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A Rapid Method to Characterize Mouse IgG Antibodies and Isolate Native Antigen Binding IgG B Cell Hybridomas. 2015

Haolin Liu, and Janice White, and Frances Crawford, and Niyun Jin, and Xiangwu Ju, and Kangtai Liu, and Chengyu Jiang, and Philippa Marrack, and Gongyi Zhang, and John W Kappler
Howard Hughes Medical Institute, Denver, Colorado, United States of America; Department of Biomedical Research, National Jewish Health, Denver, Colorado, United States of America; Department of Immunology and Microbiology, University of Colorado Denver, Colorado, United States of America.

B cell hybridomas are an important source of monoclonal antibodies. In this paper, we developed a high-throughput method to characterize mouse IgG antibodies using surface plasmon resonance technology. This assay rapidly determines their sub-isotypes, whether they bind native antigen and their approximate affinities for the antigen using only 50 μl of hybridoma cell culture supernatant. Moreover, we found that mouse hybridomas secreting IgG antibodies also have membrane form IgG expression without Igα. Based on this surface IgG, we used flow cytometry to isolate rare γ2a isotype switched variants from a γ2b antibody secreting hybridoma cell line. Also, we used fluorescent antigen to single cell sort antigen binding hybridoma cells from bulk mixture of fused hybridoma cells instead of the traditional multi-microwell plate screening and limiting dilution sub-cloning thus saving time and labor. The IgG monoclonal antibodies specific for the native antigen identified with these methods are suitable for in vivo therapeutic uses, but also for sandwich ELISA assays, histology, flow cytometry, immune precipitation and x-ray crystallography.

UI MeSH Term Description Entries
D007074 Immunoglobulin G The major immunoglobulin isotype class in normal human serum. There are several isotype subclasses of IgG, for example, IgG1, IgG2A, and IgG2B. Gamma Globulin, 7S,IgG,IgG Antibody,Allerglobuline,IgG(T),IgG1,IgG2,IgG2A,IgG2B,IgG3,IgG4,Immunoglobulin GT,Polyglobin,7S Gamma Globulin,Antibody, IgG,GT, Immunoglobulin
D006825 Hybridomas Cells artificially created by fusion of activated lymphocytes with neoplastic cells. The resulting hybrid cells are cloned and produce pure MONOCLONAL ANTIBODIES or T-cell products, identical to those produced by the immunologically competent parent cell. Hybridoma
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D000941 Antigens Substances that are recognized by the immune system and induce an immune reaction. Antigen
D001402 B-Lymphocytes Lymphoid cells concerned with humoral immunity. They are short-lived cells resembling bursa-derived lymphocytes of birds in their production of immunoglobulin upon appropriate stimulation. B-Cells, Lymphocyte,B-Lymphocyte,Bursa-Dependent Lymphocytes,B Cells, Lymphocyte,B Lymphocyte,B Lymphocytes,B-Cell, Lymphocyte,Bursa Dependent Lymphocytes,Bursa-Dependent Lymphocyte,Lymphocyte B-Cell,Lymphocyte B-Cells,Lymphocyte, Bursa-Dependent,Lymphocytes, Bursa-Dependent
D051379 Mice The common name for the genus Mus. Mice, House,Mus,Mus musculus,Mice, Laboratory,Mouse,Mouse, House,Mouse, Laboratory,Mouse, Swiss,Mus domesticus,Mus musculus domesticus,Swiss Mice,House Mice,House Mouse,Laboratory Mice,Laboratory Mouse,Mice, Swiss,Swiss Mouse,domesticus, Mus musculus
D058846 Antibodies, Monoclonal, Murine-Derived Antibodies obtained from a single clone of cells grown in mice or rats. Murine-Derived Monoclonal Antibodies,Antibodies, Murine-Derived Monoclonal,Monoclonal Antibodies, Murine-Derived,Murine Derived Monoclonal Antibodies

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