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.
References
Aug 1, 1978·Proceedings of the National Academy of Sciences of the United States of America·B LiesegangK Rajewsky
Aug 7, 1975·Nature·G Köhler, C Milstein
Feb 14, 1992·Journal of Immunological Methods·C Schwab, H R Bosshard
Aug 29, 1991·Nature·A R VenkitaramanM S Neuberger
Jan 1, 1982·Immunological Reviews·F SablitzkyK Rajewsky
Feb 1, 1981·Proceedings of the National Academy of Sciences of the United States of America·M S Neuberger, K Rajewsky
Dec 1, 1982·Proceedings of the National Academy of Sciences of the United States of America·H F BunnW A Eaton
Oct 24, 1995·Proceedings of the National Academy of Sciences of the United States of America·S J MorrisonI L Weissman
May 19, 1995·The Journal of Biological Chemistry·J A FlaspohlerC Milcarek
Jan 1, 1994·Annual Review of Immunology·G WinterH R Hoogenboom
Oct 15, 1993·Journal of Immunological Methods·G B Faguet, J F Agee
Dec 15, 1995·Structure·L C KovariM G Rossmann
Jan 15, 1997·Journal of Immunological Methods·R Karlsson, A Fält
Sep 18, 2003·RNA·Shirley R BruceMartha L Peterson
Jun 29, 2004·Molecular Immunology·Joseph M Dal PortoJohn Cambier
Feb 3, 2005·The Journal of Biological Chemistry·LaToya S JonesC Russell Middaugh
Jan 30, 2008·Cell Research·Hongliang WangChengyu Jiang
Feb 4, 2009·Journal of Immunological Methods·Paul W PriceRichard B Meagher
Aug 4, 2010·Proceedings of the National Academy of Sciences of the United States of America·Xia HongGongyi Zhang
Jan 16, 2014·The Journal of Investigative Dermatology·Christoph M Hammers, John R Stanley
Citations
Mar 11, 2017·Analytical Chemistry·Martin DippongMaren Kuhne
Aug 18, 2016·Nature Communications·Mark PogsonSai T Reddy
Jan 23, 2019·ELife·Nicolas P AndrewsJames S Trimmer
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