Thirteen human interferon-α (IFNα) subtypes were expressed in Escherichia coli and purified using an N-terminal affinity tag from the prodomain of subtilisin. IFNα 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 IFNα preparations. The IFNαs were used to produce an anti-IFNα16 antibody (MAb-1B12) that specifically neutralized the biological activity of IFNα16, but not the 12 other IFNαs. Using MAb-1B12, and a previously generated IFNAR1/IFNAR2-FChk heterodimer, an assay was developed to determine total type I IFN biological activity and IFNα16-derived biological activity in an unknown sample.