The following paper appeared in Proceedings of the forty-ninth Annual Meeting, Electron Microscopy Society of America; G. W. Bailey (Ed.). San Francisco Press, San Francisco, CA, pp. 284-285 (1991).
A NEW 1.4 NM GOLD-Fab' PROBE
James F. Hainfeld,1 Frederic R. Furuya,1 and Richard D. Powell2
1 Department of Biology, Brookhaven National Laboratory, Upton, NY 11973
2 Nanoprobes, Inc., 25 East Loop Road, Suite 113, Stony Brook, NY 11790.
A major advance in high resolution EM immunoprobes has recently been achieved: the smallest gold particles easily seen directly in the TEM have been coupled to Fab' fragments thus making them the smallest gold-antibody probe commercially available.
The gold particle, Nanogold®, is 1.4 nm in diameter with a very controlled size range, ± 10 % (Fig. 1). This is in sharp contrast to other small gold preparations, such as Auroprobe One (Janssen Life Sciences) which actually ranges from 1-3 nm.1
The Fab' conjugate (Fig. 2) has close to one gold particle per Fab' fragment. This again is different from other gold-IgG probes that have 0.2 - 10 gold particles per IgG. Another difference is that the Nanogold®-Fab' conjugates are separate molecules in solution rather than the often extensive aggregation of other colloidal gold-IgG preparations.1 The problems of ratio of goldparticles to antibody and aggregation in conventional colloidal gold conjugates were shown to be controllable by careful trial and error testing.2 By having a Fab' rather than IgG, a small gold particle, and no aggregation, the size of the probe is substantially improved by 3-10 times. This means that more antigens may be reached, penetration is improved, and the resolution of localization is increased to ~ 5 nm. No silver enhancement is necessary to see the Nanogold® (Fig. 3 shows a field of 1.4 nm gold particles in a brightfield TEM), and this simplifies procedures, reduces backgrounds, and permits use with larger probes for double labeling experiments. Preparation of 1.5 - 2.5 nm probes on Fab fragments was previously described by Baschong and Wrigley.2 However, it was necessary to bind bovine serum albumin (BSA) to the gold as well as Fab to block the nonspecific adsorption to proteins. Nanogold® does not normally bind to proteins so no BSA is required, again reducing the size of this new probe.
Fab'-Nanogold® directed agains erythrocyte surface antigens is shown in Fig. 4. Even in this 80 nm Lowicryl section, the small gold particles are clearly visible.
- Hainfeld, J. F., Proc. of XIIth Cong. for Elec. Micros., p. 954 (1990).
- Baschong, W., and Wrigley, N. G., J. Electr. Micros. Tech., 14 313 (1990).
- We thank M. N. Simon for assistance. This work was supported by USDOE and NIH Grant GM31975.
Fig. 1.--STEM darkfield micrograph of 1.4 nm gold particles (bright dots). Full width 128 nm, 500,000 X mag. Bar=20 nm.
Fig. 2.--STEM darkfield micrograph of Fab's (thick arrow) with 1.4 nm gold particles (thin arrow) attached. Full width 128 nm, 500,000 X mag. Bar=20 nm.
Fig. 3.--TEM brightfield micrograph of 1.4 nm gold particles (arrows). 300,000 X. Bar=30 nm.
Fig. 4.--TEM micrograph of human red blood cell (RBC) with Fab'-1.4 nm gold particles attached (arrow). Magnification=300,000 X. Bar=30 nm.
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