Production and characterization of monoclonal antibodies that bind to phosphatidylinositol 4,5-bisphosphate

We established a series of monoclonal antibodies (MAbs) that bound to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] which is confidently believed to play an important role in cellular signal transduction. We used complement dependent liposome lysis assay for screening MAbs and antibodies that bind to PI(4,5)P2 but not to phosphatidylinositol 4-monophosphate [PI(4)P] were selected. The reactivity of the MAbs was analyzed by complement dependent liposome lysis assay, enzyme-linked immunosorbent assay (ELISA) and inhibition of liposome lysis by soluble haptens. The MAbs exhibited three distinct reactivity profiles measured by three different assay systems and the results obtained from three typical MAbs designated AM-2, AM-7 and AM-212 are described. On liposome lysis assay, all three MAbs were highly specific with PI(4,5)P2 and no cross-reaction with other acidic phospholipids such as PI(4)P, phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE) and cardiolipin (CL) was observed. All three MAbs were able to react with as low as 0.1 mol% of PI(4,5)P2 embedded in the liposomal membrane. On ELISA, AM-2 and AM-212 bound only to PI(4,5)P2 and did not cross-react with other acidic phospholipids including PI(4)P. In contrast, AM-7 showed considerable binding to other phospholipids such as PI(4)P, PA and PE on ELISA. The reactivities of the MAbs with water soluble haptens such as inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] were examined by inhibition of liposome lysis. Among three clones examined, only AM-212 showed considerable reactivity with Ins(1,4,5)P3 while also showing weak cross-reactivity with fructose 1,6-diphosphate and inositol 1,4-bisphosphate. No cross-reaction with other structural analogs such as inositol 1-monophosphate and inositol 2-monophosphate was observed.