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

@article{citeulike:2615333, 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 106 M[-]1) and scFv:MBD (4.17 x 106 M[-]1) were close to those of Fab proteolytic fragments (9.78 x 106 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. 10.1073/pnas.261567298}, author = {Malecki, Marek and Hsu, Annie and Truong, Lynn and Sanchez, Sylvia }, citeulike-article-id = {2615333}, doi = {10.1073/pnas.261567298}, journal = {Proceedings of the National Academy of Sciences}, keywords = {electron\_microscopy, methods, microscopy, scfv}, month = {January}, number = {1}, pages = {213--218}, posted-at = {2008-03-31 09:13:33}, priority = {2}, title = {Molecular immunolabeling with recombinant single-chain variable fragment (scFv) antibodies designed with metal-binding domains}, url = {http://dx.doi.org/10.1073/pnas.261567298}, volume = {99}, year = {2002} }

TY - JOUR ID - citeulike:2615333 L3 - citeulike-article-id:2615333 TI - Molecular immunolabeling with recombinant single-chain variable fragment (scFv) antibodies designed with metal-binding domains JF - Proceedings of the National Academy of Sciences VL - 99 IS - 1 SP - 213 EP - 218 N2 - 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 106 M[-]1) and scFv:MBD (4.17 x 106 M[-]1) were close to those of Fab proteolytic fragments (9.78 x 106 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. 10.1073/pnas.261567298 KW - electron_microscopy KW - methods KW - microscopy KW - scfv AU - Malecki, Marek AU - Hsu, Annie AU - Truong, Lynn AU - Sanchez, Sylvia PY - 2002/01/08/ UR - http://dx.doi.org/10.1073/pnas.261567298 ER -