Heavy chain specificity may be confirmed by preincubation of sera in 0.1 M 2-Mercaptoethanol. The procedure is as follows: Dilute sera in PBS (four-fold dilutions) with and without 0.1 M 2-Mercaptoethanol through the endpoint and incubate for 30 minutes at 37°C. Incubate the diluted sera on the substrate slide, wash, add FITC-labeled heavy-chain specific IgG or IgM, incubate, wash, mount and observe. IgM titers in the presence of 2-Mercaptoethanol should be reduced at least two dilutions. IgG titers should be reduced little, if any.
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