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The following paper appeared in Proceedings of the fifty-fourth Annual Meeting, Microscopy Society of America; G. W. Bailey, J. M. Corbett, R. V. W. Dimlich, J. R. Michael and N. J., Zaluzec (Eds.). San Francisco Press, San Francisco, CA, pp. 892-893 (1996).

Large Cluster and Combined Fluorescent and Gold Immunoprobes


Richard D. Powell,* James F. Hainfeld,* Carol M. R. Halsey,* David L. Spector,** Shelley Kaurin,** Jennifer McCann,** Roger Craig,~ Fredric S. Fay,~ and Kathryn E. McNamara~

* Nanoprobes, Incorporated, Stony Brook, NY 11790;
** Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724;
~ University of Massachusetts Medical Center, Biomedical Imaging Group, Worcester, MA 01605.


Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm "Nanogold"1 labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand (1) and the amino-substituted ligand (2) in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of sulfo-Succinimidyl- 4-N-maleimido-cyclohexane- 1-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab' fragments against mouse IgG (obtained by reduction of F(ab')2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab' fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both (2) and its protected analog (3): the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HCl in methanol to yield the amino-substituted label. This was conjugated to goat anti-mouse Fab' fragments using the above procedure.

The fluorescein/Nanogold probe was used to label a pre-mRNA splicing factor in HeLa cell nuclei, as a secondary antibody against a monoclonal primary antibody specific for the SC35 splicing factor.2 Specific staining was observed by confocal laser scanning microscopy (Fig. 2(a)), in which measured fluorescence intensities were approximately one-half of those found with a commercially available FITC-labeled secondary IgG antibody, and by transmission electron microscopy with silver enhancement (HQ Silver, Nanoprobes)(Fig. 2(b)).

Combined fluorescein/Nanogold anti-mouse Fab' was also used to label vinculin and pericentrin in smooth muscle cells, as a secondary probe for monoclonal antibodies against these proteins.3 Specific staining was observed by fluorescence microscopy with deconvolution analysis.4 Measured fluorescence intensities were 0.4 relative to a commercially available fluorescein-labeled IgG secondary antibody. For vinculin, some specific staining was also observed by electron microscopy with silver enhancement. These results indicate that these reagents provide a unique method for visualizing the same molecules at both the fluorescence and the EM level.

Functionalized platinum and palladium clusters 2 to 3 nm in diameter were prepared by reduction of an eight-fold excess of metal acetate with a mixture of the functionalized 1,10-phenanthroline ligands (4) and (5), followed by air oxidation of the surface metal atoms.5 Platinum clusters prepared in this manner are shown in Fig. 5. These large clusters were conjugated to goat anti-mouse Fab' fragments using the procedure described above; conjugates were isolated by gel filtration HPLC (Superose-12, Pharmacia). These conjugates were readily silver enhanced: in immunoblots, they exhibited sensitivities equal to Nanogold conjugates, detecting as little as 10 pg of mouse IgG immobilized on nitrocellulose membrane. These larger labels were clearly visible in the electron microscope at 35,000X magnification in the immunolocalization of SC35 in HeLa cells; Nanogold was not visualized under these conditions.


References

  1. Hainfeld, J. F., and Furuya, F. R.; J. Histochem. Cytochem., 40, 177 (1992).

  2. Spector, D. L., Fu, X.-D., and Maniatis, T.; EMBO J., 10, 3467 (1991) .

  3. Moore, E. D. W., et al ; Nature, 365, 657 (1993).

  4. Coggins, L. W., and Fay, F. S.; in "Computer Methods and Programs in Biomedicine;" Vol. 22, p. 69; Elsevier Publishers, New York, NY (1986).

  5. (a) Schmid, G.; Morun, B., and Malm, J.-O.; Angew. Chem. Int. Ed. Eng., 28, 778 (1989); (b) Schmid, G., et al; J. Amer. Chem. Soc., 115, 2046 (1993).

  6. This work was supported by NIH grants 2R44 GM48328 and 2R44 GM49564.

[Figure 1: Ligands and configuration] (8k)

[Figures 2-4: Micrographs] (172k)

FIG. 1. (a) Ligands used for combined fluorescent /gold labels; (b) Fluorescein/Nanogold-Fab'.

FIG. 2. (a) Confocal laser scanning micrograph of HeLa cells labeled with anti-SC35 monoclonal primary antibody, detected with fluorescein/Nanogold-labeled goat Fab' anti-mouse antibody fragment (X 400) (b) Electron micrograph after silver enhancement (X 18,000).

FIG. 3. 1,10-phenanthroline ligands for preparation of large platinum/palladium cluster complexes.

FIG. 4. Transmission electron micrograph of large platinum clusters (Original mag. X 75,000).

Thanks to the San Francisco Press for allowing us to reproduce this online.


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