Production of KLH-specific IgG2 antibodies in vivo. Serially diluted prevaccination and postvaccination sera (1:500, 1:1000, 1:2000) were applied on microplates previously coated with KLH and allowed to react sequentially with the following reagents: murine biotinylated anti–human IgG subclass mAb (▪, IgG1; •, IgG2; ♦, IgG3; and □, IgG4) and peroxidase-conjugated avidin. Tetramethylbenzidine was added for color development, and the absorption (A) at 620 nm was read with an ELISA reader. Since serial dilution of the serum samples demonstrated that measurements were collected in the linear phase of the absorption curve, we subtracted the specific background value from the measured value. Mean background absorption in our assay was 0.5 (range, 0.3 to 0.7).