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The following paper appeared in Proceedings of the fifty-sixth Annual Meeting, Microscopy Society of America; Bailey, G. W.; Alexander, K. B.; Jerome, W. G.; Bond, M. G., and McCarthy, J. J., Eds.; Springer, New York, NY, 1998, pp. 992-993.

Dual-Labeled Probes for Fluorescence and Electron Microscopy


Richard D. Powell, Carol M. R. Halsey, Edmund Gutierrez, James F. Hainfeld and Frederic R. Furuya

Nanoprobes, Incorporated, Stony Brook, NY 11790;


Fluorescein and the 1.4 nm Nanogold cluster have been separately attached to polyclonal antibody Fab' fragments to generate combined fluorescent and gold secondary antibody probes,1,2 and in comparisons with Nanogold, colloidal gold and fluorescein-labeled secondary antibodies, neither label was found to be significantly compromised by the presence of the other. The proximity to a gold label with which a fluorophore retains sufficient fluorescence emission intensity to be useful is limited by non-radiative fluorescence resonance energy transfer (FRET).3 This is a function of the degree of overlap of the fluorophore emission spectrum with the metal particle absorbtion spectrum, and of the separation of the metal particle and the fluorophore;4 for an effective dual-labeled probe, separation should be greater than the Forster distance,3 at which 50 % of excited-state decay occurs by FRET.

Robinson and Vandré's finding that the fluorescent signal obtained with combined Nanogold and fluorescein probes is still present after short periods of silver enhancement2 suggests that larger, and hence more readily visualized metal clusters may still be compatible with fluorescent labels. Therefore, we have prepared new antibody probes in which both fluorophores and 2-3 nm platinum clusters are conjugated to antibody Fab' fragments and IgG molecules. The large clusters were prepared from platinum (II) acetate and stabilized with a mixture of hydorxylated and amino-derivatized 1,10-phenanthroline ligands as described previously,5 and converted to maleimides with sulfo-SMCC.1 The maleimido- clusters were allowed to react overnight at 4°C with IgG molecules or Fab' fragments in which the hinge disulfide bonds had been slectively reduced to thiols.1 Cluster conjugates were isolated by gel filtration (Superose-12 column), then then fluorescently labeled using fluorescein NHS ester (Molecular Probes) or Cy3 monofunctional NHS ester (Amersham Life Sciences) at pH 7.5; the dual-labeled conjugates were isolated by gel filtration (Superose-12).

Anti-rabbit IgG conjugates were used as secondary probes against a polyclonal rabbit anti-red blood cell antibody to label sheep red blood cells in suspension; labeling could be observed by fluorescence microscopy, as shown in Fig. 1a, although preliminary estimates indicate that fluorescence intensities were lower than with a fluorescein-labeled secondary IgG run as a control. Transmission electron microscopy showed approximately 2 nm platinum particles (Fig. 1b, c) which were readily visualized under conditions in which a comparable Nanogold conjugate was not. Immunoblot tests against a target IgG adsorbed to a nitrocellulose membrane also showed specific binding, with visualization of 0.1 ng of target IgG after silver enhancement.

The covalent attachment of metal cluster compounds has also enabled the preparation of combined fluorescent and Nanogold tracers by using starburst dendrimer macromolecules6 as carriers (Scheme 1). Mono-sulfo-NHS-Nanogold, prepared by the activation of monoamino Nanogold with bis (sulfo-succinimidyl) suberate, was coupled to eighth generation polyamidoamine dendrimers; After gel filtration to separate unbound Nanogold, 1 : 1 conjugates were separated from unconjugated dendrimer and from multiply gold-labeled derivatives by hydrophobic interaction chromatography, then labeled with Cy3 or Cy5 monofunctional NHS ester derivatives. The products were separated from excess dyes by repeated membrane centrifugation until the filtrate contained no fluorophore. Because of the high monodispersity of the dendrimer macromolecules, these new conjugates are highly regular in size. Although smaller than fluorescently labeled microspheres, they are sufficiently large that the gold cluster-fluorophore separation is greater than the Forster distance, and fluorescence is preserved.


References

  1. R. D. Powell et al., J. Histochem. Cytochem. 45 (1997) 947.

  2. J. M. Robinson and D. D. Vandr. J. Histochem. Cytochem. 45 (1997) 631.

  3. P. Wu and L. Brand. Anal. Biochem. 218 (1994) 1.

  4. Th. Forster., Ann. Physik., 2 (1948) 55.

  5. R. D. Powell et al., Proc. Ann MSA Meeting, 54 (1996) 892.

  6. D. A. Tomalia, A. M. Naylor and W. A. Goddard III, Angew. Chem. Int. Ed. Eng., 29, (1990), 138.

  7. This work was supported by NIH grants 2R44 GM49564 and 1R44 GM56090.

[Figure 1: Micrographs] (96k)

FIG. 1: (a) Sheep red blood cells labeled with rabbit anti-red blood cell primary antibody followed by platinum / fluorescein anti-rabbit secondary IgG conjugate (original magnification X 1000).

(b) Electron micrograph of platinum cluster IgG conjugate (before fluorescein conjugation), negatively stained with methylamine vanadate on formvar-coated copper grid (original magnification: X 200,000. Bar = 20 nm) (UMIC, SUNY at Stony Brook).

(c) the same conjugate negatively stained with uranyl acetate (original magnification: X 200,000. Bar = 20 nm) (UMIC, SUNY at Stony Brook).

[Gold-Fluorescent Tracer Scheme] (4k)


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