium chloride and 1 mM EDTA in a mixture of 20% DMSO and deionized water. An eight- to tenfold excess of the activated cluster label was combined with the Fab' fragments. The mixture was agitated gently for 45 min at RT, then stored at 4C overnight.
Reaction products were separated by gel filtration (Superose-12; Pharmacia) to separate monomeric, labeled Fab' fragments both from larger aggregates and from unattached gold cluster labels and smaller molecules. Of the recovered Fab', at least 80% was in the form of monomeric 1:1 conjugates. Fractions containing predominantly this species were combined, reduced to minimal volume (Centricon-30 microconcentrator; Amicon), and separated again using a second gel filtration column (GCL90; Amicon). The eluent used for both columns was 0.02 M sodium phosphate buffer, pH 7.4, with 150 mM NaCl. The fractions that comprised the first two thirds of the conjugate peak from the second separation were combined.
Conjugates were stored, after filtration through a 0.22 µm membrane filter, at a concentration of 0.08 mg/ml in 0.02 M sodium phosphate buffer, pH 7.4, with 150 mM NaCl. For storage longer than 2 weeks, 0.1% bovine serum albumin and 0.05% sodium azide were added to prevent adsorption of the Fab' to the storage vessel and to inhibit bacterial growth.
Fab' Labeling with Nanogold and Fluorescein Attached Separately
Fab' fragments were prepared as described previously. A tenfold excess of monomaleimido-Nanogold was used to label Fab' fragments; then the mixture was incubated at 4C overnight. Nanogold-Fab' was separated by gel filtration HPLC (single pass: Superose-12 column eluted with 0.02 M sodium phosphate with 150 mM NaCl, pH 7.4).
Fractions containing monomeric Nanogold-Fab' conjugates were combined, then exchanged into 0.02 M sodium phosphate with 150 mM sodium chloride, pH 8.0, and concentrated to about 0.5 ml by membrane centrifugation (centricon-30 microconcentrators, Amicon: final yield 0.5 mg labeled Fab' fragments). This solution was then combined with 5-(and 6-)carboxyfluorescein NHS ester (Molecular Probes, Eugene, OR: 0.5 mg, 100-fold excess) in DMSO (0.05 ml) and allowed to react at RT for 1 hr. The product was isolated by gel filtration (Superose-12; Pharmacia) as described above.
Conjugates were stored in 0.02 M sodium phosphate with 150 mM sodium chloride, with 0.1% bovine serum albumin (fraction V, initial fractionation by heat shock; Sigma) to prevent adhesion of the antibody to interior surfaces of the storage vial and 0.05% sodium azide to prevent bacterial growth.
Fluorescence Measurements
Fluorescence measurements in solution were made using a Turner model 415 fluorimeter equipped with a 490-nm narrow band excitation filter and 515-nm sharp cutoff emission filter. Fluorescence intensity was compared with electronic absorbance at 500 nm (the peak of the fluorescein signal from UV/visible spectroscopy). This was corrected for the absorbance of the Nanogold cluster at this wavelength by calculating the absorbance at 500 nm due to Nanogold (using its ratio to the absorbance at 420 nm where fluorescein does not absorb) and subtracting it from the absorbance value. The ratio of fluorescence:absorbance for the combined fluorescein and Nanogold conjugates was compared with that for a commercial fluorescein-labeled polyclonal F(ab')2 conjugate [goat anti-rabbit; Jackson Immunoresearch: labeled using fluorescein (5,6) N-hydroxysuccinimide ester].
Labeling of the SC35 Pre-mRNA Splicing Factor in HeLa Cells and Microscopy
HeLa cells were grown on coverslips for 2 days and fixed, washed, and permeabilized as previously described (
HQ Silver (Nanoprobes; Stony Brook, NY) was used to enhance the gold probe in the cells. HQ Silver was prepared by vortexing a 1:1:1 mixture of initiator, moderator, and activator. The backs of the coverslips were dried using filter paper and 200 µl of the silver enhancement solution was applied to the cell side of the coverslip. After approximately 15 min, or when the silver changed from clear to gray, the silver was washed off the coverslip with citrate buffer. Once silver enhancement was complete, the cells were washed extensively with citrate buffer to remove any nonspecific silver deposits and to prevent any further silver enhancement.
After washes in citrate buffer, the cells were dehydrated through a graded series of ethanol. The cells were infiltrated with a 50:50 solution of ethanol and Epon-Araldite for 18 hr followed by 100% Epon-Araldite for 8 hr. The coverslips were embedded in Epon-Araldite and placed in an oven at 60C for 48 hr to polymerize. The glass coverslips were removed using hydrofluoric acid. Silver-enhanced, gold-labeled sections were cut on a Reichert Ultracut E ultramicrotome using a Diatome diamond knife. Sections were picked up on 200-mesh copper grids and counterstained with 5% uranyl acetate for 5 min and with Reynolds lead citrate (
Immunoblot Testing of Conjugates
Conjugates were tested by immunoblots against serial tenfold dilutions of mouse IgG spotted onto hydrated nitrocellulose membrane. A modification of the procedure of Moeremans (
Buffers
PBS.
