Journal of lmmunological Methods, 123 (1989) 1-8 1 Elsevier JIM 05294 One-step immunoaffinity purification of bioactive human recombinant IL-lfl with a monoclonal antibody directed to a well-exposed domain of the protein Massimo Bigio 1, Roberta Rossi 1, Maria Giuseppina Borri 1, Maria Cristina Casagli 1, Daniele Nucci l, Cosima Baldari 2, Gianfranco Volpini 1 and Diana Boraschi 1 l Sclavo Research Center, Siena, Italy, and 2 Department of Evolution Biology, University of Siena, Italy (Received23 February1989,revisedreceived11 May 1989,accepted12 May 1989) A monoclonal antibody (mAb) specific for human recombinant IL-lfl (hu rlL-lfl) was produced by immunizing BALB/c mice with hu rlL-lfl purified with classical methods. This mAb recognizes an epitope within the highly hydrophylic fragment spanning amino acid 133-147. The affinity constant of this mAb towards IL-lfl was determined by RIA. An affinity column was prepared by covalent binding of the mAb to Sepharose CL-4B. The column was capable of selectively binding hu rlL-lfl produced in Escherichia coli directly from crude homogenates. The IL-lfl protein yield was higher than 90% with a very good recovery of IL-lfl biological activity. Moreover, the immunosorbent retained at least two thirds of its IL-lfl-binding capacity after 20 cycles of purification. Key words: Recombinant interleukin-lfl, human; Monoclonal antibody;Affinity purification Introduction The term interleukin-1 (IL-1) defines a family of protein molecules endowed with many diverse biological activities important in immune and in- flammatory responses (Dinarello, 1984; Op- penheim et al., 1986). Human recombinant IL-lfl has been produced from E. coli as a soluble cytoplasmic protein (C. Di Liegro, J.L. Telford and M. Melli, unpublished). The recombinant IL- lfl was purified by conventional chromatographic procedures (Casagli et al., 1989). However, previ- ously described methods for purification of IL-lfl generally suffer from the time-consuming multiple Correspondence to: M. Bigio, SclavoResearch Center, Via Fiorentina 1, 53100 Siena, Italy. Abbreviations: mAb, monoclonalantibody; hu rlL-lfl, hu- man recombinantinterleukin-lfl. chromatographic steps (Schmidt, 1984; Cameron et al., 1985; Krakauer, 1985; Kronheim et al., 1986; Wingfield et al., 1986; Meyers et al., 1987). Attempts of immunoaffinity purification of IL-lfl have been also performed with the use of poly- clonal antibodies which, although highly efficient, may create problems of specificity (Dinarello et al., 1977a,b; Simon and Lee~ 1986). We have thus developed an alternative method for hu rlL-lfl purification, based on the use of a monoclonal antibody (mAb) against hu rlL-lfl. In this paper we present the characterization of a selected mAb directed to the IL-lfl molecule and provide data on the use of this mAb as immuno- sorbent to purify hu rlL-lfl. We also describe the purification scheme for hu rlL-lfl starting from the soluble cell lysates of E. coli. This method combines the advantages of high yields and of a very fast procedure to obtain excellent recovery of active and homogenous IL-lfl. 0022-1759/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (BiomedicalDivision)