Molecular immunolabeling with recombinant single-chain variable fragment (scFv) 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 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, , 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 -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. -based markers into account all these facts, our attention has been attracted to should facilitate correlations of the data on avail- electron spectroscopic imaging (ESI) using the EFTEM by its able from , , 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 or ; 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 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 thin cones, mounted onto pins, and frozen in a of five glutamic acid and six histidine residues was synthesized rapid-freezer (24). For immunocytochemistry on resin sections, the (Perkin-Elmer, Shelton, CT), activated with SMCC, coupled to cells were prepared as above, but without labeling or permeabili- antibodies (26), and saturated with 3–24 nickel atoms. Polyclonal zation. Alternatively, rapidly frozen cells were freeze-substituted antibodies against peroxisomal membrane protein 3 (Pex3) (gift with acetone at Ϫ80°C, infiltrated, and embedded as described from P. Hazra and S. Subramani, Univ. of California, San Diego), above (49, 50). Ultramicrotomy of the cells in the resin blocks was catalase (CAT), superoxide dismutase (SOD) (Abcam, Cambridge, performed at room temperature with an Ultracut E (Reichert), U.K.) were IgG class raised in rabbits. IgG molecules were digested which for cryo-ultramicrotomy of frozen cells was equipped with an into FabЈ fragments. Sulfhydryl groups of FabЈ were used to FD4E chamber (49–51). covalently couple biotin (Pierce). For generating mouse monoclo- nal antibodies, BALB͞c mice were immunized through s.c. injec- Preembedding Immunolabeling. Before, between, and after labeling tions of biotin, SOD, or CAT (35). Fusion with NS-I murine steps, the cells were rinsed thoroughly through five cycles of myeloma cells was facilitated with polyethylene glycol. Based on the spinning (2,200 rpm in an Eppendorf centrifuge, 4°C) and resus- ELISA tests of supernatants, the hybridoma cell lines SSbio221, pension into HBS with BSA (0.5 mg͞ml). The cells were labeled SScat198, and SSpod311 were selected. After the BALB͞cmice with antibodies (1 ␮g͞ml) at room temperature for 30 min. To had been primed with pristane, the hybridoma cells were injected facilitate imaging after immunolabeling, NG, CG, and MBD con- intraperitoneally. From the ascites fluid, IgG fractions were puri- jugates were enhanced through autometallography (20). To quench fied on protein A͞G columns (Pierce) and digested into FabЈ. For endogenous peroxidase, the samples were incubated with 0.001 M bioengineering of scFv from the hybridomas, total mRNA was NaN3 and 0.001 M H2O2 before the antibody–enzyme step. For isolated and cDNA was generated through reverse transcription detection, the labeled cells were immersed in a solution of 0.1% (36). The HV-linker-HL coding sequence was ligated into pSTE- diaminobenzidine (DAB; Sigma) containing 0.001 M NiCl2 (31). 215(Yol) in place of 215-Vh-Yol-linker-215-Vl-core-st (gift from S. Dubel, EMBL, Heidelberg) (37, 38). The plasmid construct con- Radioimmunoassay (RIA). Effectiveness of labeling with antibodies tained the coding sequences for FLAG to facilitate the product’s carrying various reporter molecules (CG, NG, HRP, and MBD) detection and Cys to facilitate coupling with a reporter molecule was compared with RIA. Radioiodination with 125I (Amersham (39). In addition, the coding sequence for five glutamic acid and six Pharmacia) of anti-FLAG FabЈ from sheep was performed through histidine residues (MBD) was cloned into the amino or carboxyl chloramine-T oxidation followed by removal of the unconjugated terminus (40). These constructs were transformed into Escherichia 125I by gel filtration (specific activity Ϸ 1 ␮Ci͞␮g; 1 ␮Ci ϭ 37 kBq) coli XL1 Blue (Stratagene) (41). The antibodies were purified from (52). Radioiodinated antibodies were applied at 20 ng with 50,000– the periplasmic fraction on chelating Sepharose columns (Amer- 70,000 cpm for 30 min at room temperature. As a control, biotin- sham Pharmacia) saturated with nickel or cobalt (41). Specificity of ylated antibodies were absorbed to immunoplates for1hat37°C, the antibodies was checked with immunoblotting (42) from SDS͞ rinsed with HBS, and labeled with 125I-FabЈ as above. The bound PAGE (43). radioactivity was measured with a ␥-counter (Packard).

