A simplified microassay for inhibition of protein synthesis in reticulocyte lysates by immunotoxins.
We describe a simplified cell free reticulocyte lysate protein synthesis assay which has been shown to reliably demonstrate dose dependent inhibition of protein synthesis with cycloheximide and with four ricin A chain immunotoxin conjugates. Maximal [3H]leucine incorporation was observed using 40 microliter of reticulocyte lysate per microtiter well and by incubating samples for 30 minutes before harvesting with a multiple automated sample harvester. Trichloroacetic acid precipitation and hydrogen peroxide decolorization were not required with this method. This rapid, simplified assay system will facilitate testing of agents that inhibit protein synthesis, particularly immunotoxins.
D000818:Animals; D002474:Cell-Free System / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D000922:Immunotoxins / Q000494:pharmacology*; D007930:Leucine / Q000378:metabolism; D008722:Methods; D014176:Protein Biosynthesis; D011817:Rabbits; D012156:Reticulocytes / Q000166:cytology*; D012276:Ricin / Q000494:pharmacology; D014316:Tritium
D000922:Immunotoxins; D014316:Tritium; D012276:Ricin; D003513:Cycloheximide; D007930:Leucine