Thirteen human interferon-alpha (IFN alpha) subtypes were expressed in Escherichia coli and purified using an N-terminal affinity tag from the prodomain of subtilisin. IFN alpha subtypes were expressed in soluble form and purified from cell lysates or refolded and purified from inclusion bodies. Proteins produced by either protocol exhibited biological activities equal to or greater than commercially prepared IFN alpha preparations. The IFN alpha s were used to produce an anti-IFN alpha 16 antibody (MAb-1B12) that specifically neutralized the biological activity of IFN alpha 16, but not the 12 other IFNots. Using MAb-1B12, and a previously generated IFNAR1/IFNAR2-FChk heterodimer, an assay was developed to determine total type I IFN biological activity and IFN alpha 6-derived biological activity in an unknown sample. (C) 2014 Elsevier Inc. All rights reserved.