ELISA. The antibodies were adsorbed to immunoplates at 37°Cfor Immunolabeling on Sections. Serial resin and cryosections were 1 h. They were immunolabeled with HRP-conjugated anti-FLAG mounted on gold grids (50, 51) and labeled as above. Labeled antibody in Hepes-buffered saline (HBS; 0.1 M NaCl͞0.1MNa cryosections were reembedded with LR white after labeling (52). ͞ Hepes, pH 7.3 5mMNaN3) for 30 min at room temperature (44–46). 3,3Ј,5,5Ј-Tetramethylbenzidine (TMB) substrate (Pierce) Imaging. Images were acquired on JEOL JEM1200EX and Leo 912 was used and the absorbance was read at 450 nm. or 922 TEMs and processed on a stand-alone graphic station (SIS, Boulder, CO; Adobe, San Jose, CA; HyproDesign, Tempe, AZ). Surface Plasmon Resonance (SPR). Biotin was immobilized on an SA sensor chip (Amersham Pharmacia). Binding kinetics were Results measured at various concentrations of scFv between 0.1 and 1 Preembedding Immunolabeling. Preembedding labeling with scFv-NG. mM in HBS with contact times of 8 min at a flow rate of 5 ml͞min The advantage of using scFv-based labels is clear from comparing (46–48). The chip was regenerated with 1 mM HCl. images of the cells labeled with scFv and Fab (Fig. 1). scFv and Fab were conjugated with NG through covalent bonds (scFv-NG and Cells: Fixation, Freezing, and Embedding. Peroxisomes and mitochon- Fab-NG), thus the beads reflected the true distribution of the dria in yeast and mammalian cells were used as test specimens. The labels. scFv-NG and Fab-NG were used as the secondary antibodies

214 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.261567298 Malecki et al. Downloaded by guest on October 1, 2021 Fig. 1. Peroxisomes in yeast cells were first labeled with the biotinylated Fab fragment antibody against Pex3. This was followed by the second immunolabeling with scFv (A)orFab(B) against biotin conjugated to NG through MMI linker. Labeling with scFv-NG results in a much higher labeling density than with Fab-NG. Notice that the in the tight interperoxisomal spaces are heavily labeled with the scFv-, but remain unlabeled with Fab-based labels. (Horizontal field width ϭ 685 nm.)

against biotin, which was coupled to Fab fragments of antibodies results. Moreover, the background was clean in the scFv-labeled against Pex3 used as the primary antibody. Apparently, the use of samples; thus there was no false-positive labeling. scFv versus FabЈ made a huge difference in the ability of the For quantitative evaluation of the results in preembedding scFv-based labels to access the antigens. scFv-labeled peroxisomal labeling, the thickness of the sections is an important factor membrane (Fig. 1A) was covered with a dense band of NG. This contributing to the final count. With a uniform distribution of labeling was much heavier than that present in the Fab-labeled beads, a difference in the section’s thickness would result in a sample (Fig. 1B). Quantitative data are presented in Figs. 2 and 3. proportional difference in the bead count. To exclude such errors, Labeling with scFv-NG resulted in 997 beads per ␮mofthe the setting of the ultramicrotome was always at 50 nm; this section membrane cross section, and 211 with Fab, but labeling with 10-nm thickness was verified by means of fold thickness measurement and IgG CG resulted in Ϸ5 beads per ␮m, which corresponds to the with use of the film thickness monitor. To ensure that every bead bead counts on peroxisomes labeled with classical techniques as was counted, it had to stand out clearly against the background and documented in previous publications (33–37). Moreover, it is worth to generate a good signal-to-noise ratio. Two factors contributed to pointing out that only the scFv-based labels were able to penetrate this ratio: embedment and stain. The choice of postlabeling em- into the narrow spaces between peroxisomes and label the antigens bedment influenced contrast in the microscope and resistance to there (Fig. 1). Anti-Pex3 scFv directly conjugated to NG gave the the electron beam. With LR white (LRW), almost no staining was smallest radius of uncertainty, thus the highest resolution. However, necessary to reveal the ultrastructure and to visualize the beads the two-step approach resulted in a higher sensitivity as shown in sharply. In Epon-embedded cells, the unstained ultrastructure was Fig. 4. These antigens were not accessible even to Fab-NG conju- nearly invisible, but the samples were the most stable under the gates (Fig. 1B), a circumstance that could lead to false-negative beam. Clearly, LRW and Epon had distinctively different electron- CELL BIOLOGY

