Serum fractionation and confirmation of a heavy-chain IgG (HCAb) immune response following each llama immunization. (A) Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of the pre-immune and Day 42 serum fractions: G1 fraction = IgG3 HCAb, G2 fraction = IgG1 conventional antibody, A1 fraction = IgG2a HCAb, and A2 fraction = IgG2b/c HCAb. Approximately 2.0-2.5 μg of sample was loaded in each lane, except for some of the more dilute A1 and A2 fractions for which smaller, but still visualizable, amounts were loaded. IgM which sometimes co-elutes with IgG2b/c was noted in all A2 fractions examined here. In the Immunization #2 pre-immune and Day 42 A1 serum fractions, an additional band is visible below the 75 kDa marker which may consist of residual unreduced HCAb. (B) ELISAs demonstrated that Immunization #1 Day 42 G1 and G2 serum fractions showed increased recognition for the injected recombinant antigen compared to the corresponding pre-immune serum fractions. (C) An increased response was also observed for all four Immunization #2 Day 42 serum fractions.