Supramolecular Imaging of Immunolabeling with STEM EDX & EEL SI

Abstract

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.

HIV Universal Vaccine

“…After reverse transcription, these primers primers served to create cDNA templates, which were cloned into the plasmid containing CMV promoter and metal binding domains. [25–26, 45–46] Plasmids were propagated in Escherichia coli grown in the Luria-Bartani media in cultures maintained on the shakers at 37 deg. C. After purification on MaxiPreps [Qiagen], the plasmids were electroporated into the cells in cultures of myelomas, which were established from the effusions of the oncology patients, who were diagnosed with Multiple myelomas.…”

“…Chemical reactions involved in linking biomolecules and metallic entities were described in the details. [46–47]…”

“…The cells were labeled with the genomically engineered molecules (GEM) rendered superparamagnetic, as described in details elsewhere. [25–26, 29–30, 45–46] Briefly, the AVA, VR, and VUV were dissolved and all washing steps carried in phenol-free, Ca+ / Mg+- free, PIPES buffered saline solution, supplemented with 20 mM glucose, 10% human serum. The aliquots were dispensed into the magnetism-free NMR tubes (Shigemi).…”

Bioengineering of Reporter Transgenes for Integrated Imaging with Magnetic Resonance, Fluorescence, and Electron Spectroscopy.

“…Recently, we bioengineered the recombinant single-chain variable fragment (scFv) antibodies designed for simultaneous detection with magnetic resonance imaging (MRI) and positron emission tomography (PET) followed by molecular imaging with electron energy loss spectroscopy (EELS) [2]. In our previous work, we bioengineered the plasmid constructs driving organelle targeted expression of the transgene for a fusion protein with the integrated reporter molecules [3]. Here, we report the results of bioengineering of the constructs for transgenes, which products are detectable with MRI, fluorescence, and EELS.The plasmid constructs were bioengineered into the previously described frame [2] in which the scFv site was replaced with the occludin coding sequence.…”

“…Here, we report the results of bioengineering of the constructs for transgenes, which products are detectable with MRI, fluorescence, and EELS.The plasmid constructs were bioengineered into the previously described frame [2] in which the scFv site was replaced with the occludin coding sequence. The constructs were delivered into cultured human umbilical vein endothelial cells via receptor mediated gene transfer [3]. The cells were grown either as monolayers or within polystyrene sponges or as clusters injected into a nude mice.…”