Modifying the JerneNordin PFC Assay for IsotypeSpecific Responses
The above protocols measure mainly IgM-secreting plasma cells; however, the Jerne-Nordin PFC assay (see Basic Protocol 2) can be modified to measure other Ig isotypes. To do this, follow steps 1 to 4 of the Jerne-Nordin PFC assay procedure. Next, suppress the measurement of IgM by adding 50 |l diluted (e.g., 1:100; determine empirically) goat anti-IgM to the tubes into which the TNP-SRBC is added (step 5). At the same time, add 50 |l diluted (e.g., 1:200; determine empirically) rabbit antiserum specific to the desired Ig isotype (i.e., IgG1, IgG2, IgG3, or IgA). Test one or two slides as described and then proceed with slide preparation as described (steps 6 to 10). Incubate the slides 90 to 120 min at 37°C. Add guinea pig complement and complete the assay as described (steps 12 to 16).
The IgM plaques are known as direct plaques while the IgG or IgA plaques are called indirect plaques. B cells from antigen primed and boosted mice can serve as good positive controls for the indirect plaques. IgM PFCs are suppressed since the goat anti-IgM antibody cannot fix guinea pig complement.
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