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2005 Scientific Report

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  • Proteins
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  • Protein
  • Michigan
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A B mapping. A random

A B mapping. A random peptide library is constructed by genetically fusing oligonucleotides to the coding sequence of a coat protein of bacteriophage, resulting in display of the fused polypeptide on the surface of the virion. Phage display has been used to create a physical linkage between a vast library of random peptide sequences and the DNA encoding each sequence, allowing rapid identification of peptide ligands for a variety of target molecules such as antibodies. A library of phage is exposed to a plate coated with mAb. Unbound phages are washed away, and specifically bound phages are eluted by lowering the pH. The eluted pool of phage is amplified, and the process is repeated for two more rounds. Individual clones are isolated, screened by ELISA, and sequenced. We have successfully epitope-mapped a variety of important mAbs including anti-HGF/SF, anti-Met, and anti-anthrax lethal factor. We are now exploring the use of this technology on protein-protein interactions; one example is the mapping of the HGF/SF-Met binding site in an in vitro system, and several interesting peptides have been selected from the library as being potential Met antagonists. Functioning as an antibody production core facility at the Van Andel Research Institute, this lab has extensive capabilities in the generation, characterization, scaled-up production, and purification of mAbs using comprehensive cutting-edge technologies. The technologies and services available in the core include animal immunization and antigen preparation; peptide design; DNA immunization (Gene-gun technology); immunization of a wide range of antibody-producing models (including mice, rats, rabbits, human cells, and transgenic or knock-out mice); and in vitro immunization. Other services we provide include the generation of hybridomas from spleen cells of immunized mice, rats, and rabbits; hybridoma expansion and subcloning; cryopreservation of hybridomas secreting mAbs; monoclonal antibody isotyping; ELSIA screening of hybridoma supernatants; monoclonal antibody characterization by immunoprecipitation, Western blot, immunohistochemistry, immunofluorescence staining, FACS, or in vitro bioassays; production of bulk quantities of mAbs using high-density cell culture; purification of mAbs using FPLC affinity columns; generation of bi-specific mAbs by secondary fusion; conjugation of mAbs to detection enzymes (biotin/streptavidin, fluorescence reporters, etc.); and the development of detection methods/kits such as sandwich ELISA. Over the past few years, this facility has generated more than 200 different mAbs, 10 of which have been licensed to commercial companies. We have also contracted services to local biotechnology companies to generate, characterize, produce, and purify mAbs for their research/diagnostic kit development. S114 Figure 1. A) SDS–PAGE of Fab- Met-1 fragment purified by affinity chromatography. Lane 1, standard molecular weight markers; lane 2, purified Fab fragment under reducing conditions. The concentration of the running gel was 12%. B) Immunoprecipitation analysis of Fab-Met-1. Met from cell extracts was immunoprecipitated with purified Fab-Met-1 and detected by western blot analysis. Lane 1 is S114 cell lysate immunoprecipitated with C-28 (rabbit anti-human Met polyclonal antibody). Lanes 2–5 are cell lysates immunoprecipitated with Fab-Met-1: lane 2, S-114 (Met+); lane 3, MKN45 (Met+); lane 4, M14 cell (Met–); and lane 5, NIH3T3 (Met–). M14 Figure 2. The binding affinity of a selected Fab fragment (Fab-Met-1) was tested by FACS analysis. Two cell lines, S114 (Met+) and M14 (Met–), were incubated with purified Fab-Met-1. Bound Fab was detected by staining the cells with secondary goat anti-human Fab–FITC conjugate and analyzing by FACS (black lines). Green lines indicate staining with the secondary antibody only. 14

External Collaborators Zhen-qing Feng, Nanjing Medical University, China Milton Gross, Department of Veterans Affairs Medical Center/University of Michigan, Ann Arbor Xiao-hong Guan, Nanjing Medical University, China Beatrice Knudsen, Fred Hutchinson Cancer Research Center, Seattle, Washington Yi Ren, Cancer Center, Cleveland Clinic Foundation, Cleveland, Ohio Kang-lin Wan, Chinese Centers for Disease Control and Prevention, Beijing, China David Waters, Gerald P. Murphy Cancer Foundation, Seattle, Washington David Wenkert, Michigan State University, East Lansing Wei-cheng You, Beijing Institute for Cancer Research, China Recent Publications Jiao, Y., P. Zhao, J. Zhu, T. Grabinski, Z. Feng, X. Guan, R.S. Skinner, M.D. Gross, Y. Su, G.F. Vande Woude, R.V. Hay, and B. Cao. In press. Construction of human naïve Fab library and characterization of anti-Met Fab fragment generated from the library. Molecular Biotechnology. Zhang, Yu-Wen, Yanli Su, Nathan Lanning, Margaret Gustafson, Nariyoshi Shinomiya, Ping Zhao, Brian Cao, Galia Tsarfaty, Ling-Mei Wang, Rick Hay, and George F. Vande Woude. 2005. Enhanced growth of human Met-expressing xenografts in a new strain of immunocompromised mice transgenic for human hepatocyte growth factor/scatter factor. Oncogene 24(1): 101–106. Tan, Min-Han, Carl Morrison, Pengfei Wang, Ximing Yang, Carola J. Haven, Chun Zhang, Ping Zhao, Maria S. Tretiakova, Eeva Korpi-Hyovalti, John R. Burgess, Khee Chee Soo, Wei-Keat Cheah, Brian Cao, James Resau, Hans Morreau, and Bin Tean Teh. 2004. Loss of parafibromin immunoreactivity is a distinguishing feature of parathyroid carcinoma. Clinical Cancer Research 10(19): 6629–6637. Zhao, Ping, Xudong Liang, Jessica Kalbfleisch, Han-Mo Koo, and Brian Cao. 2003. Neutralizing monoclonal antibody against anthrax lethal factor inhibits intoxication in a mouse model. Human Antibodies 12(4): 129–135. From left to right: Zhu, Wang, Cao, Ferrell, Grabinski, Zhao 15

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