Molecular immunolabeling with recombinant single-chain variable fragment (scFv) antibodies designed with metal- binding domains Marek Malecki*, Annie Hsu, Lynn Truong, and Sylvia Sanchez Molecular Imaging Laboratories, University of California at San Diego, La Jolla, CA 92093 Communicated by Hans Ris, University of Wisconsin, Madison, WI (received for review June 15, 2001) To study the molecular structure and function of gene products in situ, and atomic-force imaging, are very impressive, neither their we developed a molecular immunolabeling technology. Starting with resolution nor their sensitivity is sufficiently high for identifica- cDNA from hybridomas producing monoclonal antibodies against tion of the individual immunolabeled molecules in situ (5–7). biotin, catalase, and superoxide dismutase, we bioengineered recom- Through provision of an atomic resolution, TEMs are extending binant single-chain variable fragment antibodies (scFv) and their capabilities for in situ imaging of immunolabels far beyond any derivatives containing metal-binding domains (scFv:MBD). As tested other imaging modality available at the present time. with surface plasmon resonance and enzyme-linked immunosorbent To determine spatial relationships between molecules within a assay, affinity binding constants of the scFv (5.21 ؋ 106 M؊1) and complex multimolecular bioassembly, these molecules must be scFv:MBD (4.17 ؋ 106 M؊1) were close to those of Fab proteolytic marked with unique labels that are significantly smaller than and fragments (9.78 ؋ 106 M؊1) derived from the parental IgG antibodies. easily distinguished from the molecules that are to be labeled. In After saturation of MBD with nickel or cobalt, scFv:MBD was imaged general, molecular labels must fulfill two functions: targeting and with electron spectroscopic imaging at each element’s specific energy reporting. To fulfill the labels’ targeting functions, antibodies have loss, thus generating the element’s map. Immunolabeling with been the first choice (8–11). A significant improvement of labeling scFv:MBD resulted in a significant improvement of the labeling efficiency was achieved when instead of the entire IgG (150 kDa) fidelity over that obtained with Fab or IgG derivatives, as it produced molecule, only its proteolytic fragment Fab (50 kDa) was used, but a much heavier specific labeling and label-free background. As de- even that was not small enough to penetrate into dense biomolec- termined with radioimmunoassay, labeling effectiveness with ular assemblies. Recombinant single-chain variable fragment anti- scFv:MBD was nearly the same as with scFv, but much higher than body (scFv) is a much better candidate for this task. scFv consists with scFv conjugated to colloidal gold, Nanogold, or horseradish of heavy and light variable chains constituting about 240 amino peroxidase. This technology opens possibilities for simultaneous acids with a molecular mass of 26 kDa (12–16). Therefore, scFv is imaging of multiple molecules labeled with scFv:MBD at the molec- the smallest targeting domain currently available. ular resolution within the same sample with electron spectroscopic To achieve reporting functions, quantum dots (17, 18), metal- imaging. Moreover, the same scFv:MBD can also be imaged with ligands (19, 20), colloidal gold beads (21), metal clusters (22, 23), fluorescence resonance energy transfer and lifetime imaging as well metal-binding domains (MBDs) (24), carborane clusters (25), as positron emission tomography and magnetic resonance imaging. photoconverted fluorochromes (26), and enzyme molecules (8, 9) Therefore, this technology may serve as an integrative factor in life have been used. However, features of these reporters were not science endeavors. satisfactory for simultaneous localization of multiple molecules within the same sample at molecular resolution. In most cases, the ith the rapid advances in functional genomics and proteom- reporters were big enough to create steric hindrance problems (17). Wics, there is an urgent need for structural and functional Often, the core of a small reporter had to be covered with stabilizing information on how the myriad gene products of the cell combine and functional group shells (17, 18, 23), which significantly in- and interact to form biomolecular assemblies, biomolecular ‘‘ma- creased its final size. Electrostatic charges were generating repul- chines,’’ organelles, and cells. This rapidly growing area of research sion forces (16). In multiple labeling, various antibodies could be calls for molecular imaging technology that would allow us to distinguished from each other only after being conjugated to beads pinpoint the location of many individual molecules within complex of different