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After 1 h of incubation at 37°C with peroxidase-conjugated rabbit anti-human IgG (1:10,000) or IgM (1:5000) antibody (in PBS-M, Jackson Immuno Research), four washes with PBS-T, ortho-phenylenediamine (0.5 mg/ml; Sigma) in 0.1 M citrate buffer, pH 5.6, containing 0.08% H2O2 was used to develop the reaction.
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