To study the molecular structure and function of gene products in situ, we developed a molecular immunolabeling technology. Starting with cDNA from hybridomas producing monoclonal antibodies against biotin, catalase, and superoxide dismutase, we bioengineered recombinant single-chain variable fragment antibodies (scFv) and their derivatives containing metal-binding domains (scFv:MBD). As tested with surface plasmon resonance and enzyme-linked immunosorbent assay, affinity binding constants of the scFv (5.21 ؋ 10 6 M ؊1 ) and scFv:MBD (4.17 ؋ 10 6 M ؊1 ) were close to those of Fab proteolytic fragments (9.78 ؋ 10 6 M ؊1 ) derived from the parental IgG antibodies. After saturation of MBD with nickel or cobalt, scFv:MBD was imaged with electron spectroscopic imaging at each element's specific energy loss, thus generating the element's map. Immunolabeling with scFv:MBD resulted in a significant improvement of the labeling fidelity over that obtained with Fab or IgG derivatives, as it produced a much heavier specific labeling and label-free background. As determined with radioimmunoassay, labeling effectiveness with scFv:MBD was nearly the same as with scFv, but much higher than with scFv conjugated to colloidal gold, Nanogold, or horseradish peroxidase. This technology opens possibilities for simultaneous imaging of multiple molecules labeled with scFv:MBD at the molecular resolution within the same sample with electron spectroscopic imaging. Moreover, the same scFv:MBD can also be imaged with fluorescence resonance energy transfer and lifetime imaging as well as positron emission tomography and magnetic resonance imaging. Therefore, this technology may serve as an integrative factor in life science endeavors.

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Development of supramolecular resolution immunolabeling using derivatized IgG class antibody (Ab) technology has been a longstanding, but largely unrealized goal. Limitations of existing hybridoma and recombinant IgG-based approaches include steric hindrance in accessing the antigens, limited number of accessible receptors, random incorporation/attachment of metal ions and colloids, reduced affinity of the derivatized Ab toward the targeted ligand or receptor. Recently, we pioneered a unique technology of recombinant fusion proteins consisting of variable fragment (Fv) antibodies as targeting domains and metal coordination sites as reporting domains [1].The objective of this work is development of metal-tagged molecular markers, which are capable of targeting biomolecules, using bioengineered recombinant antibodies.The antibodies were bioengineered using two complementary routes: (a) natural immunization, affinity purification; (b) preparation of the libraries with expression in yeast Pichia Pastoris and bacteria Escherichia Coli, followed by selection through biopanning and affinity purification. The antibodies were derivatized with various metal ions and colloids (Fe, Co, Gd, etc). Affinity testing was performed using Western blotting, surface plasmon resonance, ELISA, and immunofluorescence. For molecular analysis, glioma cells were labeled with antibodies, rapidly frozen, freeze-substituted, infiltrated with K4M at low temperature with UV polymerization, ultra thin sectioned. Imaging was performed on VG HB501 STEM with Gatan electron energy loss (EEL) and Noran energy dispersive X-ray (EDX) detectors. Spectral imaging (SI) mapping was conducted with Emispec software.The antibodies retained affinity towards targeted receptors even at high levels of their derivatization. Not only cell ultrastructure was visible through zero loss imaging with EEL SI (fig.A), but also metal tagged antibody distribution on cell receptors was demonstrated through EEL and/or EDX SI based upon tagged metal elemental mapping (fig.B) [2]. ScFv recombinant antibody fragments have much smaller size (29kDa), than natural IgG (155kDa), which improves their hydrodynamic diameter and volumetric density. Absence of Fc fragment reduces non-specific binding. Presence of metal tags not only allows us supramolecular imaging, but also opens avenues toward molecular imaging of our recombinant antibodies in vivo. [3] [1] M. Malecki et al., Proc. Nat. Acad. Sci. USA 99 (2002) 213-219 [2] Figure legends. A. EEL image of the glioma cell. B. Elemental map of the metal coordinated within the structure of recombinant antibody, while labeling transferrin receptor. Horizontal field width 300 nm. [3] Supported by the NIH Grant.

In life sciences, the essential part of the functional molecular analysis is the unambiguous identification of biochemical composition of the observed structures. This analysis is expected to create a bridge between functional data pouring from biochemistry and molecular biology laboratories with the molecular architecture data available from ultrastructural images. This goal can be attained by application of various ultrastructural tags (See: Albrecht et al. 1992).A new promising approach for high fidelity labeling is offered by molecular cloning and expression of molecules containing metal binding sites making them suitable for electron spectroscopic imaging (ESI) (Malecki 1995). A truly enormous potential of ESI relies in its ability for mapping of various elements within the same sample. Interactions of electrons with an atom result in the electrons specific energy loss. Based upon these energy losses distribution of the elements within the sample can be mapped.

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