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

PloS One
Haolin LiuJohn W Kappler

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.

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Citations

Aug 18, 2016·Nature Communications·Mark PogsonSai T Reddy
Dec 8, 2019·The Journal of Immunology : Official Journal of the American Association of Immunologists·Giuseppe A SauttoTed M Ross
Aug 5, 2020·Proceedings of the National Academy of Sciences of the United States of America·Haolin LiuGongyi Zhang
Aug 21, 2020·The Journal of Clinical Investigation·Jinmin LeeAimee S Payne

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Methods Mentioned

BETA
ELISA
x-ray crystallography
surface plasmon resonance
chip
single
PCR
flow cytometry
ELISAs

Software Mentioned

BIAcore

Related Concepts

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