Total concentrations of IgM and IgG in each fraction were quantified with ELISA against ChromPure human IgM and IgG standards (Jackson ImmunoResearch). Standards and IgM- and IgG-purified fractions were coated in serial dilutions in 96-well flat bottom MaxiSorp plates (Nunc) at 4°C overnight in coating buffer (0.1 M sodium carbonate and 0.1 M sodium bicarbonate). Plates were washed in PBS-Tween 0.05% three times and blocked with 1% casein in PBS for 2 hours at 37°C, and IgG and IgM were detected with goat anti-human IgG horseradish peroxidase (HRP; Merck Millipore) and sheep anti-human IgM HRP (Chemicon Australia). Binding was detected with TMB (3, 3´,5,5´-tetramethylbenzidine) substrate (Sigma-Aldrich), and reaction was stopped using 1 M HCl. The optical density was read at 450 nm.

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