The heavy chain isotype switch is reported to occur at a rate of 10-6 to 10-5 in B cell hybridomas. Because of the rare occurance, usually two or three rounds of cell sorting and subcloning are needed to isolate these variants, which is labor and time consuming. We find that the switch rate of γ2a variants in a γ2b antibody producing B cell hybridoma, S212, is around 5*10-6 by ELISPOT assay. Using the Alexa Fluor 647 conjugated goat anti-mouse IgG2a heavy chain specific antibody, we first enriched γ2a switched variants in S212 with the use of anti-AF647 microbeads. We then used the FACS to sort the single AF647 positive cell from this enriched population into a single well to ensure the single cell cloning. 18 cells were sorted into 18 wells, and there were hybridoma cells growing in 12 wells after 10 days of culture. We tested the isotype of antibody produced in the supernatant by passing it through the four channels of a CM5 chip which were respectively coated with goat anti-mouse IgG1, IgG2a, IgG2b and IgG3 heavy chain specific antibody in a Biacore instrument. The result showed that all of the 12 antibodies were IgG2a and none of them secreted IgG2b antibody any more. This method will be useful in isolating downstream Ig class switch variants of monoclonal antibodies to obtain antibodies with different Fc functions.
- Copyright © 2013 by The American Association of Immunologists, Inc.