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ACPA + RF− sera were incubated with active pepsin (Sigma Aldrich) at a 20:1 ratio (serum:enzyme) for 1 hr at 37 °C. F(ab’)2 fragments were negatively selected using Protein G affinity columns (Thermo Fisher) and filtered over a 30 kDa membrane (Millipore) to remove low molecular weight contaminants.
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