diameters, which caused changes in their labeling biomolecular assemblies. Ideally, the same markers should be efficiency (8, 9, 13). Moreover, enhancement and enzymatic reac- detectable with various research instruments, allowing us direct tions often led to the products’ drift or diffusion (27, 28). Taking interdisciplinary correlation of the data. Antibody-based markers into account all these facts, our attention has been attracted to should facilitate correlations of the data on gene expression avail- electron spectroscopic imaging (ESI) using the EFTEM by its able from biochemistry, molecular biology, and molecular imaging ability to map multiple elements within the same sample (29–32). laboratories, as well as the location and functions of the gene Exploiting this unique feature, we bioengineered labels containing products. In this sense, molecular imaging should also serve as an integrative factor in life science endeavors (1). Efficient pursuit of CELL BIOLOGY Abbreviations: TEM, transmission electron microscope or microscopy; EFTEM, energy- biomolecular imaging depends on resolution of the instruments and filtering TEM; scFv, recombinant single-chain variable fragment antibody; MMI, monoma- accuracy of the labels. leimido; CG, colloidal gold; NG, Nanogold; HRP, horseradish peroxidase; Pex3, peroxisomal The repertoire of imaging instruments capable of resolving membrane protein 3; CAT, catalase; SOD, superoxide dismutase; MBD, metal-binding separate, multiple, individually labeled molecules in situ, within domain; ESI, electron spectroscopic imaging; SMCC, sulfosuccinimidyl-4-(N-maleimido- methyl)-1-carboxylate; SPR, surface plasmon resonance. complex bioassemblies and cells is practically limited to the trans- *To whom reprint requests should be sent at present address: 1675 Observatory Drive, mission electron microscope (TEM) and its ‘‘offspring,’’ the energy- University of Wisconsin, Madison, WI 53706. E-mail: [email protected]. filtering TEM (EFTEM). High-resolution TEMs can reach a The publication costs of this article were defrayed in part by page charge payment. This spatial resolution of 0.89 Å (2–4). Although recent advances in article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. other imaging techniques, including scanning near-field optics §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.261567298 PNAS ͉ January 8, 2002 ͉ vol. 99 ͉ no. 1 ͉ 213–218 Downloaded by guest on October 1, 2021 MBDs and incorporated atoms of selected elements into them so yeast cells were grown in LB medium supplemented with 0.5% that the distribution of the labels could be pinpointed on the basis methanol to induce peroxisome proliferation (a gift from P. Hazra). of the maps of the elements gathered through ESI (24, 33, 34). The neuronal and glial primary cultures were grown in Dulbecco’s In this publication, we report a molecular labeling technology medium supplemented with 10% bovine serum (gift from L. Ogden that is based on bioengineered scFv antibodies designed with and D. Berg, Univ. of California, San Diego, and G. Konat, West MBDs. Virginia Univ., Morgantown). For preembedding labeling, the cells were fixed with 4% formaldehyde͞0.1% glutaraldehyde buffered Materials and Methods ͞ ͞ with 1 M sorbitol 0.1 M Na Hepes, pH 7.3 5mMNaN3 (SHA). Antibodies and Reporters. Colloidal gold beads (CG) (Ϸ3.4nmin The cells were then permeabilized with 0.1% digitonin in SHA, diameter) were prepared by condensation of metallic gold from a blocked with glycine, and labeled with antibodies in SHA contain- supersaturated solution (10). The beads were adsorbed to antibod- ing 4% skimmed milk. The labeled cells were spun down into 0.4% ies above their isoelectric points. Monomaleimido-Nanogold gelatin and cut into Ϸ0.5-mm3 cubes in ice-cold HBS. These were (MMI-NG) (15 kDa) (Nanoprobes, Yaphank, NY; 1.4-nm diam- dehydrated with 66% ethylene glycol at 4°C, followed by 80% eter core consisting of 67 gold atoms within a 3.4-nm organic shell) ethanol at Ϫ15°C, and then infiltrated by gradually increasing the was mixed with the buffer containing antibodies. The product was concentration of embedding media: Epon and LR white (Ted Pella, purified by gel filtration and ion-exchange chromatography (17). Redding, CA) (49, 50). For immunolabeling on thawed sucrose- Horseradish peroxidase (HRP) (40 kDa) (Sigma) was activated infused cryosections, the cells were fixed with formaldehyde͞ through the sulfosuccinimidyl-4-(N-maleimidomethyl)-1-carboxy- glutaraldehyde, rinsed and pelleted into the gelatin cushion, and late (SMCC) hetero-bifunctional linker (Pierce) in sodium borate. infused with buffered 330 mM sucrose (51). The samples were After purification, it was coupled to antibodies. An MBD consisting shaped into