Fig. 2. Effectiveness of different immunolabeling strategies for CAT and SOD was evaluated on the basis of calculation of NG bead density per unit of the cell volume (CAT: 100% ϭ 89,657 beads per ␮m3; SOD: 100% ϭ 10,246 beads per ␮m3). After labeling of the cells with the biotinylated primary Fab against CAT and SOD, conjugates of NG with Fab and scFv were used for immunolabeling in pre-embedding (@PE), on-resin-section (@ORS), and on-thawed-sucrose-infused-cryosection (@OTCS) approaches.

Malecki et al. PNAS ͉ January 8, 2002 ͉ vol. 99 ͉ no. 1 ͉ 215 Downloaded by guest on October 1, 2021 Fig. 3. Sensitivity of immunolabeling in neuronal cells was assessed either in one step with anti-CAT and anti-SOD scFv covalently conjugated to NG (scFv-NG)orin two steps in which the first anti-CAT and anti-SOD scFv biotinylated antibodies (scFv-B) were followed by the second anti-biotin scFv conjugated with NG (scFv-NG). Immunolabeling efficiency was evaluated by counting the beads and calculating labeling density, NG beads per unit of volume, and is plotted as a percentage. The two-step method has high sensitivity. The one-step method offers high resolution.

scattering cross sections. Another factor that contributed greatly to illustrated in Fig. 5A. The distribution of antibodies containing efficient imaging was the choice of staining. Osmium tetroxide, nickel atoms imaged with ESI at the nickel’s specific edge is known to erode shells of silver enhancements, was avoided alto- presented in Fig. 5B. It is worth mentioning here that the location gether. Sodium vanadate, potassium ferricyanide, and uranyl ace- of the metal definitely pointed to the location of the antibody, tate were used. This modification of classical staining and embed- because the MBD was a part of the fusion protein. As such, it could ding protocols affected the appearance of peroxisomes and not drift away from the antibody. Fab covalently linked to MBD had mitochondria. The faint staining was just sufficient to outline the identical reliability. The two images can be superimposed to match labeled ultrastructure and expose the labels. the label with the ultrastructure. scFv offered the smallest possible Preembedding labeling with scFv-HRP. Biotinylated anti-Pex3 size of the label’s targeting portion. It achieved labeling effective- followed by anti-biotin scFv-HRP resulted in a very distinct band ness close to that of scFv alone (Fig. 4). along peroxisomal membrane. In the case of enzyme-based report- ers, labeling efficiency should translate into local concentration of Comparison of Affinity Constants and Labeling Fidelity. To evaluate the reaction products, which are generated by the antibody-bound contribution of antibody affinity to labeling fidelity, binding affinity . In practice, these products often drifted and͞or diffused. constants were calculated from ELISA and SPR data. Binding Therefore, labeling intensity was hard to quantify, and the radius of affinity constants for Fab, scFv, scFv:MBD (MBD at amino ter- uncertainty was increased. Attempts to improve ultrastructural minus), and scFv:MBD (MBD at carboxyl terminus) were, respec- resolution through reduction of the precipitate’s volume resulted in ϫ 6 Ϫ1 ϫ 6 Ϫ1 ϫ 6 Ϫ1 reduced contrast, making faint diaminobenzidine precipitates tively, 9.78 10 M ,5.21 10 M ,4.17 10 M , and not barely distinguishable from the structures. Therefore, quantitative measurable. evaluation of the labeling effectiveness could be made through RIA For comparison of labeling fidelity of antibodies conjugated to (Fig. 4). different reporter molecules, these antibodies were detected in situ. Preembedding labeling with scFv:MBD. For ESI, the samples had After the last step of immunolabeling for electron microscopy was to meet two requirements: sections had to be uniformly thin to completed, all of the reporter-carrying antibodies that were marked ensure a single scattering event (30, 50, and 80 nm thick for work with FLAG peptide were labeled with 125I-anti-FLAG antibodies. at 80, 120, and 200 kV, respectively), and reporting atoms had to be In a control test involving labeling of the reporter-carrying anti- present in a sufficient number to meet the minimum detectable bodies adsorbed to immunoplates, the presence of covalently linked number criterion (MDN) (3). This was accomplished with reporter molecules did not interfere with efficiency of labeling with scFv:MBD (Fig. 5). radioiodinated antibodies. The relative radioactivity counts are An ultrastructural overview with ESI at the zero energy-loss is listed in Fig. 4.

