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PLoS One. 2015 Aug 28;10(8):e0136613. doi: 10.1371/journal.pone.0136613. eCollection 2015.

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

Author information

1
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.
2
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.
3
State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China.

Abstract

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.

PMID:
26317987
PMCID:
PMC4552657
DOI:
10.1371/journal.pone.0136613
[Indexed for MEDLINE]
Free PMC Article

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