AN AUTOMATED HIGH-THROUGHPUT FILTRATION ASSAY - APPLICATION TO POLYMERASE INHIBITOR IDENTIFICATION

A high throughput filtration assay was developed and automated for scr eening compounds and natural product extracts using 96-well filterplat es. The selection of a hydrophobic membrane and appropriate capture co nditions enabled us to perform the enzymatic reaction, capture of prod ucts, vacuum filtration, and detection in a single filtration micropla te, without any transfer step. The hydrophobic membrane has the advant age of preventing any loss of aqueous solution during the enzymatic re action. The results are comparable with those obtained in a solid plat e format followed by transferring the reaction mixture to a traditiona l hydrophilic filterplate. This assay is being used to screen for micr obial RNA polymerase inhibitors as potential new drugs for treatment o f emerging antibiotic-resistant bacterial and fungal infections. The f iltration setup can be applied to a variety of assay systems. (C) 1997 Academic Press.