A multi-well filtration assay for quantitation of inositol phosphates in biological samples

The quantitation of inositol phosphates (IPs), mediators of certain signal transduction processes, typically involves laborious and time consuming con ventional ion-exchange chromatography procedures. We have developed a high throughput microtiter plate-based IP assay that utilizes vacuum rather than gravitational flow and has significant advantages over existing methods. T he response of recombinant HEK-293 cells expressing human LHRH receptor cDN A to LHRH agonists was used as a model system to develop the assay conditio ns. Cell lysates containing labeled IPs were applied in 96-well plates fitt ed with filtration discs containing regenerated Dowex AG1-X8 resin. Specifi cally bound inositol phosphates were eluted with 1 M ammonium formate in 0. 1 M formic acid directly into a fresh 96-well plate and an aliquot of the e luate from each well is transferred into a 96-well plate and counted. The r esults were comparable to those obtained with the conventional column metho d and the variation among replicates was significantly improved. This assay facilitates rapid quantitation of inositol phosphates from a large number of samples with relative ease and reduced generation of radioactive waste. (C) 1999 Elsevier Science B.V. All rights reserved.