Fig. 4. Fidelity of immunolabeling with the antibodies carrying various reporter molecules was evaluated on samples of 100,000 neuronal cells, which were labeled in three steps: (i) with biotinylated anti-CAT antibody; (ii) the secondary anti-biotin scFv containing FLAG peptide only (scFv), or additionally containing MBD at the amino terminus (scFv:MBD), or conjugated with NG (scFv-NG), CG (scFv-CG), or HRP (scFv-HRP); (iii) with 125I-radioiodinated anti-FLAG Fab antibodies. Measurements of radioactivity detected (cpm) in a ␥-counter were calculated as percentage of the cpm registered with scFv:MBD as 100%. Cryoimmobilization protocol (Cryo.) results in higher labeling effectiveness than chemical fixation (Chem. Fix.). scFv:MBD results in much higher labeling than the other reporters.

216 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.261567298 Malecki et al. Downloaded by guest on October 1, 2021 Fig. 5. Peroxisomes in yeast cells were labeled with the biotinylated Fab fragment antibody against Pex3. This was followed by secondary immunolabeling with chimeric scFv:MBD saturated with nickel (scFv:MBDNi). Peroxisomal ultrastructure is visualized at the zero-loss (A). The distribution of the immunolabels is visualized at the nickel’s spectroscopic map (B). The distribution of the labels can be referred to the peroxisomal ultrastructure. (Horizontal field width ϭ 702 nm.)

Immunolabeling on Sections. On-thawed-sucrose-infused-cryo-section metal-binding insert’s location in scFv were essential for retaining immunolabeling (OTCS). The cells were chemically fixed and frozen, the paratope affinity, as discussed earlier (44). Therefore, it can be followed by cryosectioning and labeling on thawed sections. The assumed that the molecular size and organic nature of the labels scFv-based labeling effectiveness was significantly higher than that described here facilitated their penetration, leading to increased obtained with Fab and IgG conjugates (Fig. 2) (53–55). The labeling efficiency with scFv compared with Fab (Fig. 1). In ELISA, background was free of unspecific labeling. It can be assumed that immunoblotting, or SPR, where the antibodies had unrestricted the bioengineered scFv was small enough to penetrate into the access to antigens, neither the size of the antibody nor the properties peroxisomal bioarchitecture contained within the depth of the of the reporter mattered. However, these tests were informative sections through a molecular-sieve-like network of chemically concerning affinity and specificity of binding. Usefulness for im- cross-linked molecules. This was in contrast to other labels munocytochemistry could be tested only on cells for labeling cell (Fab-NG, Fab-CG, or Fab-HRP), which were retained on the surface receptors (e.g., CD68, transferrin, etc.) and far more section surface on a manner similar to that reported earlier challenging immunocytochemistry of intracellular compartments (51, 52, 56). (e.g., Pex3, titin, etc.). On-resin-section immunolabeling (ORS). In addition to the den- For two reasons the labels should be small and neutral. First, sity of the native structure and networking induced by cross-linking labeling small densely packed can be accomplished only if fixative, embedment created an additional obstacle for antibodies to the labels can be packed proportionally (e.g., actin polymer), that reach antigens within sections. Increasing hydrophilic features, is, if they can get close to each other. It was demonstrated—e.g., porosity of resins, their etching, extraction, swelling, or partial with 10-nm CG—that electrostatic repulsion forces and label size polymerization might improve accessibility of antigens (49, 50, 52). significantly reduced labeling density, as compared with 1 nm (23). However, heavily cross-linked matrix was still the pri- Second, if the molecules located deep within a dense cellular matrix mary barrier retaining the IgG-based labels on the section’s surface were targeted through preembedding or embedment-free section (51, 52, 56). That matrix kept even the smallest 1-nm ImmunoGold labeling, then steric hindrance and repulsion forces prevented large on the section’s surface without any penetration into its depth (52, labels from entering the structure and significantly reduced the 56). To obtain efficient labeling, neither extraction of embedment, label count (56, 57). However, avidity of the monovalent Fab and nor rinsing off sucrose, nor treatment with detergents was very scFv was lower than that of polyvalent IgG and IgM. Nevertheless, efficient in achieving penetration of the labels. Reducing the size of small monovalent labels of lower avidity not only reflected quan- the labels was necessary and sufficient to achieve this goal. titatively the real occurrence of the labeled molecules but also visualized the lifelike distribution of these molecules with a higher Discussion fidelity. In essence, this technology increases the sensitivity of The most striking result of the work presented here is the significant detection of molecules (i.e., some molecules that would remain increase in labeling efficiency achieved with scFv-based labels over undetected and gave false-negative result with the classical proto- that obtained with IgG- or Fab-based labels. This increase was cols have been revealed with the present method). Moreover, this observed after employing four major reporter molecules. More- method increases the specificity of immunolabeling, because it over, it was achieved with use of two major immunocytochemical excludes the presence of reporters in the structures with a known approaches: pre- and postembedding immunolabeling. Molecular absence of antigens (i.e., false indication that the molecules are labeling patterns were demonstrated at the ultrastructural level with present in sites where they are not: false-positive). Finally, this TEM and ESI using EFTEM. method gives the highest resolution when the scFv antibodies are

Maximum sensitivity, specificity, resolution, and signal strength directly conjugated to reporter molecules. CELL BIOLOGY are sought in immunocytochemical methods to reveal a lifelike For efficient detection of the labels, a strong signal was essential. molecular organization of a biostructure with high fidelity. In If the reporters are to be detected through the same imaging essence, it can be accomplished only if all and only the targeted modality as the labeled structure, then they have to be big enough, molecules are labeled and detected. Improved immunolabeling or accumulated, or enhanced to a sufficient electron density to fidelity described in this publication has its origins in molecular boost the signal of the labels above the signal from the stained interactions that occurred between the antibodies and the labeled structures (e.g., 10-nm gold bead Ϸ cluster of 10 beads of 1 nm Ϸ structures. They are analyzed below. silver͞gold-enhanced 1-nm bead). All these approaches took place Affinity constants of scFv and its parental Fab were nearly at the expense of resolution or fidelity. Therefore, the approaches identical. It is worth stressing here that the linker’s sequence and the presented in this project, in which the reporters were detected at

Malecki et al. PNAS ͉ January 8, 2002 ͉ vol. 99 ͉ no. 1 ͉ 217 Downloaded by guest on October 1, 2021 their energy-loss edges and the labeled structure at the zero loss, It opens avenues for the correlation of research data at the allowed us to avoid that compromise. Our imaging approach molecular level. The scFv-MBD labels may help with the molecular allowed us to increase not only sensitivity (two-step scFv labeling interpretation of the phenomena involved in binding and turnover approach) but also resolution of immunolabeling (primary antibody of antibodies used in PET or MRI. Additionally, new markers can carrying the reporter molecule). Bioengineering of chimeric pro- be tested in cell cultures at the molecular level with EFTEM or teins with MBDs, which were identical in size and charge, but FRET, until the exact mechanisms of the markers’ turnover are different in metal cargo, opened new possibilities for multiple known. Thereafter, they can be safely transferred to clinical practice labeling. These chimeric immunolabels could be detected at the involving MRI. Therefore, this labeling technology will potentially very specific energy losses. have a great impact not only on progress in functional genomics and Finally, we developed the technology for imaging MBDs with proteomics but also on the advances in immunology-based diag- molecular resolution in situ within cells. Other articles have shown nosis and therapy. that organometallic clusters are suitable for magnetic resonance imaging (MRI), positron emission tomography (PET), fluores- We thank Dr. Joyce Sexton (Univ. of Wisconsin) for editing of the cence resonance energy transfer (FRET), and lifetime imaging manuscript and Dr. Ray Egerton (Univ. of Alberta) for calculations of (58–60). Therefore, the scFv-MBD has become a universal marker the spectra. We acknowledge with thanks the generous support that we spanning a whole spectrum of imaging modes, from the structure have received from the National Science Foundation as Grants 9420056, of a biomolecule to its distribution within the entire human body. 9522771, 9902020, and 0094016 to M.M.

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