Preparation of an antibody that recognizes and neutralizes Dictyostelium differentiation-inducing factor-1

Abstract

In the development of the cellular slime mold Dictyostelium discoideum, the differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) plays an important role in the regulation of cell differentiation and chemotaxis; however, the cellular signaling systems involving DIF-1 remain to be elucidated. To obtain a probe for DIF-1, we synthesized a DIF derivative (DIF-1-NH 2; 6-amino-1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one), and prepared an anti-DIF-1 antibody using a DIF-1-NH 2-conjugated macromolecule as the immunogen. A 100-fold dilution of the antibody bound to DIF-1-NH 2-conjugated resin, and this binding was inhibited by co-addition of 20 μM DIF-1 or DIF-1-NH 2. In a monolayer culture of HM44 cells, a DIF-deficient D. discoideum strain, 0.5 nM exogenous DIF-1 induced stalk cell formation in ∼60% of the cells; this induction was dose-dependently inhibited by the antibody (diluted 12.5- or 25-fold). Furthermore, this inhibition by the antibody was recovered by co-addition of 2.5 or10 nM DIF-1. The results indicate that the anti-DIF-1 antibody recognizes DIF-1 and neutralizes its function.