Sodium phosphate 0.01 M with 150 mM sodium chloride, pH 7.4.
Wash Buffer. PBS with 0.8% w/w bovine serum albumin (fraction V by heat shock), 0.1% w/w gelatin, type B from bovine skin, approximately 60 bloom, and 2 mM sodium azide.
Blocking Buffer. Wash buffer with additional bovine serum albumin, total BSA content 4%.
Incubation Buffer. Wash buffer with 1% w/w normal goat serum.
Labeling of Sheep Red Blood Cells and Fluorescence Measurements
A simple cell-labeling assay was developed to evaluate the fluorescence of bound antibodies. Fixed sheep red blood cells suspended to 10% hematocrit in PBS buffer (as above) were diluted to 5% hematocrit with deionized water (final volume 10 µl), then mixed with 100 µl of rabbit anti-sheep red blood cell stroma (IgG fraction) (18.5 mg/ml in 0.01 M PBS, pH 7.2) and incubated with gentle shaking for 1 hr at RT. The cells were pelleted (centrifuge, 5 min at 1900 x g), the supernatant was removed, and the cells were washed twice in PBS containing 1% bovine serum albumin (PBS-BSA). After pelleting again, the cells were resuspended in 100 µl of mouse anti-rabbit IgG (H+L) (Jackson: 1.7 mg/ml in 0.01 M sodium phosphate with 0.25 M NaCl, pH 7.6) and incubated with gentle shaking at RT for 1 hr further. After pelleting, and washing twice with PBS-BSA as described above, the cells were resuspended in 50 µl of PBS-BSA. Labeling with the combined Nanogold-fluorescent probes (goat Fab' anti-mouse IgG) was conducted in glass tubes previously blocked with blocking buffer (as described above for blotting) at 40C. Into each tube was placed 10 µl of the red blood cell suspension and 25 µl of (a) the labeled experimental anti-mouse Fab' conjugate at a concentration of 80 µg/mL, or (b) a commercial fluorescein NHS ester-labeled anti-mouse IgG at a concentration of 100 µg/ml. After shaking gently for 1 hr, the cells were washed twice with PBS-BSA buffer as described above and suspended in PBS. Their fluorescence was then measured in the fluorimeter.
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Results |
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Labeling with each of the components of the label was calculated from the UV/visible spectra of the conjugate, the UV/visible spectrum of the unattached cluster, and the spectrum of an analogous gold cluster without incorporated fluorescein. In general, the contribution of the components to the UV/visible spectrum was found to be additive. Both the unconjugated fluorescein-Nanogold cluster label and Fab' conjugates yielded UV/visible absorption spectra in which the spectral features arising from each component were clearly defined. Spectra for Nanogold (with no attached fluorophores), fluorescein-conjugated Nanogold, and a fluorescein-Nanogold-Fab' conjugate are shown in Figure 3. From comparison of the UV/visible spectrum of the fluorescein-substituted cluster with that of an identical but unsubstituted compound prepared using only nonfluorescent ligands, it was calculated that each gold cluster incorporated up to 3 fluorescein moieties but that in the labels that were conjugated to antibodies, 1-2 fluoresceins were typically incorporated per Nanogold.
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From these spectra and measurements of fluorescence emission intensity, the degree to which fluorescence intensity was preserved was calculated using
Figure 4A shows a fluorescence micrograph of HeLa cells in which the SC35 pre-mRNA splicing factor is labeled with the new probe as described above. Figure 5A shows an electron micrograph of a cell from the same preparation. Both are shown with controls in which the monoclonal primary antibody was absent (Figure 4B and Figure 5B). These micrographs were obtained with probes prepared by Nanogold conjugation followed by fluorescein attachment.
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Similar results were obtained from the same procedure using Fab' fragments conjugated with the combined label with the fluorescein attached directly to the Nanogold. In the electron microscope, similar low levels of background staining were found with both types of probes. Because the immunofluorescence staining required a higher concentration of the new probe than would be required for Nanogold labeling, the buffers and washing procedure were carefully optimized for the most effective removal of unbound labeled conjugates. As a result, background staining was found to be similar to that found in previous studies using nonfluorescent Nanogold conjugates.
The staining pattern is consistent with the distribution of SC35 found in previous studies (SC35 antibody recognizes a 35,000 MW protein which has been shown to be required for assembly of spliceosomes and for pre-mRNA splicing. Previously, this protein was found to localize with small nuclear ribonucleoprotein particles (snRNPs) only in the nuclear speckles and not in the surrounding nucleoplasm (
Fluorescence intensities, obtained by fluorimetry, were found to be preserved for suspensions of sheep red blood cells labeled with a tertiary probe in which fluorescein was conjugated to a preformed Nanogold-Fab' conjugate as described above. Fluorescence intensities were typically close to 0.25 relative to those obtained with a commercial fluorescein-labeled IgG used as a positive control. Some of the difference might be due to the larger number of fluoresceins conjugated to the IgG compared with the experimental Fab' probe. However, probes prepared using the combined Nano-gold/fluorescein label (in which the fluorescein is attached directly to the Nanogold) showed substantial reductions in fluorescence intensities. More detailed studies of their fluorescence properties are in progress to determine the exact nature of the interactions between the components of this label.
In immunoblots, conjugates prepared using both approaches successfully visualized 10 pg of rabbit IgG after silver enhancement; both the sensitivity and the level of background staining were similar to those found with nonfluorescent Nanogold in the same blot test, indicating that the gold particle is conjugated to the antibody and that antibody targeting is preserved.
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Discussion |
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Colloidal gold probes are the preferred label for electron microscopy (
An alternative method for fluorescence and electron microscope labeling with a single probe has been reported, based on the photo-oxidation of fluorescent dyes (
The stepwise probe synthesis, with chromatographic separation of the desired product after each step, confirms the attachment configuration of the probe components. With the fluorescein-Nanogold label, because only fluorophores conjugated to the gold particle are used for labeling the antibody and unattached fluorescent groups are removed chromatographically before antibody labeling, the fluorescence of the antibody conjugate must arise from the dual label, and its properties can be directly compared both with fluorescently labeled conjugates and with dual-labeled probes in which the components are separately attached. When a preformed Nanogold-Fab' conjugate is treated with amine-reactive fluorophores, these can react only with the antibody, and this provides a control for the properties of probes prepared using the combined label. The covalent crosslinking rationale enables the molecular definition to be preserved, and it is feasible, using different crosslinking reagents, to use this approach to label a wide variety of proteins and other targeted biomolecules in a site-specific manner.
The combined Nanogold-fluorescein probes behaved in a similar manner to separate fluorescent and immunogold probes. Labeling was imaged by widefield fluorescence and electron microscopy using the same procedures used for conventional fluorescent probes and 1.4-nm Nanogold-labeled probes. Neither component of the label is significantly compromised by the other. Fluorescence intensity measurements, which yielded intensities of approximately 0.4 relative to comparable commercially available fluorescein-labeled IgG conjugates, are consistent with negligible fluorescence quenching by the gold cluster. The higher fluorescence intensity values obtained with IgG likely arise because the IgG conjugate contains more fluorescein groups (3 to 4 per IgG) than the fluorescein-Nano-gold-Fab' conjugate (1 to 2).
Features labeled with nonfluorescently conjugated Nanogold probes are not directly visible by light microscopy or on blots, although they can be visualized for both by silver enhancement. However, the degree of silver enhancement required is greater for light microscopy than for electron microscopy, and once developed for light microscopic observation, such samples are generally overdeveloped for electron microscope use.
In studies of luminescent molecules near metal surfaces (
The described probes provide a significant advantage to correlative fluorescent and electron microscopic studies. This approach can be extended to other fluorescent moieties, which can be incorporated into gold cluster labels using similar approaches, and a number of such studies are now in progress. In the electron microscope, the characteristics of the combined probe were found to be indistinguishable from those of nonfluorescent Nanogold-labeled Fab' fragments. Penetration was the same, and silver enhancement rates and the distribution of silver-enhanced particles were essentially identical. Because the immunofluorescence staining required a higher concentration of the new probe than would be required for Nanogold labeling, the buffers and washing procedure were carefully optimized for the most effective removal of unbound labeled conjugates. As a result, background staining was found to be similar to that with nonfluorescent Nano-gold conjugates.
In summary, we describe a novel reagent that effectively labels cellular antigens for both fluorescence and electron microscopy in a single immunostaining procedure. This enables direct correlative investigations at the cellular and ultrastructural levels before the more extensive processing for electron microscopy is performed on the same sample. The probe preparation methods allow the use of many commonly used fluorescent labels, and the new reagents should be useful for a variety of applications in biological microscopy.
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Acknowledgments |
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Supported by a grant from the National Institute of General Medical Sciences (no. 2R44 GM48328-02) and by a grant from the Center for Biotechnology, State University of New York at Stony Brook.
We thank Dr James F. Marecek for helpful discussions.
Received for publication November 4, 1996; accepted February 13, 1997.
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