Preparation of an antibody that recognizes and neutralizes Dictyostelium differentiation-inducing factor-1.
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 microM 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 approximately 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.
Kubohara
Yuzuru
Y
Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan. kubohara@showa.gunma-u.ac.jp
Kikuchi
Haruhisa
H
Nakamura
Koji
K
Matsuo
Yusuke
Y
Oshima
Yoshiteru
Y
D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology* / Q000302:isolation & purification; D000913:Antibodies, Protozoan / Q000276:immunology* / Q000302:isolation & purification; D004023:Dictyostelium / Q000254:growth & development; D005260:Female; D006588:Hexanones / Q000032:analysis* / Q000276:immunology*; D006843:Hydrocarbons, Chlorinated / Q000032:analysis* / Q000276:immunology*; D011817:Rabbits
D057134:Antibodies, Neutralizing; D000913:Antibodies, Protozoan; C490570:Differentiation-inducing factor-1, Dictyostelium; D006588:Hexanones; D006843:Hydrocarbons, Chlorinated