Molecular immunolabeling with recombinant single-chain variable fragment (scFv) antibodies designed with metal-binding domains

Abstract

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 x 10(6) M(-1)) and scFv:MBD (4.17 x 10(6) M(-1)) were close to those of Fab proteolytic fragments (9.78 x 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.

Figure 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) or Fab (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 antigens in the tight interperoxisomal spaces are heavily labeled with the scFv-, but remain unlabeled with Fab-based labels. (Horizontal field width = 685 nm.)

Figure 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% = 89,657 beads per μm³; SOD: 100% = 10,246 beads per μm³). 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.

Figure 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) or in 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.

Figure 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 γ-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.

Figure 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:MBD_Ni). 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 = 702 nm.)